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Post-translational modifications and expression stability of gpi-anchored and secreted forms of a recombinant… Morrison, Charlotte J. 1997

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P O S T - T R A N S L A T I O N A L M O D I F I C A T I O N S A N D E X P R E S S I O N S T A B I L I T Y O F G P I - A N C H O R E D A N D S E C R E T E D F O R M S O F A R E C O M B I N A N T M E T A L L O P R O T E I N A S E by C H A R L O T T E J. MORRISON B.Sc, The University of Surrey 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Microbiology and Immunology Biotechnology Laboratory We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1997 © Charlotte J. Morrison, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Abstract The majority of recombinant proteins are produced as soluble secreted proteins. An alternative approach involves expressing recombinant proteins on the cell surface as glycosylphosphatidylinositol (GPI)-anchored proteins and harvesting with phosphatidylinositol-phospholipase C. Leishmania GP63 is a GPI-anchored zinc metalloproteinase that is synthesized as a preproprotein and has three potential sites for N-linked glycosylation. Recombinant GP63 was produced in Chinese hamster ovary (CHO) cells as both a GPI-anchored and secreted protein, to investigate the differences in expression levels, stability and post-translational modifications. The expression of both proteolyrically active GP63 (GP63WT) and an active site mutant of GP63 (GP63E265D) was investigated. Flow cytometry was used to follow the stability of GP63WT and GP63E265D expression on the cell surface. Expression of GP63WT was found to be unstable whether or not methotrexate (MTX) selection was maintained. In contrast expression of GP63E265D was stable under M T X selection. In the absence of selection the decline in GP63E265D expression was more gradual than the loss of GP63WT expression. Loss of GP63 expression resulted in higher growth rate nonproducer populations. Expression of recombinant GP63 on the surface of CHO cells enabled a quantitative approach to evaluate expression stability during long-term culture. Comparison of producing and nonproducing populations has given insight into the mechanisms involved in loss of recombinant protein expression. Secreted GP63 production rates were over 20 fold higher than membrane expressed protein, however the post-translational modifications of secreted and membrane expressed proteins were different. GP63 was secreted in a latent form that retained the pro-region normally ii Abstract processed during cell surface expression. Fast atom bombardment-mass spectrometry of the N-linked glycans isolated from both secreted and membrane expressed GP63 showed that the predominant structures were complex biantennary types, however the glycan profiles showed dramatic differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained poly-N-acetyllactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. These changes in post-translational modifications represent important factors to be considered when modifying membrane expressed protein for secreted production. iii Table of Contents Table of Contents Abstract ii Table of Contents iv List of Tables viii List of Figures ix Acknowledgments xii Chapter 1 Introduction 1 1.1 Recombinant Protein Production in Mammalian Cells 1 1.2 Stability of Recombinant Protein Expression in Mammalian Cells 2 1.3 Leishmania GP63 4 1.3.1 Active Site of GP63 6 1.3.2 Activation of GP63 9 1.4 Role of the Pro-region 12 1.5 Processing in Mammalian Cells : 12 1.6 Glycosylation in Mammalian Cells 13 1.6.1 N-linked Glycosylation 14 1.6.2 O-linked Glycosylation 17 1.6.3 Heterogeneity in Glycosylation 17 1.6.4 Effect of Glycosylation on Protein Properties 18 1.6.5 Factors Influencing Glycosylation 19 1.7 Glycosylphosphatidylinositol Anchors 22 1.7.1 GPI-anchor Addition 23 iv Table of Contents 1.7.2 GPI-anchor Function 25 1.7.3 Internalization of GPI-anchored Proteins in Mammalian Cells 25 1.7.4 Phosphatidylinositol-phospholipase Cleavage of GPI-anchors 26 1.8 Secretion Pathway of Membrane bound and Soluble proteins 27 1.9 Thesis objectives 28 Chapter 2 Materials and Methods 30 2.1 Vector Construction 30 2.1.1 Expression of Secreted and Membrane Expressed GP63 30 2.1.2 Expression of a Mature Form of GP63 Lacking the Pro-region 31 2.2 Mutation of the Active Site of GP63 32 2.3 Transfection of CHO Cells 33 2.4 Cell Counts and Viability Determination 33 2.5 Cell Culture 33 2.5.1 Stability of GP63WT(M) and GP63E265D(M) Expression 33 2.5.2 Secretion and Continuous Harvesting of GP63E265D 34 2.5.3 Purification of GP63E265D(M) and GP63E265D(S) for FAB-MS Analysis 35 2.6 Harvesting of GP63WT(M) and GP63E265D(M) from the Cell Surface using PI-PLC..35 2.7 Protein Purification 35 2.8 ELISA 36 2.9 Flow Cytometric Analysis 37 2.10 Analytical SDS-PAGE 37 2.11 Western Blot Analysis 37 2.12 GP63WT(M) & GP63E265D(M) Proteinase Activity 38 Table of Contents 2.13 GP63WT(S) and GP63E265D(S) Activation and Proteinase Activity 38 2.14 Pulse Chase Experiments ; 39 2.14.1 GP63WT(S) and matGP63WT(S) Clones 39 2.14.2 GP63E265D(S) and GP63E265D(M) Clones 39 2.15 Immunoprecipitation 40 2.16 Endo H Digestion 40 2.17 Mass Spectrometry (MS) Sample Preparation and Analysis 40 2.18 Reverse Transcription-Polymerase Chain Reaction 41 2.19 Northern Blot Analysis 42 2.20 Southern Blot Analysis 42 Chapter 3 Expression of GP63 in CHO Cells 44 3.1 Gene Construction and Transfection of CHO Cells 44 3.2 Site Directed Mutagenesis of the L. major GP63 Active site 46 3.3 Expression of a Mature Form of GP63 48 3.4 Membrane Expression and Harvesting with PI-PLC of GP63WT(M) and GP63E265D(M) 48 3.5 Secretion of GP63WT and GP63E265D 51 3.6 Discussion 54 Chapter 4 Stability of Proteolytically Active GP63 and the Active Site Mutant of GP63 Membrane Expression in CHO Cells 57 4.1 Stability of Cell Surface Expression of GP63WT(M) and GP63E265D(M) 58 4.2 Determination of Specific Growth Rates and Model Simulation 62 4.3 RT-PCR Analysis of GP63 mRNA 66 vi Table of Contents 4.4 Southern Blot Analysis 66 4.5 Discussion ; 69 Chapter 5 Comparison of Membrane Expression versus Secretion Production Rates.. .73 5.1 Continuous Harvest of Membrane Expressed GP63E265D(M) 74 5.2 Secretion of GP63E265D(S) 79 5.3 Cell Specific Production of GP63E265D(M) versus GP63E265D(S) 79 5.4 Southern Blot Analysis to Determine Gene Copy Numbers 79 5.5 Northern Blot Analysis of GP63E265D mRNA Levels 84 5.6 Discussion 87 Chapter 6 Comparisons of the Post-translational Modifications of Membrane Expressed and Secreted GP63 91 6.1 Processing of Pro-region and Activation of Cell Surface Expressed GP63WT(M) 92 6.2 Comparison of GP63WT(S) and GP63E265D(S) Proteinase Activation 94 6.3 Role of the Pro-region of GP63 in Secretion 99 6.4 Purification of Recombinant GP63E265D for FAB-MS analysis of Glycans 99 6.5 FAB-MS Analysis of Glycans of Membrane Expressed and Secreted GP63E265D 102 6.6 FAB-MS Analysis of Neuraminidase Treated N-linked Glycans 110 6.7 Pulse chase of Clones Expressing GP63E265D(M) or GP63E265D(S) 110 6.8 Discussion 118 Chapter 7 General Discussion 124 Nomenclature 128 References 131 vii List of Tables List of Tables Table 1. Abbreviations used for GP63 constructs 47 Table 2. Growth rates of clones before and after loss of GP63 expression in the presence and absence of methotrexate 64 Table 3. Cell specific production and mRNA levels of clones expressing GP63E265D(M) and GP63E265D(S) 88 Table 4. Assignments of FAB-MS peaks observed for the molecular and fragment ions of the permethylated N-linked glycans released from membrane and secreted GP63 109 Table 5. Relative proportions of fucosylated and non-fucosylated glycans from FAB-MS peaks observed for the molecular ions of neuraminidase treated permethylated N-linked glycans released from membrane and secreted GP63 112 viii List of Figures List of Figures Figure 1. Leishmania GP63 6 Figure 2. Active site of GP63 8 Figure 3. Activation of GP63 10 Figure 4. Processing of N-linked glycans 15 Figure 5. N-link glycan structures 16 Figure 6. GP63 GPI structure 24 Figure 7. GP63 constructs and the pNUT expression vector 45 Figure 8. Construction of an active site mutant of GP63 (GP63E265D) and characterisation of proteinase activity 49 Figure 9. Flow cytometric analysis of cell surface expression of proteolytically active and inactive GP63 50 Figure 10. PI-PLC harvesting of GP63 52 Figure 11. Secretion of proteolytically active GP63 WT(S) and the active site mutant GP63E265D(S) 53 Figure 12. Flow cytometric analysis of cell surface expression of the active site mutant GP63E265D(M) (clone 29.8.6) 59 Figure 13. Flow cytometric analysis of cell surface expression of proteolytically active GP63WT(M) (clone 9.4.4) 60 Figure 14. Flow cytometric analysis of cell surface expression of proteolytically active GP63WT(M) (clone 41.9.8) 61 Figure 15. Stability of GP63WT(M) expression in the presence and absence of M T X 65 i x List of Figures Figure 16. RT-PCR analysis of clones before and after loss of GP63 expression 67 Figure 17. Southern blot analysis of clones before and after loss of GP63 expression 68 Figure 18. Continuous harvest of GP63E265D(M) from the cell surface of clone 29.8.6 - flow cytometry profdes 75 Figure 19. Continuous harvest of GP63E265D(M) from the cell surface of clone 29.8.6 76 Figure 20. Continuous harvest of GP63E265D(M) from the cell surface of clone 51.2.8 77 Figure 21. Continuous harvest of GP63E265D(M) from the cell surface of clone 65.12.10 78 Figure 22. Secretion of GP63E265D(S) by clone 39.10 80 Figure 23. Secretion of GP63E265D(S) by clone 55.13 81 Figure 24. Secretion of GP63E265D(S) by clone 66.1 82 Figure 25. Cell specific production of GP63E265D 83 Figure 26. Southern blot analysis and of clones that express GP63E265D(M) on the cell surface or secrete GP63E265D(S) 85 Figure 27. Northern blot analysis of clones that express GP63E265D(M) on the cell surface or secrete GP63E265D(S) 86 Figure 28. Processing of GP63WT(M), GP63E265D(M) and GP63WT(S) 93 Figure 29. Proteinase activity of GP63 WT(M) and GP63E265D(M) purified from the cell surface of CHO cells 95 Figure 30. Processing and proteinase activity of purified secreted GP63 WT(S) and GP63E265D(S) 98 Figure 31. Pulse chase to follow expression of GP63WT(S) in clone 49.3 100 Figure 32. Pulse chase to follow expression of matGP63WT(S) in clone 1.4.6.1 101 Figure 33. Production of secreted and membrane expressed GP63E265D 103 List of Figures Figure 34. Purification of GP63E265D(S) and GP63E265D(M) 104 Figure 35. Scheme for analysis of N & O-linked glycan structures by fast atom bombardment-mass spectrometry (FAB-MS) 105 Figure 36. FAB mass spectra of permethylated N-glycans from GP63 in the molecular ion region (mass range 1950-3400) 107 Figure 37. FAB mass spectra of permethylated N-glycans from GP63 in the molecular ion region (mass range 2550-4400) 108 Figure 38. FAB mass spectra of neuraminidase treated permethylated N-glycans from GP63 in the molecular ion region (mass range 1950-3500) I l l Figure 39. Pulse chase to follow expression of GP63E265D(M) in clone 29.8.6 115 Figure 40. Pulse chase to follow expression of GP63E265D(S) in clone 55.13 117 xi Acknowledgments Acknowledgments I thank my supervisor Jamie Piret for his advise, support and guidance throughout the project. Special thanks also to Rob McMaster for major contributions to the project and to my thesis committee Doug Kilburn, Ross MacGillivray and Frank Tufaro for their advise and support. I gratefully acknowledge Anne Dell for her collaboration. I thank the University of British Columbia for the financial support of a University Graduate Fellowship. xii Introduction Chapter 1 Introduction 1.1 Recombinant Protein Production in Mammalian Cells Many recombinant proteins require complex post-translational modifications found only in higher eukaryotic cells. Many protein modifications are performed on entry into the endoplasmic reticulum (ER) and during transit through the ER and the Golgi. These include proteolytic processing, glycosylation, disulphide bond formation, amino acid modifications and glycosylphosphatidylinositol (GPI) anchor addition. In many cases these modifications are critical for protein function. In the case of recombinant therapeutic proteins, it is usually desirable to produce protein with consistent modifications similar to those of the native protein. Chinese hamster ovary (CHO) cells are used widely for the production of recombinant proteins because of their relatively stable integration of amplified cDNA (Schimke, 1984), similar glycosylation patterns to human cells (Jenkins et al., 1996b) and ease of culture in stirred suspension bioreactors. Large scale recombinant protein production by mammalian cells is usually performed in serum-free perfusion or fed batch suspension cultures (Hu and Piret, 1992; Kyung et al., 1994; Xie and Wang, 1994). Most recombinant proteins are produced as secreted proteins. It is a common practice to produce membrane proteins such as adhesion molecules and receptor molecules, as recombinant soluble proteins by removing sequences that encode the signal for GPI attachment or transmembrane regions (Spellman et al., 1991; Ierino et al., 1993; Wojczyk et al., 1995; Bloom et al., 1996). An alternative approach is to produce recombinant membrane proteins on the cell surface as GPI-anchored proteins (Caras et al., 1987; Lin et al., 1990; Scallon et al., 1992; Kennard et al., 1993). For expression of transmembrane or secreted proteins this 1 Introduction involves genetically engineering a GPI signal sequence (Lin et al., 1990; Kennard et al., 1997). A process termed "controlled release" (Kennard et al., 1993) was developed for harvesting of the proteins from the cell surface using a bacterial enzyme phosphatidylinositol-phospholipase C (PI-PLC) specific for GPI anchors (Low et al., 1986). This approach was shown to have many advantages for downstream processing including the separation of the harvest phase from the growth and production phase. Cells were cultured in serum-free medium and harvesting with PI-PLC was performed in small volumes of phosphate buffered saline (PBS). A product concentration of up to 35 u.g/mL and 30% purity was achieved after the initial harvest (Kennard et al., 1993). A number of harvesting regimes for GPI-anchored proteins were investigated including: i) cyclic harvesting, in which cells were harvested every 2 days after recovery of recombinant protein expression, ii) repeated harvesting which was similar to cyclic harvesting but the same enzyme solution was used for each harvest so that increased product concentration was achieved and iii) continuous harvesting of protein from cells cultured in the presence of PI-PLC which released recombinant protein into the culture supernatant (Sunderji, 1994; Sunderji et al., 1997). A process was developed using high density CHO cells on porous microcarriers (Kennard and Piret, 1994) or spheroids (Kennard and Piret, 1995). Harvest concentrations of 100 ug/mL and 35% purity were recovered from cells grown on microcarriers. Harvesting was carried out every 2 days for 40 days. This process facilitated the alternation between growth and harvesting media and could be performed in a suitably designed bioreactor. 1.2 Stability of Recombinant Protein Expression in Mammalian Cells Large scale recombinant protein production involves prolonged culture periods with many cell divisions. In addition, for the production of therapeutic proteins, the presence of selective agents 2 Introduction such as methotrexate (MTX) is not acceptable to regulatory agencies. Loss of protein expression reduces productivity and more importantly, can lead to protein heterogeneity. For instance, the fraction of gamma-carboxylated recombinant Protein C was increased as much as 3-fold in baby hamster kidney clones expressing at 10-fold lower cell specific rates (Guarna et al., 1995). Instability of protein expression in recombinant cell lines can therefore be an important constraint on production processes. The instability of hybridoma and transfectoma cell lines has been investigated by a number of groups (Frame and Hu, 1990; Heath et al., 1990; Ozturk and Palsson, 1990). These studies have shown a general decline in antibody secretion levels over long-term culture, that in many cases was attributed to loss of heavy chain expression (Bae et al., 1995; Couture and Heath, 1995). In some studies flow cytometry was used to estimate the fraction of producers within a population from the cell associated antibody (Merritt and Palsson, 1993; Kromenaker and Srienc, 1994; Bae et al., 1995). Nonproducing populations with increased growth rates were reported to cause the decline in expression (Merritt and Palsson, 1993; Kromenaker and Srienc, 1994). Hybridomas are a product of cell fusion and are inherently unstable, chromosome loss has frequently been reported in these cells (Castillo et al., 1993). Studies of expression stability of heterologous proteins have primarily been concerned with instability in highly amplified cells. Earlier work by Schimke (1984) showed that CHO cells with large numbers of endogenous dihydrofolate reductase (DHFR) genes were obtained by amplification in the presence of increasing amounts of MTX. However these cells were often unstable when cells were grown in the absence of MTX. Co-amplification of transfected DHFR genes and genes of heterologous proteins has been used to obtain amplified CHO cell lines often containing between 100-1000 heterologous gene copies. The expression stability of the amplified 3 Introduction genes of a number of heterologous secreted proteins has been investigated. Amplified heterologous genes were found to be unstable in some cell lines when selective pressure was removed (Kaufman et al., 1985; Wiedle et al., 1988; Pallavicini et al., 1990). However in other cell lines stable protein expression levels were reported for up to 40 population doublings (PD) in the absence of M T X (Kaufman et al., 1985). There have also been some reports of decreasing average recombinant protein production rates in the presence of selective pressure (Cossons et al., 1991; Raper et al., 1992). The mechanisms by which loss of heterologous protein expression can occur in unamplified cell lines, even in the presence of selection have not been extensively studied. 1.3 Leishmania GP63 Leishmanolysin or GP63 is the major surface glycoprotein of the parasitic protozoan Leishmania. Leishmania causes a complex group of diseases in man termed collectively as leishmaniasis. These diseases range from a self-curing skin lesion referred to as cutaneous leishmaniasis to the more severe forms of diffuse cutaneous, mucocutaneous and visceral leishmaniasis. The latter form may be fatal. Drugs administered for treatment of the disease are antimony based and highly toxic. The latest figures from the World Health Organization indicate that there are 12 million cases of leishmaniasis world wide with 0.4 million new cases each year. Endemic areas are restricted to the range of the sandfly vector that transmits the parasite to humans and other mammalian hosts. There are two distinct life stages of Leishmania: the promastigote, an extracellular flagellated form found in the insect host and the amastigote, an obligate intracellular form that resides within the phagolysomal compartment of the mammalian host macrophage. 4 Introduction With the aim of developing a vaccine, the immune response to Leishmania has been extensively characterised. A protective immune response to leishmaniasis has been demonstrated to be T-cell mediated rather than a humoural response. In mice a protective response was shown to be mediated through CD4 + T-cells via a Thl response and interferon-y (IFN-y) production (reviewed by Reed and Scott, 1993). A number of candidate molecules for vaccine development have been identified in both the promastigote and amastigote life stages. These include GP63 (Connell et al., 1993), L A C K antigen (Mougneau et al., 1995), GP46 (McMahon-Pratt et al., 1993) and other membrane and cytoplasmic proteins. GP63 is present at approximately 5 x 105 copies per cell (Etges et al., 1986) in the promastigote life stage of all Leishmania species and is also found on the cell surface of the amastigote life stage in some species (Medina-Acosta et al., 1989; Frommel et al., 1990). Human peripheral blood T-cells from patients with leishmaniasis or from cured patients were demonstrated to respond to purified GP63 from L. major and to recombinant GP63 produced in Escherichia coli (Russo et al., 1991). Purified recombinant GP63 from E. coli (McMaster and Reiner, 1996) and GP63 expressed in a bacille Camlette-Guerin vaccine strain (Connell et al., 1993) or in Salmonella (Yang et al., 1990) have been demonstrated to elicit protective immune responses in mice. As a result GP63 is a prime candidate to form part of a defined subunit vaccine against leishmaniasis. GP63 is a 63 kDa GPI-anchored zinc metalloproteinase (Etges et al., 1986). Based on the gene sequence, L. major GP63 was predicted to be synthesized as a 602 amino acid pre-pro-protein (Fig. 1). The precursor protein contains a 39 amino acid pre or leader sequence at the amino-terminus for targeting to the ER and a 25 amino acid signal sequence at the carboxy terminus which directs GPI attachment. There is also a regulatory pro-region of 61 amino acid that in L. major is processed to leave a 477 amino acid mature and active proteinase. There are 5 Introduction three potential sites for attachment of asparagine-linked glycans (Asp X Ser/Thr) (Button and McMaster, 1988). The structure of N-linked glycan in L. amazonensis GP63 were predominantly biantennary complex oligomannose types and O-linked glycans were not detected (Olafson et al., 1990). The function of GP63 in Leishmania has not been fully identified, although it was shown to play a role in resistance to complement (Brittingham et al., 1995). PRE | PRO | MATURE I GPI 39 61 ^ 25 HEMAH Z n 2 + (CHO) n (CHO) n (CHO) n Figure 1 The predicted structure of L. major GP63 showing the pre region, pro-region, the GPI signal sequence and the mature proteinase. The position of the active site (HEMAH) and three N-linked glycosylation sites (CHO)n are indicated. 1.3.1 Active Site of GP63 GP63 is an endopeptidase with a broad substrate specificity and a wide pH range (Bouvier et al., 1989). A large number of substrates including casein, gelatin, albumin, haemoglobin and fibrinogen were readily hydrolyzed by GP63 (Chaudhuri et al., 1989; Bouvier et al., 1989; Bouvier et al., 1990; Ip et al., 1990; Tzinia and Soteriadou, 1991). There is no consensus sequence for sites cleaved by GP63. Using synthetic peptides GP63 was shown to preferentially cleave peptides at sites with hydrophobic residues in the P,' position and basic amino acids in the P 2' and P 3' positions (Bouvier et al., 1990). In addition tyrosine residues were frequently found at 6 Introduction the P, position. However cleavage was not restricted to sites containing these residues. GP63 also cleaved a synthetic peptide that contained the last four residues of the pro-region and first five of the mature protein (ARSV/VRDVN) at the predicted site between two valine residues (Bouvier et al., 1990). GP63 purified from L. major contained an equal molar ratio of zinc (Bouvier et al., 1989) and based on the predicted protein sequence (Button and McMaster, 1988) contains an active site (HEMAH) that conforms to the consensus sequence (HEXXH) of other well characterised zinc metalloproteinases (McMaster et al., 1994). Based on the crystal structure determined for thermolysin (Matthews et al., 1972; Matthews, 1988) it was proposed that the active site of GP63, like thermolysin, contains a catalytic glutamic acid residue (Glu2 6 5) and two histidine (His) residues involved in zinc co-ordination (Fig. 2) (McMaster et al., 1994). In thermolysin the third zinc binding ligand is a Glu residue located 23 amino acids on the carboxy side of the catalytic Glu (reviewed by Hooper et al., 1994). In the metalloproteinase astacin, this third residue was a His located 9 amino acids from the catalytic Glu within the extended motif (HEXXHXXGXXH) of the astacin family (Bode et al., 1993). The third residue involved in zinc co-ordination in GP63 has not been identified. Located 45-78 amino acids on the carboxy side of the catalytic Glu are 5 Glu residues. The nearest His residue on the carboxy side is separated by 68 amino acids. These residues are all conserved in 9 predicted GP63 protein sequences from 5 species of Leishmania and are all potential candidates for the third zinc binding ligand (McMaster et al., 1994). The mechanism of action of thermolysin was proposed based on the structural studies (Hanguar et al., 1984). In thermolysin the zinc is tetrahedrally co-ordinated with the three ligands and a water molecule. The incoming substrate is presumed to displace the water molecule towards the catalytic Glu forming an ion pair. This ionization allows the 7 Introduction G l u 265 Figure 2. Schematic of the proposed active site of GP63. Based on the active site of thermolysin, the active site of GP63 contains a catalytic glutamic acid (Glu 2 6 5) and two histidine residues (His 2 6 4 & His 2 6 g) involved in zinc co-ordination. The third zinc binding ligand has not been identified in GP63. Substitution of the catalytic glutamic acid with aspartic acid at position 265, maintains a negative charge within the active site but the position is retracted due to the loss of a methylene group. This figure was adapted from a figure by Le Moual et al. 1992. 8 Introduction nucleophilic attack of the zinc bound water molecule on the carbonyl group of the sissile peptide bond. The effects on the proteinase activity of amino acid substitutions of the catalytic Glu in the active site, were investigated for a number of zinc metalloproteinase including stromelysin (Sanches-Lopez et al., 1988), neutral endopeptidase (Devault et al., 1988) and gelatinase A (Crabbe et al., 1994). Replacing the catalytic Glu with a number of different amino acids including glycine, valine, alanine and glutamine resulted in non-functional proteinases. A more conservative substitution of aspartic acid was investigated in neutral endopeptidase and gelatinase A (Devault et al., 1988; Crabbe et al., 1994). This substitution maintained the negative charge within the active site but its position was retracted by approximately 1.4 angstrom due to the loss of a methylene group (Fig. 2). A reduced catalytic activity of at least 400-fold for neutral endopeptidase (Devault et al., 1988) and 100-fold for gelatinase A (Crabbe et al., 1994) were observed for the mutant proteinases compared to the wild type enzymes. Synthetic substrate studies showed that these Glu to Asp mutations did not disrupt substrate binding (Devault et al., 1988; Crabbe et al., 1994). This provided indirect evidence that zinc co-ordination and the tertiary structure of the active site were not disrupted. 1.3.2 Activation of GP63 GP63 shows similarities to members of the family of matrix metalloproteinases (McMaster et al., 1994; Macdonald et al., 1995). One criterium for family members is that they are secreted in a latent form that can be activated via a pathway termed the "cysteine switch" (Fig. 3). According to this model, latency of the proteinase is maintained by an interaction the free sulphydryl of a cysteine residue in the pro-region with the active site zinc. This interaction is disrupted in the 9 Introduction Figure 3. Schematic of the in vitro activation of GP63 via the "cysteine switch" pathway. The histidine (H) residues involved in zinc co-ordination are shown, the third residue (X) has not been identified in GP63. (A) Latent secreted GP63 showing the proposed interaction of the free sulphydryl of the cysteine residue in the pro region and the active site zinc. (B) Activation of GP63 proteinase and the processing of the pro-region occurs after disruption of the interaction between the pro-region cysteine and the zinc, and following co-ordination of the zinc with water. 10 Introduction presence of a number of compounds, such as HgCl 2 , that bind to the sulphydryl of the cysteine residue and allow the co-ordination of the zinc with water. The role of the zinc is thus switched from a non-catalytic to a catalytic one (Springman et al., 1990; Van Wart and Birkedal-Hansen, 1990). The pro-region of GP63 contains a single cysteine and recombinant GP63 expressed in the baculovirus system was secreted in a latent form which, in a manner analogous to the cysteine switch, was activated by the addition of HgCl 2 (Button et al., 1993; Macdonald et al., 1995). Time course experiments showed that after the addition of HgCl 2 to latent GP63, a progressive step-wise removal of the pro-region occurred. By 10 min the predominant species observed was protein with the predicted mature amino-terminus, however four intermediate sequences were detected that disappeared upon further incubation (Macdonald et al., 1995). A similar progressive removal of the pro-region was observed for some matrix metalloproteinases after activation with mercurial compounds (Grant et al., 1987; Nagase et al., 1990; Crabbe et al., 1992). In Leishmania GP63 is expressed on the cell surface as an active proteinase (Etges et al., 1986). Amino acid sequencing of purified GP63 from L. major showed that GP63 was present in a processed form that lacked the putative pro-region (Button and McMaster, 1988). GP63 has also been demonstrated to be active when expressed on the cell surface of COS cells as either a GPI-anchored or a transmembrane protein (Macdonald et al., 1995). The mechanism by which GP63 is processed in Leishmania or COS cells is unknown, activation may occur as a result of an autoprocessing event or alternatively it may involve specific processing enzymes. 11 Introduction 1.4 Role of the Pro-region Many proteins are synthesized as latent precursors containing pro-regions that require cleavage to liberate the mature and active form of the protein. Included in this group are proteinases as well as growth factors, receptors, hormones and plasma proteins. The pro-regions of proteins can be amino-terminal extensions, carboxy-terminal extensions or a combination of both. Amino-terminal extensions are the most common and they can range in size from 40 to over 750 amino acids. One function of the pro-regions of proteinases is to maintain the proteinase in a latent state. Extracellular processing of latent proteinases is often brought about in a controlled hierarchical proteolytic cascade such as in matrix metalloproteinases and plasminogen/plasmin systems (reviewed by Vassalli and Pepper, 1994). The presence of the pro-region was also required by a number of proteins to obtain correctly folded and secreted protein, examples include the bacterial proteinases subtilisin (Ikemura et al., 1987) and a-lytic proteinase (Silen and Agard, 1989), as well as activin A and transforming growth factor-pi (TGF-pi) expressed in a human cell line (Gray and Mason, 1990). Pro-regions are thought to work by actively promoting folding (Baker et al., 1993). Pro-regions have also demonstrated in wide range of proteinases, to act as inhibitors of their respective mature proteinases (reviewed by Baker et al., 1993). Other functions attributed to pro-regions include directing y-carboxylation in plasma proteins (Handford et al., 1991) and multimer assembly in von Willebrand factor (Wise et al., 1988). 1.5 Processing in Mammalian Cells The first proteolytic processing step of secreted and membrane expressed proteins involves the removal of a signal peptide by signal peptidase, that occurs co-translationally on entry into the 12 Introduction ER. A diverse group of proteins such as the pro-enzymes and pro-proteins already described, are synthesized as inactive precursors that require further processing steps to achieve full biological function. These also include virtually all neuropeptides and peptide hormones. Proteolytic cleavage of these precursors is initiated by endoproteolytic cleavage at specific single or paired basic residues (reviewed by Smeekens, 1993). Subsequent processing events may then take place. These processing events occur within the Golgi or in transport vesicles en route to the plasma membrane for the constitutive secretory pathway or within secretory granules of the regulated secretory pathway. Processing in mammalian cells is catalyzed by a set of precursor convertases, serine proteinases that belong to the subtilisin/kexin family. To date seven members have been identified that can be placed in four groups based on their tissue distribution. The most widely expressed are furin and PC7, some overlap in distribution occurs with Pace 4 and PC5, while PCI and PC2 are restricted to endocrine and neuroendocrine cells and PC4 is found only in spermatids (reviewed by Denault and Leduc 1996). Furin is the most characterised of the enzymes. It is a transmembrane protein localized primarily in the Golgi and has been detected in a number of different cell types including CHO and COS cells (Hatsuzawa et al., 1992; Rehemtulla and Kaufman, 1992). Interestingly furin is itself produced as a pro-enzyme with a dibasic cleavage site and has been shown to undergo intramolecular autoprocessing in the ER (Creemers et al., 1994). 1.6 Glycosylation in Mammalian Cells The biological importance of glycosylation and its effects on the properties of proteins are well established (Paulson, 1989; Cumming, 1991a; Varki, 1993). Glycosylation can have a profound effect on the physical properties of a protein as well as the biological function. As a result, the 13 Introduction importance of producing recombinant proteins with consistent glycosylation has been recognized (Jenkins and Curling, 1994). Glycosylation can be classified into three groups according to the type of glycan-peptide linkage: i) N-linked glycans attach to asparagine residues in the consensus sequence Asp-X-Ser/Thr, ii) O-linked glycans attach to the hydroxyl group of serine or threonine residues and iii) GPI membrane anchor oligosaccharides. Glycoproteins frequently possess more than one glycan chain per molecule, these may be both N- & O-linked glycans and the structures at each site may vary. 1.6.1 N-linked Glycosylation N-linked glycosylation of a protein begins with the attachment of a lipid-linked oligosaccharide moiety (Glc3Man9GlcNAc2-P-P-Doli) (Fig. 4) to the asparagine residue. This transfer by oligosaccharyltransferase occurs co-translationally in the ER and is followed by a series of trimming reactions catalysed by glucosidase I & II and a-mannosidase I. Trimming continues in the Golgi by the action of a-mannosidase I and is followed by a series of processing steps brought about by a set of very specific glycosyltranferase enzymes (Fig. 4) (Komfeld and Komfeld, 1985). The action of glycosyltransferases leads to a diverse array of N-linked glycan structures that share a common core structure of (Man3GlcNAc2) (Fig. 5). These structures are divided into three groups according to the type of side chains present, high mannose, hybrid and complex. In hybrid and complex types variation arises due to the oligosaccharide composition of the arms or antennae. In complex types there may be two (Biantennary) three (triantennary) or four (tetraantennary) arm structures present. Hybrid types may contain up to three antennae (Fig. 5). 14 Introduction ENDOPLASMIC RETICULUM MEDIAL-GOLGI TRANS-GOLGI TRANS-GOLGI NETWORK > EXIT Figure 4. A potential pathway for processing of N-linked glycans. The glycosyltransferases are as follows: (1) oligosaccharyltransferase, (2) ct-glucosidase I, (3) a-glucosidase II, (4) ER al,2-mannosidase I, (5) Golgi cd,2-mannosidase I, (6) N-acetylglucosaminyltransferase I, (7) Golgi ocl,2-mannosidase II, (8)N-acetylglucosaminyltransferase II, (9) fucosyltransferase, (10) galactosyltransferase, (11) sialyltransferase. The symbols indicate: N-acetylglucosamine (filled squares), mannose (open circles), glucose (filled diamonds), fucose (open diamonds), galactose (closed circles) and sialic acid (open squares). Dol-P-P is dolichyldiphosphoryl. This figure is from Gooche and Monica, 1990 and was adapted from a figure in Komfeld and Komfeld, 1985. 15 High Mannose Hybrid Complex Biantennary Triantennary Biantennary Triantennary Tetraantennary Figure 5. N-linked glycan structures in mammalian cells. Symbols represent: • = N-acetylglucosamine, O = mannose, (V) = variable presence of fucose (O) = variable number of mannose and X = variable oligosaccharide structure. Introduction 1.6.2 O-linked Glycosylation There is no specific consensus sequence for O-linked glycosylation which begins in the Golgi with the addition of N-acetylgalactosamine. However the amino acids surrounding serines or threonines are known to influence site occupancy (Hansen et al., 1996). O-linked glycans can be divided into five groups based on five different core structures (reviewed by Furukawa and Kobata, 1992). 1.6.3 Heterogeneity in Glycosylation Heterogeneity in glycosylation arises from variable site occupation and from the presence of different glycan structures at a given site. Different glycoforms correspond to identical polypeptide chains with distinct glycan structures (Rademacher et al., 1988). However the major source of structural diversity, referred to as microheterogeneity, arises from the variation in terminal oligosaccharides. Typical terminal oligosaccharides are ce-linked sialic acid, fucose, galactose, N-acetylgalactosamine and N-acetylglucosamine. There are over 20 known terminal oligosaccharide sequences and in combination this leads to great diversity. Site heterogeneity and microheterogeneity was observed in the production of recombinant IFN-y by CHO cells and resulted in up to 12 variants compared to the two predominant natural forms (Curling et al., 1990). Heterogeneity in glycosylation can lead to functional diversity, for example variable in vivo bioactivity was shown for glycoforms of gonadotrophic/thyrotrophic hormones (Baenziger et al., 1988; Szkudlinski et al., 1993). When producing recombinant therapeutic proteins it is necessary to establish the degree of heterogeneity that will not effect the efficacy or safety of the protein (Cumming, 1991b). 17 Introduction 1.6.4 Effect of Glycosylation on Protein Properties Glycosylation has been shown to influence the physical properties of a protein such as solubility and tendency towards aggregation. Deglycosylation of the interleukin-4 receptor resulted in increased aggregation and reduced solubility of the receptor (Rajan et al., 1995). The presence of sialic acid residues alter the charge and generally increase the solubility of a protein (Berman & Lasky, 1985). Since most proteins are active in aqueous solution, lack of carbohydrate can have important consequences. Glycosylation has been shown to facilitate the proper folding and stabilization of proteins (Paulson, 1989). For a number of proteins such as erythropoeitin (EPO) (Nielson et al., 1987), influenza virus haemaglutin (Gallagher et al., 1988), lipoprotein lipase (Semenkovich et al., 1990) and rabies virus glycoprotein (Wojczyk et al., 1995) lack of carbohydrate resulted in aggregation of non-glycosylated protein within the ER. For other proteins, such as IFN-y, lack of carbohydrate had no effect on secretion (Conradt et al., 1989). Glycosylation is one of the key factors in determining the efficacy of many therapeutic proteins (Hansen et al., 1988; Takeuchi et al., 1989; Szkudlinski et al., 1993; Flesher et al., 1995). For many proteins such as granulocyte/macrophage colony stimulating factor (Mooren et al., 1987) and tissue plasminogen activator (Hansen et al., 1988) the non-glycosylated form had higher in vitro activity, however in vivo it was rapidly cleared from the circulation. Clearance mechanisms exist for a number of glycan determinants. For example the asialoglycoprotein receptor that recognizes terminal galactose and N-acetylglucosamine, the mannose receptor and a receptor for terminal sulphated N-acetylgalactosamine are all found on hepatocytes (reviewed by Ashwell and Hartford, 1982; Jenkins and Curling, 1994). Presentation of these determinants leads to a rapid clearing from the blood stream (Ashwell and Hartford, 1982). For example, glycoforms of EPO produced in CHO cells bearing branched and highly sialylated residues had a 18 Introduction much longer half life in circulating blood (Takeuchi et al., 1989) than desialylated EPO that was rapidly cleared (Fukuda et al., 1989). Recombinant proteins produced in yeast and insect cells also have greatly reduced half lives due to the presence of oligosaccharides with terminal mannose (Goochee et al., 1991). Glycosylation has important effects on antigenicity of a protein. Carbohydrates can modulate the immunogenic potential of a glycoprotein in a number of ways. They may define all or part of an antigenic determinant of the protein or they may mask potential antigenic sites (Cumming, 1991a). The presence of glycans also influences the overall folding of the protein and may therefore effect antigenic determinants at sites remote from the glycosylation site (Berman and Lasky, 1985). While particular moieties of glycoproteins and glycolipid oligosaccharides are known to be immunogenic (Feizi and Childs, 1987) non-glycosylated proteins tend to aggregate and this can also cause increased immunogenicity (Goochee et al., 1991). 1.6.5 Factors Influencing Glycosylation A number of factors are known to influence glycosylation. The protein itself exerts an influence on the type of glycan structures present at a given site. For example, human tissue plasminogen activator (t-PA) has three N-linked glycosylation sites, in both native and recombinant t-PA the glycan structure at the first N-linked glycosylation site (Asn 117) in the kringle domain was found to be high mannose with complex types exclusively at the next two sites. However mutations amino-terminal to Asn 117, that perhaps disrupted the protein structure, resulted in the presence of complex rather that high mannose type glycans at Asn 117 (Wilhelm et al., 1990). Glycosylation is known to be both species dependent and cell-type dependent. Careful selection of the host cell type for recombinant protein expression is of extreme importance 19 Introduction (Jenkins et al., 1996b). The influence of cell type on glycosylation has been shown to be primarily dependent on presence, concentration, kinetic characterization and compartmentalization of individual glycosyltransferases and glycosidases (Goochee et al., 1991). A comparison of the same protein produced in a number of different cell lines highlights the influence cell type can have on glycosylation. Kagawa et al. (1988) compared the glycan structures of native human interferon-p (IFN-P) to recombinant protein produced in three different cell types. Approximately 35% and 40% of the glycan structures of IFN-P produced in human PC8 cells and mouse CI27 cells respectively, were not found in the native protein. While in CHO cells only 5% of structures were not found in the native protein, but the proportions of common structures were different and some structures in the native IFN-P were absent. Comparison of EPO expressed in different cell lines gave similar results (Tsuda et al., 1988; Takeuchi et al., 1988). Site occupancy can also vary with cell type, interleukin-2 (IL-2) has one O-linked glycosylation site, recombinant IL-2 produced in CHO cells was 90% glycosylated compared to 50% in baby hamster kidney (BHK) cells and a mouse fibroblast cell line (Conradt et al., 1989; Riske et al., 1991). CHO cells are the most frequently used cell line for the production of therapeutic recombinant proteins because the glycosylation is very similar to that of human cells (Jenkins et al., 1996b). However differences between the glycosylation patterns of CHO and human cell lines do exist. For example CHO cells lack a functional oc2,6-sialyltransferase and exclusively synthesize a2,3-linked terminal sialic acids. In contrast human cells contain both enzymes. In addition most CHO lines lack a functional al,3-fucosyltransferase found in human cells. As more glycosyltransferases have been cloned (Field and Wainwright, 1995) the glycosylation repertoire of cell lines has been manipulated by the transfection of glycosyltransferase genes (Lee et al., 1989; Potvin et al., 1990; Minch et al., 20 Introduction 1995; Monaco et al., 1996). Also an increasing number of CHO mutant cell lines capable of producing only a specific type of glycosylation have been identified and characterised (Stanley and Ioffe, 1995). The cell culture environment and the physiological state of the cell were shown to have important influences on recombinant protein glycosylation (Goochee and Monica, 1990; Anderson and Gooche, 1994). An increase in monoclonal antibody (mAb) containing glycans with terminal sialic acid and galactose residues, was observed when hybridomas were grown in serum-free cultures compared to serum-containing cultures (Patel et al., 1992). Increased ammonium concentration in CHO cultures caused a reduction in the sialylation of O-linked glycans in recombinant granulocyte colony stimulating factor (Anderson and Goochee, 1995) and an overall decrease in N-linked glycosylation of mouse placental lactogen I (Boyrs et al., 1994). A similar effect was observed for mouse placental lactogen I produced at extreme culture pH conditions (below 6.9 and above 8.2) (Boyrs et al., 1993). CHO cells grown under conditions of glucose starvation, either attached smaller precursor oligosaccharides or left sites unglycosylated (Davidson et al., 1985; Chapman and Calhoun, 1987). The physiological state of the cells was shown to greatly influence the glycosylation of IFN-y (Hayter et al., 1991). In CHO batch cultures grown for over 100 h, the decline in cell viability was accompanied by a decrease in the proportion of IFN-y with complex biantennary glycans and an increase in IFN-y with oligomannose and truncated structures (Hooker et al., 1995). A similar result was observed for the production of mAb by a mouse myeloma cell line grown in fed-batch cultures (Robinson et al., 1994). Another cause of glycosylation heterogeneity resulted from the presence of glycosidases in cell culture supernatants (Sliwkowski et al., 1992; Gramer et al., 1995). A number of 21 Introduction glycosidase activities including sialidase, fucosidase, p-galactosidase and P-hexosaminidase were detected in cell culture supernatants of CHO, myeloma and hybridoma cells (Gramer and Goochee, 1993 & 1994). Sialidase activity in CHO culture supernatants was reported to result from the release of a cytoplasmic form of the enzyme after cell lysis (Munzert et al., 1996). A sialidase (Warner et al., 1993) and fucosidase (Gramer et al., 1994) were isolated from CHO culture supernatants. The sialidase had an optimum pH of 5.9 and showed similarities to a rat cytoplasmic sialidase (Warner et al., 1993). Sialidases from CHO culture supernatants removed sialic acid residues from recombinant human deoxyribonuclease (Sliwkowski et al., 1992) and recombinant gpl20 (Gramer et al., 1995). The fucosidase isolated had optimal activity at neutral pH and although it was able to act on a synthetic substrate, it was unable to cleave fucose from intact glycoproteins (Gramer et al., 1994). 1.7 Glycosylphosphatidylinositol Anchors In eukaryotes many diverse proteins are attached to the cell surface via a GPI anchor. Over 50 GPI-anchored proteins have been identified in mammalian cells (Takeda and Kinoshita, 1995). These include proteins such as decay accelerating factor (DAF) and Thy-1 that are present at approximately 5 x 105 (Metz et al., 1994) and 1 x 106 (Williams et al., 1977) molecules per cell respectively. GPI-anchored proteins are particularly common in parasites, in which most of the major cell surface proteins are GPI-linked. Examples include Trypanosoma brucei procyclin, expressed in the procyclic form at 5 x 106 molecules per cell and the variant surface glycoproteins (VSG), expressed in the blood stream form at 1 x 107 molecules per cell (McConville and Ferguson 1993). The basic structure of GPI anchors is highly conserved in eukaryotes and consists of: ethanolamine-P04-6manal-2manal-6manal-4GlcNal-6myo-22 Introduction inositol-l-P04-lipid. Species specific variations in this common core sequence occur, particularly with respect to the glycan structures (McConville and Ferguson, 1993). The GPI anchor of GP63 conforms to the conserved structure (Fig. 6) (Schneider et al., 1990). 1.7.1 GPI-anchor Addition For GPI addition to occur a protein requires both a leader sequence for entry into the ER and a 20-50 amino acid GPI signal sequence. There is no specific consensus for the signal sequence however site directed mutagenesis identified regions within the sequence that are important. Within these regions the type of amino acids present and the length of the sequences are critical for GPI addition to occur (Lowe, 1992; Coyne et al., 1993). The regions include: i) a 12-20 amino acid hydrophobic region at the carboxy terminus that acts as a transient membrane anchor prior to GPI addition, ii) a relatively hydrophilic spacer region of 8-12 amino acids that is followed by, iii) a cleavage/attachment site that consists of amino acids with small side chains such as serine, alanine, glycine, asparagine and aspartate. In particular the presence of amino acids with small side chains either side of the cleavage site are essential (Moran et al., 1991; Gerberetal., 1992). The addition of the pre-formed GPI anchor occurs post translationally in the ER shortly after chain termination (reviewed by Tartakoff and Singh, 1992). The enzyme thought to be responsible is a transamidase that cleaves the signal sequence and covalently attaches the GPI anchor to the carboxy terminus of the protein (Doering et al., 1990). Failure to cleave the GPI signal sequence of DAF expressed in CHO and COS cells was reported to result in ER retention and ultimately degradation of the protein (Moran and Caras, 1992; Field et al., 1994). 23 Introduction c=o Etnl Figure 6. Schematic diagram of the GPI anchor of GP63. Etn = ethanolamine, P = phosphate, Man = mannose GlcN = glucosamine, I = inositol. PI-PLC cleavage site indicated by arrow. 24 Introduction A number of proteins have been expressed on the cell surface by the introduction of a signal sequence from a GPI-anchored protein. Thus signal sequences are able to direct GPI attachment of heterologous proteins (Caras et al., 1987; Keller et al., 1992; Lowe, 1992; Moran and Caras, 1992; Kemble et al., 1993; Harrison et al., 1994). Also modification or removal of the signal sequence from proteins that are normally GPI-anchored results in secretion of the protein (Lowe 1992; Button et al., 1993). 1.7.2 GPI-anchor Function A number of specialized functions have been proposed for GPI anchors and as reasons for their involvement in cell membrane attachment (reviewed by McConville and Ferguson, 1993). These include enhanced motility on the cell surface compared to proteins anchored via transmembrane regions, isolation of the membrane expressed protein from the cytoplasm and susceptibility to cleavage from the cell surface by endogenously expressed phosphatidylinositol (Pl)-specific phospholipases. In mammalian cells, GPI anchors have been shown to play a role in intracellular targeting of proteins and in cell signaling via cross-linking of GPI anchored proteins. In parasites GPI-anchoring is used for high density packing of proteins on the cell surface that together with the expression of other GPI-linked molecules such as lipophosphoglycan in Leishmania results in the formation of a protective glycocalyx. 1.7.3 Internalization of GPI-anchored Proteins in Mammalian Cells A number of reports suggest that in mammalian cells internalization of GPI-anchored proteins occurs by a clathrin-independent pathway distinct from the clathrin dependent-pathway of transmembrane proteins (Bamezai et al., 1992; Keller et al., 1992). Complete endocytosis of 25 Introduction some GPI-anchored proteins such as Thy-1 has been reported to occur very slowly (Lemansky et al., 1990). Studies of CD4 expressed as a GPI-anchored protein in CHO cells by replacement of the transmembrane region with the GPI signal sequence of DAF, showed that endocytosis of the GPI-linked molecule was three time slower than for the transmembrane protein (Keller et al , 1992). Pseudoendocytosis or potosis in membrane invaginations resembling caveolae has also been reported for GPI-anchored proteins, such as the folate receptor (Rothberg et al., 1990). Caveolae are membrane invaginations rich in the protein caveolin which transiently close without pinching off. It is thought that this effect concentrates trace ligands such as folate, prior to membrane translocation (Anderson et al., 1992). 1.7.4 Phosphatidylinositol-phospholipase Cleavage of GPI-anchors GPI anchors are susceptible to cleavage by a set of enzymes that specifically hydrolyze PI. An example is the bacterial enzyme PI-PLC that is produced in a number of bacteria including Bacillus thirungensis (reviewed by Englund, 1993). PI-PLC cleaves the GPI-anchor at the phosphodiester bond between the phosphoinositol group and the lipid portion of the anchor (Fig. 6). The released protein retains the inositol glycan portion of the anchor and diacylglycerol is left in the cell membrane. The kinetics of cleavage of GPI-anchored proteins from the cell surface were shown to be approximated by Michaelis-Menton kinetics (Sunderji et al., 1997). Rapid non-specific adsorption of PI-PLC to the cell surface was demonstrated and shown to be reversible (Sunderji et al., 1997). Resistance to PI-PLC has been observed for some GPI anchored proteins such as alkaline phosphatase expressed in human cell lines (Wong and Low, 1992). In most cases resistance to PI-PLC was shown to be caused by acylation of the phosphatidylinositol (reviewed by Ferguson and Williams, 1988). Mammals also have a PI 26 Introduction specific phospholipase, PI-PLD, that is present in plasma (Davitz et al., 1987). Endogenous PI-PLD activity in HeLa cells and bone marrow stromal cells was reported to release DAF into cell culture supernatants (Metz et al., 1994). 1.8 Secretion Pathway of Membrane Bound and Soluble Proteins Little is known about whether the secretion pathway of membrane bound and soluble proteins differ. Evidence suggests that transport of GPI-anchored proteins to the plasma membrane may be distinct from the transport of both soluble and transmembrane proteins. Association of GPI proteins in microdomains with sphingolipids is thought to occur in the Golgi (Brown and Rose, 1992; Simons and Ikonen, 1997). GPI anchors have been shown to act as sorting signals for targeting proteins to the apical membranes in polarised epithelial cells. It has been postulated that sorting may occur as a result of association with sphingolipids and transport within distinct vesicles targeted to the apical membrane (Brown and Rose, 1992; Garcia et al., 1993). The effect on the post-translational modifications of proteins being membrane bound or soluble within the lumen of the ER and Golgi, has not been well established. Many processing enzymes such as the glycosyltransferases and some precursor convertases are themselves transmembrane proteins and whether this influences the accessibility of membrane expressed or secreted proteins remains to be established. Post-translational modifications such as glycosylation have been analysed in great detail for numerous recombinant secreted proteins and some recombinant membrane proteins. However there are few studies in which the post-translational modifications of the same protein expressed on the cell surface and secreted in the same cell line have been compared. 27 Introduction 1.9 Thesis Objectives Post-translational modifications can have profound effects on the properties and function of a protein. The primary goal of this study was to identify the influences of membrane versus secreted recombinant protein production on both the expression level and the post-translational modifications. The recombinant protein investigated in this study was GP63, the major cell surface glycoprotein of the parasitic protozoan Leishmania. Proteolytically active GP63 and an active site mutant of GP63 were produced as either GPI-anchored membrane expressed or secreted proteins in CHO cells to achieve the following objectives: i) To compare the expression levels of membrane expressed and secreted production of GP63. ii) To investigate the pro-region processing of membrane expressed and secreted proteolytically active GP63 and the active site mutant of GP63. iii) To identify structural differences in the N-linked glycosylation of membrane expressed and secreted GP63. iv) To determine the dynamics of the secretory pathway of membrane bound and soluble protein. Differences identified in both expression levels and post-translational modifications of a membrane expressed and secreted protein produced in the same cell line should be considered when choosing an expression route for other recombinant proteins. Stability of protein expression is an important factor in large-scale recombinant protein production, however the kinetics and mechanisms of protein expression loss in unamplified heterologous cell lines are poorly defined. A second major goal of this study was to investigate the expression stability of membrane expressed GP63 and elucidate the mechanisms involved in 28 Introduction loss of protein expression. Cell surface expression of GP63 greatly facilitated the analysis, by flow cytometry, and enabled a quantitative approach to: i) Determine the distribution of protein expression within the population every 6 days for periods over 140 days. ii) Determine the effect of selective pressure on expression stability. iii) Investigate the correlation between proteinase activity and instability by comparing the expression of the active site mutant with the proteolytically active GP63. iv) Identify nonproducing populations and gain insight into the molecular and dynamic mechanisms involved in loss of protein expression. Understanding the mechanisms involved in loss of protein expression, could provide the basis for alternative approaches to avoid protein expression instability. 29 Material and Methods Chapter 2 Materials and Methods 2.1 Vector Construction 2.1.1 Expression of Secreted and Membrane Expressed GP63 The L. major GP63 gene (Button and McMaster, 1988) and a gene in which the region encoding the GPI signal sequence had been removed (Button et al., 1993) were modified by replacement of the DNA encoding the Leishmania leader sequence with DNA encoding the rat CD4 leader sequence (Clark et al., 1990). This was achieved using two complementary synthetic oligonucleotides ( 5 ' - G G G G G G A T C C A T G T G C C G A G G C T T C T C T T T C A G G C A C T T G C T G C C GCTGCTGCTCCTGCAGCTGTCAAG-3' ) and ( 5 ' - A G T C T T G A C A G C T G C A G G A G C A G C A G C G G C A G C A A G T G C C T G A A A G A G A A G C C T C G G C A C A T G G A T C C C C C C - 3 ' ) that encoded 20 amino acid of the new leader sequence. The oligonucleotides were annealed leaving a 3' overhang Hinf I site. A Hinf I - Eco RV fragment encoding the remaining 7 amino acids of the new leader sequence and amino acids 40-190 of GP63 (commencing at the start of the pro-region) was generated by polymerase chain reaction (PCR) and subsequent restriction enzyme digestion of the PCR product. PCR was performed for 30 cycles using a Perkin Elmer 9600, each cycle consisted of 30 s at 94°C, 30 s at 55°C and 2 min at 72°C. PCR reactions were 100 uL and contained lx buffer (Perkin Elmer), 10% DMSO (BDH), 0.2 mM of each dNTP (USB), 2.5 units Taq (Perkin Elmer) 100 ng of template and 25 pmol of each primer. The following primers were used: 5' primer ( 5 ' - C A T A T G G A C T C C T A G T C A C C C A A G T C A C C C A A G G A C A C G C C G G T G CGCTGCAGCA-3') that encoded 7 amino acids of the new leader sequence and the first 7 amino acids of the pro-region of GP63 and introduced a Hinf I restriction site by conserved mutation (Lys to Arg) at position 20 within the leader sequence and the 3' primer 53 (Macdonald et al., 30 Material and Methods 1995). The annealed oligonucleotides, the PCR fragment and the remaining portion of the GP63 gene were ligated using the Hinf I and Eco RV sites, resulting in the generation of a modified GP63 gene containing the new 27 amino acid leader sequence. The modified portion of the gene was sequenced by double stranded DNA sequencing using a "Sequenase" kit according to the manufacturer's instructions (USB). The modified GP63 genes for membrane expression and for secretion referred to as GP63WT(M) and GP63WT(S) respectively, were cloned into the pNUT expression vector (Palmiter et al., 1987) and used to transfect CHO-K1 cells (ATCC). 2.1.2 Expression of a Mature Form of GP63 Lacking the Pro-region The approach described in Section 2.1.1 was used to generate a construct that encoded a mature form of GP63 lacking the pro-region and the GPI signal sequence. The only difference in the approach was that the Hinf I - Eco RV fragment generated by PCR and subsequent restriction enzyme digestion of the PCR product, encoded the remaining 7 amino acids of the new leader sequence and amino acids 129-190 of GP63 (commencing at the mature amino-terminus). The PCR primers used were (5' - C A T A T G G A C T C C T A G T T G T C A C C C A A G G A G T G C G C G A C G T GAACTGGGGC-3') and the 3' primer 53 (Macdonald et al., 1995). The modified portion of the gene was sequenced by double stranded DNA sequencing using a "Sequenase" kit according to the manufacturer's instructions (USB). The modified GP63 gene referred to as matGP63WT(S) was cloned into a human cytomegalovirus (HCMV) expression vector (Kettleborough et al., 1991) and used to transfect CHO-K1 cells (ATCC). 31 Material and Methods 2.2 Mutation of the Active Site of GP63 Site directed mutagenesis of the L. major gene was carried out by PCR, using the conditions described in Section 2.1. The L. major GP63 gene was used as template and two sets of primers were used to introduce the mutation into the active site and to create a new Ava I restriction site for subcloning. 5'-primer I, T G A A G G T G C A G C A G G T G C and 3'-primer I (complimentary strand), G C C G C T G A A C C C G A G C G C G T G C G C C A T G T C G T G C G T G A C were used to introduce the mutation of G A G (Glu2 6 5) to G A C (Asp2 6 5) (in bold) and to introduce a 3' A v a l restriction site (underlined) by silent mutation (CCG to CCC). These primers generated a 307 bp fragment (PCR product I). A second product of 169 bp was generated using the 5' primer II C A C G C G C T C G G G T T C A G C G G and the 3' primer II (complimentary strand) T C C T C C A C C T C C A G A T A C T C C that overlapped with the 3' end of PCR product I and contained a 5' Ava I restriction site (underlined). The Ava I site in PCR fragments I & II and internal restriction sites at the 5' end of PCR product I (Eco RV) and at the 3' end of PCR product II (Bel I) were used to subclone the PCR products into the modified GP63WT genes replacing the 327 bp Eco RV-Bcl I fragment of the L. major GP63 gene. 5'-primer I and 3' primer II were used to sequence the modified portion of the gene by double stranded DNA sequencing using a "Sequenase" kit according to the manufacturer's instructions. The resulting active site mutant constructs for membrane expression and for secretion referred to as GP63E265D(M) and GP63E265D(S) respectively, were cloned into the pNUT expression vector (Palmiter et al., 1987) and used to transfect CHO-K1 cells (ATCC). 32 Material and Methods 2.3 Transfection of CHO Cells Cells were resuspended in 0.8 mL PBS (137 mM NaCl 2 , 2.7 mM KC1, 4.3 mM Na 2HP0 4 .7H 20, 1.4 mM K H 2 P 0 4 , pH 7.4) at 1 x 107 cells/mL and 10u,g of cesium chloride purified plasmid DNA was added. Cells were electroporated using a gene pulser (BioRAD) at 1900 V, 25 f^ F capacitance (Kettleborough et al., 1991). The cells were left at room temperature for 10 min after electroporation then resuspended in 48 mL of Dulbecco's modified essential medium (DMEM), containing lx non-essential amino acids (Life Technologies) and 10% Fetal Clone II serum (Hyclone) and plated in 6 tissue culture plates (75 cm2) (Falcon). After incubation at 37°C for 24 h, M T X (David Bull Laboratories) was added to a final concentration of 50-250 |^M. Foci were picked after 14-21 days and screened for cell surface expression of GP63 by flow cytometry or secretion of GP63 by enzyme linked immunosorbent assay (ELISA). Positive clones were recloned by limiting dilution and maintained in medium containing MTX. 2.4 Cell Counts and Viability Determination Cells were counted on a haemocytometer and viability assessed by trypan blue dye exclusion using phase contrast microscopy. At least 100 cells were counted at each determination. 2.5 Cell Culture 2.5.1 Stability of GP63WT(M) and GP63E265D(M) Expression Cells were maintained in D M E M containing 10% Fetal Clone II serum and lx non-essential amino acids with and without 50 uM M T X at 37°C in a humidified atmosphere containing 5% CO2. For the repeated batch culture every six or seven days cells were detached using versene or trypsin (Life Technologies) and between 0.75 -2x10^ cells were seeded in 150 cm^ flask 33 Material and Methods (Falcon). Lower seeding levels were used for clones with higher growth rates. Cell counts and viability were determined at each passage. The specific growth rates were determined before and after loss of GP63WT(M) and GP63E265D(M) expression, cells were seeded at 6 x 104 cells per well (9 cm2) of a 6-well plate (Falcon) in 4 mL D M E M containing 10% Fetal Clone II serum, lx non-essential amino acids with and without 50 uM M T X . At each time point duplicate wells were harvested, cells counted and viability determined. Statistical difference between the producing and nonproducing populations for each clone were determined using a two-tailed Student t-test. The conversion rate (a) was determined for clone 9.4.4 GP63WT(M) by Marquardt-Levenberg fitting of the percent producer population data in terms of the number of population doublings (NPD). 2.5.2 Secretion and Continuous Harvesting of GP63E265D Cells adapted for growth in suspension were seeded at 105 cells/mL in 75 cm2 tissue culture flasks (Falcon) containing 30 mL serum-free medium (CHO-S-SFM II, Life Technologies) and grown at 37°C in a humidified atmosphere containing 5% CO2. Clones secreting GP63E265D(S) were cultured in duplicate. For the clones with membrane expression of GP63E265D(M) 3 mU/mL of PI-PLC (kindly provided by Dr. M . Kennard) was added to duplicate cultures, while a third culture was grown without PI-PLC as a control. Each day, 1-2 mL samples were removed and the following parameters were monitored: cell number and viability; cell culture glucose concentration using a N O V A biomedical analyser; GP63 accumulation by ELISA and, for cell surface expression, the mean fluorescence of cultures with and without PI-PLC using flow cytometry. 34 Material and Methods 2.5.3 Purification of GP63E265D(M) and GP63E265D(S) for FAB-MS Analysis Cells were seeded at 1-2 x l O 5 cells/mL in spinners (Bellco) containing 200 mL serum-free medium (CHO-S-SFM II) and grown at 37°C in a humidified atmosphere containing 5% CO2. Cells expressing membrane GP63 were harvested in late-log phase after approximately 96 h (>90% viable). Cells secreting GP63 were harvested either in mid-log phase (48 h) or late-log phase (96 h) (>90% viable). The cells were centrifuged at 1600g and the supernatant containing GP63 removed and stored at -20°C. 2.6 Harvesting of GP63WT(M) and GP63E265D(M) from the Cell Surface Using PI-PLC To harvest cell surface GP63 the cells were washed twice in PBS, pH 7.4 and then resuspended at 108 cells/mL in 250 mU/mL PI-PLC (Oxford Glycosystems) in PBS and placed at 37°C for 60 min with constant agitation. Cells were then centrifuged at 1600g, supernatants recovered and stored at -20°C. The percentage of GP63 removed from the cell surface was determined by flow cytometry. 2.7 Protein Purification Cell surface expressed and secreted GP63 were purified using a monoclonal antibody affinity column. A monoclonal antibody specific to GP63 (mAb 96) (Macdonald et al., 1995) was coupled to cyanogen bromide activated Sepharose 4CL beads (Pharmacia) as described in McMaster (1984). CHO cell culture supernatants or PI-PLC harvested supernatants were centrifuged at 15000g for 20 min before loading under gravity onto a mouse IgG column (non-35 Material and Methods specific antibody) in tandem with the mAb 96 affinity column. The column was washed with 200 mL of Tris buffered saline (TBS) (20 mM Tris-HCl, 150 mM NaCl 2 , pH 7.5) and 1 mL fractions were eluted in elution buffer (0.15 M N a C l 2 , 0.05 M diethylamine-HCl, pH 11.5). The column was then neutralised with 20 mL 1 M Tris-HCl pH 8 and washed in TBS as described in McMaster (1984). Fractions containing recombinant GP63 were pooled and dialysed against TBS. GP63 was quantified by ELISA and total protein by the BioRAD protein assay (BioRAD). The purified protein was analysed by silver staining of SDS-PAGE. 2.8 ELISA Immunoplates (96-well) (Nunc) were coated with 100 uL/well of 10 ixg/mL solution of purified mAb 96 (Macdonald et al., 1995) in PBS at 4°C overnight. Plates were then blocked for 2 h at room temperature (RT) by the addition of 300 uL/well of blocking buffer, 3% bovine serum albumin (BSA) (Sigma) in PBS. Plates were then washed 3 times in PBS. Samples and standards (purified CHO GP63 of known concentration) were diluted in blocking buffer and 100 uL was added to each well for 1 h at RT. Plates were then washed 3 times in PBS. Affinity purified rabbit antibody specific to GP63 was diluted in blocking buffer at 1 u.g/mL and 100 uL was added to each well for 1 h at RT. Plates were then washed 3 times in PBS. Goat anti-rabbit IgG alkaline phosphatase conjugated antibody (BioRAD) was diluted 1:1000 in blocking buffer and 100 uL was added per well for 1 h at RT. Plates were then washed 3 times in PBS. Alkaline phosphatase substrate (Sigma) was diluted at 1 mg/mL in 10% diethanolamine buffer (10%) diethanolamine, 0.5 mM MgCl 2 .6H 2 0, 0.02% NaN 3, pH 9.8) and 100 uL was added to each well for 30 min at RT. Absorbance was then read at 405 ran using a V m a x kinetic microtitre plate reader (Molecular Devices). 36 Material and Methods 2.9 Flow Cytometric Analysis Flow cytometry was performed using a Becton Dickinson FACScan. Cells (10^ per sample) were labeled with 100 uL supernatant containing mAb 96 specific to GP63 (Macdonald et al., 1995) for 1 h at 4°C. Cells were washed 2 times in wash buffer (PBS containing 0.1% BSA) and then labeled for 1 h at 4°C in a 1:50 dilution of goat anti-mouse IgG fluorescein isothiocyanate conjugated antibody (GAM-FITC) (BioRAD). Negative controls were labeled with the second antibody only. Cells were washed once in wash buffer, resuspended in 500 uL PBS and analysed immediately or fixed by the addition of 500 uL of 3% para-formaldehyde (JBS) in PBS for later analysis. 2.10 Analytical SDS-PAGE Protein was analysed by SDS-PAGE (Laemmli, 1970) using 10% separating gels. Protein was visualised by silver stain. Gels were fixed in 50% methanol, 10% acetic acid for 30 min, then rehydrated by microwaving for 1 min in 10% methanol, 10%) acetic acid and washed for 5 min in distilled H 2 0 . The gels were then microwaved for 1 min in 33 uM dithiothreitol (DTT) and washed in distilled H 2 0 . The gels were incubated for 15 min in 0.1 % AgN0 3 , washed in distilled H 2 0 and developed with 3% Na 2 C0 3 , 0.037%) formaldehyde. The reaction was stopped in 5% acetic acid. 2.11 Western Blot Analysis Following SDS-PAGE (Laemmli, 1970) using 10% separating gels, proteins were transferred to Immobilon P (Millipore) using a semi-dry transfer cell (BioRAD) for 40 min at 15 V according 37 Material and Methods to the manufacturer's instructions. Western blots were probed for 1 h at RT with constant agitation with either 1:10 dilution of supernatant containing mAb 139 specific to GP63 (Button et al., 1991) or 1:50 dilution of an affinity purified rabbit polyclonal antibody to the pro-region of GP63 (Macdonald, 1995) diluted in antibody dilution buffer, TBS containing 0.5% BSA and O.P/o Tween 20 (Sigma). Blots were washed 3 times for 5 min in wash buffer, TBS containing 0.1%o BSA and 0.1 % Tween 20. The blots were then probed for 1 h at RT with constant agitation, with 1:3000 dilution in antibody dilution buffer of either goat anti-mouse or goat anti-rabbit IgG alkaline phosphatase conjugated antibodies (BioRAD). Blots were washed as before and developed with BCIP/NBT reagents (Life Technologies) diluted 1:300 in alkaline phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl 2 , 50 mM MgCl 2 .6H 2 0, pH 9.5). 2.12 GP63WT(M) & GP63E265D(M) Proteinase Activity Purified GP63WT(M) & GP63E265D(M) were concentrated using a centricon-10 (Amicon). Proteinase activity was assessed by zymogram using a 10% SDS-PAGE gel that contained 0.1 % gelatin instead of 0.25%) fibrinogen described in Button et al. (1993). Western blot analysis with a mAb 96 specific to GP63 was performed as described in Section 2.11. 2.13 GP63WT(S) and GP63E265D(S) Activation and Proteinase Activity Samples of purified GP63WT(S) and GP63E265D(S) were either incubated for 24 h at 4°C, 37°C, or 37°C in the presence of 6.4 uM HgCl 2 . Western blot analysis using antibodies specific to GP63 and to the pro-region of GP63 was performed as described in Section 2.11 and zymogram analysis as described in Section 2.12. The proteinase activity of GP63WT(S) and GP63E265D(S) was also assessed using a "Quanticleave" kit (Pierce). Duplicate samples were 38 Material and Methods incubated with succinylated casein in 50 mM borate buffer pH 8.5 at 37°C for 1.5 h as described in the manufacturer's protocol. 2.14 Pulse Chase Experiments 2.14.1 GP63WT(S) and matGP63WT(S) Clones Cells were seeded at 3 x 105 cells per well in 6-well plates (Falcon) and incubated at 37°C in 4 mL culture medium, D M E M with lx non-essential amino acids (Life Technologies) and 10% Hyclone II serum (Hyclone). After 48 h, attached monolayers were washed 3 times with PBS and cells were incubated for 1 h at 37°C in 2 mL pre-incubation medium, methionine and cysteine free D M E M (Life Technologies) with 10% dialysed fetal calf serum (Hyclone). Cells were then pulsed for 40 min at 37°C in 1 mL of pulse medium, pre-incubation medium containing 100 uCi of Trans 35S-label (35S methionine and cysteine) (ICN). Cells were washed 2 times with PBS warmed to 37°C and incubated at 37°C in 1 mL of culture medium supplemented with 2 mM methionine and 4 mM cysteine (Sigma). At each chase time point the supernatant was removed, cells were washed 3 times in PBS at 4 ° C and then lysed in 0.5 mL lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl 2 , l%nonidet P-40, 1% deoxycholate) with 0.25 mM phenylmethylsulfonyl fluoride (PMSF). Cell extracts and supernatants were stored at -20°C. 2.14.2 GP63E265D(S) and GP63E265D(M) Clones Pulse chase experiments were performed as described in Section 2.14.1 except cells were pre-incubated for 3 h at 37°C and pulsed for 30 min at 37°C. For experiments in which GP63 39 Material and Methods was cleaved from the cell surface 500 mU/mL of PI-PLC (Oxford Glycosystems) was added to the pre-incubation, pulse and chase media. 2.15 Immunoprecipitation Cell lysates and culture supernatants were pre-cleared by the addition of 5 uL (settled bead volume) of recombinant protein A beads (Repligen). The beads were removed by centrifugation (lOOOg). Affinity purified rabbit polyclonal antibody specific to GP63 or an irrelevant rabbit polyclonal antibody control were added at 1 jj.g per 125 uL of cell lysate or 250 u.L of culture supernatant and incubated overnight at 4°C. Protein A beads which had been blocked in BSA (0.1% BSA in TBS for 30 min at 4°C) were added at 5 uL (settled bead volume) per sample, to the lysates or supernatants and incubated at 4°C for 2 h. The beads were collected by centrifugation (lOOOg) and washed 3 times in TBS. Protein was eluted from the beads in 20 uL of SDS-PAGE loading buffer or 20 uL of 2x Endo H buffer (100 mM sodium acetate pH 5.5, 0.2% SDS, 5% p-mecaptoethanol) for 10 min at 95°C. 2.16 Endo H Digestion Each sample was divided into two tubes containing 10 uL of distilled H 2 0 . The samples were digested or mock digested for 18h at 37°C by the addition of 1 uL Endo H or 1 uL H 2 0 respectively. Samples were then subjected to SDS-PAGE and autoradiography. 2.17 Mass Spectrometry (MS) Sample Preparation and Analysis Samples were prepared and analysed by fast atom bombardment (FAB)-MS as described in detail in Dell et al. (1993). In brief, approximately 3 nmol of membrane expressed and secreted 40 Material and Methods GP63 from mid and late-log phase cultures were reduced in DTT (Sigma) and carboxymethylated with iodoacetic acid (Sigma) followed by dialysis against 50 mM ammonium bicarbonate buffer pH 8.4. The samples were digested with porcine pancreas trypsin (Sigma) and then digested for 16 h at 37°C with 0.6 U PNGase F (Boehringer Mannheim). The released glycans were separated by chromatography using a C18-Sep Pak (Waters). After lyophilization 1/3 of the purified glycans from each sample were resuspended in 50 mM ammonium acetate buffer at pH 5.5 and treated for 24 h at 37°C with 25 mU Vibrio cholerae neuraminidase (Boehringer Mannheim). This was followed by lyophilization. Glycans were permethylated using the NaOH procedure described in Dell et al. (1993) and purified by C18 Sep-Pak chromatography. 2.18 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) RNA was isolated (Chomczynski and Sacchi, 1987) from cells in exponential growth phase. Total RNA (2 ixg per sample) was DNase I (Life Technologies) treated for 15 min at room temperature followed by heat inactivation at 70°C for 10 min. Reverse transcription was carried out on 1 u,g total RNA using an oligo DT probe (Perkin Elmer) and Superscript II reverse transcriptase (Life Technologies) according to the manufacturer's instructions. A control reaction was performed with 1 u,g of total RNA and no reverse transcriptase. PCR was performed using 50 uL reactions and conditions described in Section 2.1.1. GP63 specific primers 5'(AGCACGGCAGTGGCGAAG) and 3'(TTGCCCTGGCACACCTCC), as well as CHO p-tubulin primers 5 ' (AATGCCGACCTCCGAAAG) and 3' (ATGCGCTTGAAGAGCTCC) were used. The amplified products were resolved on a 1% agarose gel and visualised by staining in 0.5 ug/mL ethidium bromide. 41 Material and Methods 2.19 Northern Blot Analysis RNA was isolated (Chomczynski and Sacchi, 1987) from cells in exponential growth phase. The RNA was resolved on 1% agarose gels containing 0.6 M formaldehyde using lx MOPS/EDTA buffer (20 mM MOPS pH 7, 5 mM sodium acetate, 1 mM E D T A pH 8). Samples were prepared for electrophoresis by the addition to 4.5 uL RNA of 2 uL of lOx MOPS/EDTA buffer, 2 uL of 1 Ox glycerol loading buffer, 10uX formaldehyde and 3.5 uL of formamide as described in Strauss et al. (1987). RNA samples were then boiled for 5 min and chilled on ice before loading. After electrophoresis at 60 V for 2-3 h, gels were stained in 0.5 p.g/mL ethidium bromide to visualise the RNA. Gels were washed 3 times in DEPC-treated distilled H 2 0 , before transferring to a positively charged nylon membrane (Amersham) by capillary transfer according to the manufacturer's instructions. A 0.92 Kb Pst I fragment of GP63 and a 0.4 Kb PCR fragment of the murine actin gene (Clontech) were used as probes. These probes were labeled with 32p using a "Prime it kit" (Stratagene) and hybridisation was performed at 50°C. The blots were washed under stringent conditions O.lx SSC (3 M NaCl 2 , 0.3 M sodium citrate) at 65°C and exposed to X-ray film (Amersham) for 2 and 5 days at -70°C with intensifying screens. Bands were analysed using a Fast Scan Computing densitometer (Molecular Dynamics). 2.20 Southern Blot Analysis. Genomic DNA was isolated from cells (Strauss, 1987) and digested with Pst I (New England Biolabs) which cleaves three times within the DNA sequence of GP63 and once within the pNUT vector. Digested DNA was resolved on a 0.7% agarose gel and transferred to a positively charged nylon membrane (Amersham) by alkali transfer according to the manufacturer's instructions. A 0.92 Kb Pst I fragment of GP63 and a 2.9 Kb fragment containing the murine 42 Material and Methods DHFR cDNA derived from the pNUT vector were used as probes. These probes were labeled and the blots hybridised, washed and exposed as described for Northern blots in Section 2.19. Bands were analysed using a Fast Scan Computing densitometer (Molecular Dynamics) and gene copy numbers calculated as described in Guama et al. (1995). 43 Chapter 3 Expression of GP63 in CHO Cells Chapter 3 - Results Many recombinant proteins require the complex post-translational modifications found in higher eukaryotic cells. CHO cells are one of the mammalian cell lines most frequently used for the production of recombinant proteins for therapeutic and vaccine use. Recombinant proteins are usually produced in a secreted form and purified from cell culture supernatants or, in the case of proteins that have GPI anchors, they can be expressed on the cell surface and harvested by cleavage with PI-PLC. In this study recombinant Leishmania GP63 was produced in CHO cells as a GPI-linked membrane expressed protein and as a secreted protein, in order to compare the post-translational modifications and production levels of protein produced by these pathways. Production of GP63 in CHO cells also provided a continuous source of protein in native conformation for use in structural, functional and vaccine studies. The expression of an active site mutant of GP63 (GP63E265D), that had previously been expressed and characterised in COS cells (Macdonald et al., 1995), was also investigated. 3.1 Gene Construction and Transfection of CHO Cells GP63 was produced in membrane expressed and secreted forms in CHO cells. For expression in mammalian cells the DNA encoding the Leishmania leader sequence of both constructs was replaced with DNA encoding the rat CD4 leader sequence (Clark et al., 1987) using synthetic oligonucleotides and PCR (Fig. 7 A). This leader sequence was chosen because rat CD4 has been expressed at high levels in CHO cells (Davis et al., 1990). The construct for secretion was modified at the 3' end by PCR to delete 23 amino acids of the signal sequence required for GPI 44 Chapter 3 - Results C H O C H O C H O Leader 61 Sequence Z n 2 + Figure 7. GP63 constructs and the pNUT expression vector. (A) For expression in CHO cells the Leishmania leader sequence was replaced with the rat CD4 leader sequence and GP63 constructs with and without the 25 amino acid signal sequence for GPI attachment were cloned into the pNUT expression vector. A third form of GP63 that lacked both the 61 amino acid pro-region and the signal sequence for GPI attachment was constructed. (B) GP63 was expressed under the metallothionine promoter in pNUT. This vector contains a mutant form of the DHFR gene under an SV40 promoter for selection in high levels of MTX. 45 Chapter 3 - Results attachment and membrane expression (Fig. 7 A). The cell surface GP63WT(M) construct and secreted GP63WT(S) construct were sequenced, cloned into a high level expression vector pNUT (Palmiter et al., 1987) and used to transfect CHO cells (Fig. 7 B). GP63 was expressed under the transcriptional control of a metallothionine promoter. For selection the pNUT vector contains a mutant form of the DHFR gene with a reduced binding affinity for the DHFR inhibitor M T X (Simonsen and Levinson, 1983). This allows the selection of high copy number cells even in the presence of endogenous wild type DHFR by using high levels of MTX. Clones were selected by culturing in the presence of high levels of M T X (50-250 uM). Clones transfected with GP63 genes were screened for expression of secreted and membrane bound GP63 by enzyme linked immunosorbent assay (ELISA) or fluorescence activated flow cytometry respectively. Positive clones were recloned by limiting dilution. A list of constructs and the abbreviations used in this study is outlined in Table 1. 3.2 Site Directed Mutagenesis of the L. major GP63 Active site An active site mutant of the L. major GP63 gene was generated by site directed mutagenesis (Fig. 8 A) (Macdonald et al., 1995). The G A G codon for Glu265 in the active site was modified to G A C encoding aspartic acid using a PCR based mutagenesis procedure described in the Materials and Methods, Section 2.2. A second silent mutation was made to create an Aval restriction site for subcloning. The DNA sequence of the mutant GP63 gene, referred to as GP63E265D, was verified by DNA sequence analysis. Constructs for cell surface expression GP63E265D(M) and secretion GP63E265D(S) were cloned into the pNUT vector and used to transfect CHO cells as described in Section 3.1. The proteinase activity of secreted GP63WT(S) and GP63E265D(S) was determined by zymogram analysis (Fig. 8 B). A small band of clearing 46 Chapter 3 - Results Table 1. Abbreviat ions used for GP63 constructs Abbreviation Construct G P 6 3 W T ( M ) W i l d Type GP63 wi th GP I GP63WT(S ) W i l d Type GP63 without GP I GP63E265D(M) Act ive Site Mutant (Glu265 to Asp265) wi th GP I GP63E265D(S) Ac t ive Site Mutant (Glu265 to Asp265) without GP I matGP63WT(S) Mature Fo rm without Pro Reg ion or GP I \ 47 Chapter 3 - Results indicating proteinase activity was detected for GP63WT(S) (lane 2). Incubating the purified protein at 37°C increased the intensity of the band of clearing (lane 3) and incubating at 37°C with the addition of HgCl 2 caused further enhancement of the intensity of the band (lane 4). GP63WT(S) was secreted from CHO cells in a latent form that retains the pro-region but could be readily activated (discussed in Chapter 6). In contrast, no detectable proteinase activity was observed for GP63E265D(S) incubated at 37°C, even with the addition of HgCl 2 (lane 5 & 6). 3.3 Expression of a Mature Form of GP63 A GP63 gene construct for the expression of a mature form of GP63, matGP63WT(S), lacking the 61 amino acid pro-region had previously been generated. This construct contained the rat CD4 leader sequence and was targeted for secretion by removal of the GPI signal sequence as described for the wild type gene in Section 2.1. The construct was expressed in CHO cells using an H C M V vector (Kettleborough et al., 1991) and selection in G418. Clones expressing matGP63WT(S) were selected by Western blot analysis of cell lysates. 3.4 Membrane Expression and Harvesting with PI-PLC of GP63WT(M) and GP63E265D(M) Clones expressing GP63WT(M) and GP63E265D(M) on the cell surface were identified by flow cytometry, representative clones are shown in Figure 9. Clones with approximately 10-fold higher average cell expression based on mean fluorescence, were detected for GP63E265D(M) expression compared to GP63WT(M) expression. The level of M T X at which the clones expressing GP63E265D(M) were selected did not appear to effect the level of GP63 expression obtained. Clones expressing GP63E265D(M) selected at 50 uM M T X (Fig. 9 D & E ) had 48 Chapter 3 - Results A 262 263 264 265 266 267 268 269 270 271 272 V a l Thr His Glu Met A l a His A l a Leu G l y Phe GTC ACG CAC GAG ATG GCG CAC GCG CTC GGC TTC • I GTC ACG CAC GAC ATG GCG CAC GCG CTC GGG TTC V a l Thr His Asp Met A l a His A l a Leu G l y Phe B 1 2 3 4 5 6 Figure 8. Construction of an active site mutant of GP63 (GP63E265D) and characterisation of proteinase activity. (A) PCR was used to introduce two point mutations into the GP63 gene. The first mutation (underlined) was in the codon for Glu 2 6 5 , this resulted in an amino acid substitution within the active site (Glu 2 6 5 to Asp 2 6 5). The second was a silent mutation (underlined and bold) that created a new Ava I restriction site (bold) for subcloning. (B) Proteinase activity of GP63WT(S) and GP63E265D(S) were determined by Zymogram analysis. Lanes were loaded with 200 ng of GP63WT(S) incubated for 24 h at, 4°C (lane 2), 37°C (lane 3), 37°C with 6.4 urn HgCl 2 (lane 4) and 200 ng/lane GP63E265D(S) incubated for 24 h at, 37°C (lane 5) and 37°C with 6.4 urn HgCl 2 (lane 6). Control: 30 uL of supernatant from CHO cells transfected with pNUT vector only (lane 1). 49 Chapter 3 - Results 400 B B 200 1 c 200 #41.9.8 - I l mean fl. = 23 • " W r f M l l l #43.9 1 " 1 mean fl. - 21 > l nr inf*-- ' 1 D 200 E 100 #29.8.6 mean fl. = 353 i | T I i I fin #51.2.8 mean fl. = 206 #65.10.12 mean fl. = 106 0 10° 101 102 103 104 10° 101 102 103 104 Fluorescence Figure 9. Flow cytometric analysis of cell surface expression of proteolytically active GP63WT and the active site mutant GP63E265D. Clones expressing GP63WT(M) (A-C) and GP63E265D(M) (D-F). Cells were labeled with mAb 96 against GP63 and GAM-FITC (heavy line) and controls with GAM-FITC only (light line). Clones were selected at 50 uM (A-E) or 250 uM (F) M T X . The clone number and mean fluorescence of the expressing cells is indicated in each panel. In panel B cells in the first channel were not included in the calculation of the mean, therefore the mean fluorescence of the control cells was slightly overestimated and the mean fluorescence of the expressing cells was slightly underestimated. 50 Chapter 3 - Results comparable levels of expression to clones selected at 250 uM M T X (Fig. 9 F). Clones expressing GP63E265D(M) were adapted for suspension growth in serum-free medium. However clones expressing GP63WT(M) were found to be unstable and could not be adapted to growth in serum-free medium before loss of GP63 WT(M) expression occurred (discussed in Chapter 4). The conditions for harvesting GP63 from the cell surface were determined by incubating clone 29.8.6 expressing GP63E265D(M) with increasing concentrations of PI-PLC in PBS at 37°C (Fig. 10 A). The percent of cleavage from the cell surface was determined by flow cytometry and increased with concentration of PI-PLC up to 250 mU/mL, after which it leveled off. A concentration of 250 mU/mL was selected for subsequent work, since doubling the amount of PI-PLC to 500 mU/mL increased the cleavage by only 2%. An incubation time of 60 min was selected as this resulted in maximal cleavage without loss of viability (data not shown). Expression of GP63E265D(M) in clone 29.8.6, was estimated to be approximately 40-fold higher than expression of GP63WT(M) in clone 9.4.4 (Fig. 10 B). This result was consistent with the 32-fold difference in GP63-associated mean fluorescence of these clones (Fig. 9 A & D). 3.5 Secretion of GP63WT(S) and GP63E265D(S) Clones secreting GP63WT(S) and GP63E265D(S) were identified by ELISA of cell culture supernatants. The highest producing clones were adapted for suspension growth in serum-free medium. For larger scale batch production cells expressing GP63WT(S) and GP63E265D(S) were grown in spinners (Fig. 11 A & B). 51 Chapter 3 - Results ° — % Cleavage • — • % Viability 1-40 ^ ? 20 O N 200 400 600 800 1000 Concentration PI-PLC (mU/mL) B Construct Clone [GP63]/106 cells (ng) GP63 molecules / cell GP63WT(M) #9.4.4 1.2 11 500 GP63E265D(M) #29.8.6 50.0 500 000 Figure 10. PI-PLC harvesting of GP63. (A) PI-PLC harvesting of GP63E265D(M) from the cell surface of clone 29.8.6. Flow cytometry was used to determine the percent cleavage of cell surface GP63E265D(M) with increasing concentrations of PI-PLC (solid line). The viability of the cells was determined after treatment with PI-PLC (dashed line). (B) The levels of expression of GP63WT(M) and GP63E265D(M) were estimated in two clones, based on a single time point harvest. 52 Chapter 3 - Results o . 1—I S-H CD O o U H-l s CO o O B o CD O a o U CD U 3.0 .-I on o O 100 h80 •-[Cell] • - -Viabilityf o-[GP63] < 60 g . 40 r-20 0 20 40 60 80 100 120 140 Time (h) 6 D u - u - - . . . 2.5-0 100 80 < i-6o g: - • - [Ce l l ] --•--Viabilityf -Q-[GP63] 60~ ~80~ r-40 20 o 2 n « ft O 3 1-0 •2.0 M.5 o ON 1 0 3 g n a* hO.5 0.0 ft o 3 100 Time (h) Figure 11. Secretion of proteolytically active GP63WT and the active site mutant GP63E265D. Spinner cultures of cells adapted for suspension growth in serum-free medium producing: (A) GP63WT(S) and (B) GP63E265D(S), cell growth (filled squares) viability (open squares) and GP63 production (open circles) are shown. 53 Chapter 3 - Discussion 3.6 Discussion A number of different GP63 constructs were expressed in CHO cells (Table 1). These included the expression of proteolytically active wild type GP63, both as a GPI-anchored and as a secreted protein, and also the expression of a "mature" construct of GP63 lacking both the pro-region and the signal sequence for GPI attachment. Expression of the latter construct is discussed in Chapter 6. Previously, it was shown that to obtain secretion of GP63 in the baculovirus expression system it was necessary to modify the Leishmania leader sequence by replacement with a baculovirus signal sequence (Button et al., 1991). Without modification, intracellular accumulation of GP63 was observed. Enhanced expression was observed in COS cells when the modified leader sequence was used compared to the Leishmania leader sequence (Macdonald, 1995). For expression in CHO cells, the extended Leishmania leader sequence of 39 amino acids was replaced with the 27 amino acid leader sequence of rat CD4, a protein expressed at high levels in CHO cells (Davis et al., 1990). Expression of GP63 on the cell surface as a GPI-anchored protein indicates that the Leishmania signal sequence for GPI attachment is recognized in mammalian cells. In contrast the GPI signal sequence from two other parasitic proteins Trypanosma brucei V S G and the Plasmodium bergei circumsporozoite protein were found to function poorly in COS cells (Moran and Caras, 1994). The amino acid requirements for a fully functional cleavage site were apparently different in COS cells and these parasites. An active site mutant of GP63 was generated by a single amino acid substitution of the catalytic glutamic acid in the active site (Glu2 6 5) by aspartic acid (Macdonald et al., 1995). This mutation results in a decrease by one carbon atom in the side arm of the residue at position 265. Indirect evidence supports that this conservative mutation in other zinc metalloproteinase, such as neutral endopeptidase (Devault et al., 1988) and gelatinase A (Crabbe et al., 1994), does not 54 Chapter 3 - Discussion disrupt zinc co-ordination or the tertiary structure of the active site. Previously zymogram analysis showed that in contrast to the expression of proteolytically active wild type GP63 on the cell surface of COS cells, no detectable proteinase activity was observed in cell extracts of COS cells expressing GP63E265D(M) (Macdonald et al., 1995). Zymogram analysis of secreted GP63E265D(S) from CHO cells also showed no detectable proteinase activity (Fig. 8 B). Based on the mean fluorescence determined by flow cytometry, clones were detected with higher membrane expression levels of GP63E265D(M) compared to GP63WT(M) (Fig. 9). Clones expressing GP63E265D(M) also had increased stability of protein expression compared to the expression of GP63WT(M) (discussed further in Chapter 4). A PI-PLC harvesting protocol was developed based on previous studies (Kennard et al., 1993; Sunderji, 1994). This protocol was used for subsequent harvesting of membrane expressed GP63 for analysis and purification. The number of molecules of GP63WT(M) and GP63E265D(M) were estimated in two clones based on single time point harvests. The expression level of GP63WT(M) in clone 9.4.4 was very low approximately 11 500 molecules per cell, while in clone 29.8.6 GP63E265D(M) was expressed at 500 000 molecules per cell (Fig. 10 B). This 40-fold difference in expression level of clones 29.8.6 and 9.4.4 was consistent with the difference on the basis of mean fluorescence obtained by flow cytometry. Low levels of production in BHK cells was previously reported for the expression of plasminogen, the precursor of the serine proteinase plasmin (Busby et al., 1991). It was concluded that the deleterious effects on expression levels were due to the activation of plasminogen within the cells. Production levels were enhanced by expression of an active site mutant of plasminogen. Expression levels of GP63E265D(M) were comparable to reported levels of expression of other GPI-anchored proteins such as DAF, in mammalian cells (Metz et al., 1994). Also, the 55 Chapter 3 - Discussion number of molecules per cell of GP63E265D(M) expressed in CHO cells was equivalent to the number of molecules per cell reported for GP63 expression in Leishmania (Etges et al., 1986). However the surface density of molecules was approximately 10-fold lower in CHO cells (1000 molecules peruM 2) due to the increased size of CHO cells. To facilitate cell culture manipulation and protein purification, cells expressing GP63E265D(M) on the cell surface and clones secreting both GP63E265D(S) and GP63WT(S) were adapted for suspension growth in serum-free medium. Clones secreting GP63WT(S) and GP63E265D(S) were grown in spinners and batch expression levels were approximately 4 u,g/mL and 1.5 irg/mL respectively (Fig. 11). While expression levels of recombinant GP63 were not high, with the exception of GP63WT(M) and matGP63WT(S) discussed in Chapters 4 and 6 respectively, they were sufficient to provide enough protein for the following studies. 56 Chapter 4 - Results Chapter 4 Stability of Proteolytically Active GP63 and the Active Site Mutant of GP63 Membrane Expression in CHO Cells For large scale production of recombinant therapeutic proteins, expression instability can be an important constraint that limits the acceptable duration of production processes. However the mechanisms involved in loss of recombinant protein expression in unamplified cell lines are not well known. Expression of proteolytically active GP63WT(M) on the cell surface appeared to be unstable in all the independent clones isolated. The kinetics and mechanisms of this protein instability of GP63WT(M) were investigated. Cell surface expression of GP63WT(M) in CHO cells greatly facilitated the analysis, since by flow cytometry the distribution of protein expression within the population could be accurately determined without limiting dilution cloning. The effect on stability of expressing an active proteinase with broad substrate specificity was investigated by following the expression of both the proteolytically active GP63WT(M) and the active site mutant GP63E265D(M). The effect of selective pressure on expression stability was also investigated by culturing cells in the presence and absence of M T X . A dynamic population model was used to determine the conversion rate of GP63WT(M) producer to nonproducer cells. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis were used to analyse the molecular mechanisms involved in the loss of GP63 expression. 57 Chapter 4 - Results 4.1 Stability of Cell Surface Expression of GP63WT(M) and GP63E265D(M) The stability of GP63WT(M) and GP63E265D(M) expression was followed by flow cytometric analysis for over 138 days in the presence and absence of M T X . Two independent clones expressing GP63WT(M) (#9.4.4 and #41.9.8) and one clone expressing GP63E265D(M) (#29.8.6) were grown in repeated batch culture. These cell lines had been selected in 50 u.M M T X and recloned twice by limiting dilution. On the final redone, all clones assayed were positive. Expression of the active site mutant GP63E265D(M) in the presence of M T X was stable for 138 days (Fig. 12 B). In parallel cultures without M T X (Fig. 12 A) the average cell specific expression of GP63E265D(M) gradually declined due to the appearance by 84 days of a population with 10-fold lower expression. A distinct population of apparent nonproducers was also observed that increased in number as shown at 128 and 161 days. An accompanying decline in the high expressing population was observed. Apparently complete loss of GP63E265D(M) expression occurred by 249 days. However this loss was reversed by placing the cells grown out of M T X back into selection medium, full recovery of expression was observed by day 295 (i.e. within 46 days) (Fig 12 C). Clone 9.4.4. expressing proteolytically active GP63WT(M) appeared to exhibit stable expression for up to approximately 51 days in culture with and without M T X (Fig. 13 A & B). After this time a population of nonproducers was distinguished that gave rise to a bimodal distribution of cells expressing GP63. This was followed by an increase in nonproducers and an accompanying decline of the expressing population. By 138 days in the presence of M T X and 111 days in the absence of M T X , the expressing population had decreased to less than 17% and 11% of the total population, respectively. A similar pattern for loss of GP63WT(M) was observed for a second clone 41.9.8 grown both with and without M T X (Fig. 14 A & B). Unlike 58 Chapter 4 - Results Fluorescence Figure 12. Flow cytometric analysis of cell surface expression of the active site mutant GP63E265D(M). Cells labeled with mAb 96 against GP63 and GAM-FITC (heavy line) and GAM-FITC only (light line). Clone 29.8.6: grown without M T X (panel A), grown with M T X (panel B), placed back in M T X containing medium after loss of GP63E265D(M) expression (panel C). Days indicate time in culture. 59 Chapter 4 - Results Fluorescence Figure 13. Flow cytometric analysis of cell surface expression of proteolytically active GP63WT(M). Cells labeled with mAb 96 against GP63 and GAM-FITC (heavy line) and GAM-FITC only (light line). Clone 9.4.4: grown without M T X (panel A), grown with M T X (panel B). Days indicate time in culture. 60 Chapter 4 - Results Fluorescence Figure 14. Flow cytometric analysis of cell surface expression of proteolytically active GP63WT(M). Cells labeled with mAb 96 against GP63 and GAM-FITC (heavy line) and GAM-FITC only (light line). Clone 41.9.8: grown without M T X (panel A), grown with M T X (panel B). Days indicate time in culture. 61 Chapter 4 - Results GP63E265D(M), the loss of GP63WT(M) expression in the absence of M T X could not be reversed by placing cells back into selection medium. To confirm that these observations were not clone specific, the stability of GP63 expression in a further six GP63E265D(M) clones and five GP63WT(M) clones was investigated in the presence of MTX. All six GP63E265D(M) clones exhibited stable expression for over 100 days, while loss of GP63WT(M) expression was observed in all the wild type clones within 100 days. 4.2 Determination of Specific Growth Rates and Model Simulation Exponential phase growth rates were determined for both wild type and mutant clones before and after loss of GP63 expression. A significant increase in growth rate was observed after loss of both GP63WT(M) and GP63E265D(M) expression (Table 2). No change was observed in the growth characteristics of clone 29.8.6 that exhibited stable expression of GP63E265D(M) when grown in M T X for 138 days. A dynamic population mathematical model was used to investigate the kinetics of GP63WT(M) production stability in clone 9.4.4. This model was derived from the previous analysis of hybridoma production stability (Frame and Hu, 1991; Lee et al., 1991; Kromenaker and Srienc, 1994) and contained the assumptions that (i) the ratio of the producer (uP, h"1) and nonproducer (p.N, h"1) growth rates was constant, (ii) the death rate was negligible and (iii) the net conversion rate of producers to nonproducers could be characterised by a first order constant, a (h"1). These assumptions yielded the following equations: 62 Chapter 4 - Results dX p — = L i p X p - a X p ( 1 ) d X N - ^ = L i N X N + a X p (2) where X P and X N were the numbers of viable producers and nonproducers, respectively and t was time (h). Such a model was appropriate for the case of wild type clones which exhibited an apparently bimodal distribution of producer and nonproducer cells (Fig. 13 & 14). Using the single cloned producer cell as the initial condition (X P = 1 and X N = 0 at t = 0), these equations were integrated to obtain the predicted percent of producer cells out of the total population as a function of the producer and nonproducer growth rates, time and the parameter a: % Producer = 100[Xp/(XP+XN)] (3) where X p / ( X p + X N ) = (4) [e (^-a ) ,] + [ (e^ -e ( ^ a ) ' ) ] "M'N + a " l l p Using the independently measured growth rates of the producer and nonproducer cells, the conversion rate (a) was determined by Marquardt-Levenberg fitting of the percent producer GP63WT(M) population data in terms of the number of population doublings (NPD) for cultures with and without MTX. The results are shown in Figure 15. Conversion rates of 3.2 X 106 h"' (± 0.2 X 10"6) and 1.0 X 106 h 1 (+ 0.1 X 10"6) were determined for cells grown with and without M T X , respectively. 63 Chapter 4 - Results Table 2. The exponential phase specific growth rates (n.) were determined for clones before and after the loss of GP63 in the presence and absence of M T X . A statistically significant (p<0.05) increase in growth rate was observed after the loss of both GP63WT and GP63E265D. A significant change in the growth rate was not observed when clone 29.8.6 GP63E265D was grown in the presence of M T X . The growth rate of the parental untransfected CHO cells was also determined (u. = 0.044 h"1). Clone +GP63 - GP63 + GP63 - GP63 Specific Growth Rates (LI) h + M T X - M T X #9.4.4 GP63WT 0.025 0.028 0.024 0.028 #41.9.6 GP63WT 0.029 0.035 0.039 0.045 #29.8.6 GP63E265D 0.042 N / A 0.041 0.050 64 Chapter 4 - Results 100. 80J •g 60J P H 6s- 40J 20J 0 B 6 2"0 4'0 60 8'0 lOO ' Number of Population Doublings 0 20 40 60 8"0 ldO Number of Population Doublings Figure 15 Stability of GP63WT(M) expression in the presence and absence of MTX. Experimental data obtained for clone 9.4.4 (square symbols) and model simulation (line) (A) with MTX (B) without MTX. 65 Chapter 4 - Results 4.3 RT-PCR Analysis of GP63 mRNA Cells were analysed for the presence of GP63 specific mRNA by RT-PCR to evaluate the correlation between GP63 mRNA and protein expression. GP63 mRNA (0.8 Kb) was detected in both wild type and mutant clones at the start of the experiments (Fig. 16 lanes 1, 4 & 7). For wild type clones that had lost GP63 protein expression no GP63 mRNA was detected (Fig. 16 lanes 2, 3, 5 & 6). The presence of GP63 mRNA was detected in clone 29.8.6 grown without M T X at 161 days when the cells were still expressing a low level of GP63E265D(M) (Fig. 16 lane 9). The presence of p-tubulin mRNA (0.41Kb band) was detected in all samples. No bands were detected in control reactions in which reverse transcriptase was omitted. 4.4 Southern Blot Analysis To determine whether loss of GP63 protein expression correlated with a decrease in gene copy number, Southern blot analysis was performed on genomic DNA isolated from cells at the beginning and end of the long-term culture experiments (Fig. 17). Hybridisation of Pst 1 digested DNA from all clones at the start of the experiments with a GP63 specific probe, resulted in the expected 0.9 Kb band (Fig. 17 A lanes 1,4&7). Titration of plasmid standards was used to estimate the gene copy number for clones expressing GP63WT(M) and GP63E265D(M). The gene copy numbers were found to be low in all clones (< 10 copies per cell) (data not shown). Bands corresponding to GP63 were not detected in lanes containing DNA from wild type clones that had lost GP63 protein expression (Fig. 17 A lanes 2, 3, 5 & 6). In contrast, loss of GP63E265D(M) expression in the absence of M T X did not result in a measurable reduction in GP63 gene copy number (Fig. 17 A lane 9 & 10) when normalised with respect to actin. Cells expressing GP63E265D(M) grown in the presence of M T X also maintained stable gene copy 66 Chapter 4 - Results Figure 16. RT-PCR analysis of clones before and after loss of GP63 protein expression. Samples were analysed for the presence of GP63 mRNA (0.80 Kb DNA band) and p-tubulin mRNA (0.41 Kb DNA band). Total RNA was isolated from cells grown with or without M T X at times given in days. Clone 9.4.4 GP63WT(M): t = 0 (lane 1); t = 111 + M T X (lane 2); t = 138 - M T X (lane 3). Clone 41.9.8 GP63WT(M): t = 0 (lane 4); t = 138 + M T X (lane 5); t = 100 - M T X (lane 6). Clone 29.8.6 GP63E265D(M): t = 0 (lane 7); t = 138 + M T X (lane 8); t = 161 - M T X (lane 9). Cells transfected with pNUT vector (lane 10). Untransfected CHO cells (lane 11). PCR control containing no template (lane 12). Position and size (Kb) of markers is indicated on left. 67 Chapter 4 - Results 1 2 3 4 5 6 7 8 9 10 11 12 13 B B 5.0-3.5-2.0-1.6-1.0-0.5-1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 17. Southern blot analysis of clones before and after loss of GP63 protein expression. Genomic DNA was isolated from cells grown with or without M T X at times given in days and was digested with Pst I. Clone 9.4.4 GP63WT(M): t = 0 (lane 1); t = 111 + M T X (lane 2); t = 138 - M T X (lane 3). Clone 41.9.8 GP63WT(M): t = 0 (lane 4); t = 138 + M T X (lane 5); t = 100 -M T X (lane 6). Clone 29.8.6 GP63E265D(M): t = 0 (lane 7); t = 138 + M T X (lane 8); t = 161 -M T X (lane 9); t = 249 - M T X (lane 10); t = 295 placed back in M T X (lane 11). Cells transfected with pNUT vector (lane 12). Untransfected CHO cells (lane 13). (A) 20 ug of each DNA hybridised with GP63 probe. (B) 10 u,g of each DNA hybridised with DHFR probe. Position and size (Kb) of markers is indicated on left. 68 Chapter 4 - Results numbers (Fig. 17 A lane 8). Parallel Southern blots were hybridised with a probe to DHFR. The expected 2.9 Kb band corresponding to DHFR genes from the pNUT vector, was detected in lanes containing DNA from cells at the start of the experiments (Fig. 17 B lanes 1, 4 & 7). Both wild type clones showed a reduction in gene copy number of DHFR after loss of GP63WT(M) expression (Fig. 17 B lanes 2, 3 5 & 6). A reduction in the DHFR copy number was not observed for clone 29.8.6 after loss of GP63E265D(M) expression (Fig. 17 B lane 9 & 10). The bands observed in lane 11 of Figure 17 A & B were less intense due to under loading of the DNA as determined by actin controls (data not shown). 4.5 Discussion In this study the expression stability of recombinant GP63 in unamplified CHO cells was investigated in long-term cultures. Most production processes involve secreted proteins, but the analysis of population heterogeneity for such cases involves multiple long and arduous limiting dilution clonings to estimate expression variability within the population. Cell surface expression of GP63 greatly facilitated this investigation by enabling the use of flow cytometry to accurately determine the expression distribution within the population. At each time point large numbers of cells, routinely 20 thousand, were analysed. Expression of proteolytically active GP63WT(M) was unstable and loss of expression appeared to be independent of selective pressure. Loss of recombinant protein expression in the presence of selection has been reported previously. The overall secretion rates in the presence of M T X declined after 60 days for IFN-y (Cossons et al., 1991) and after 100 to 200 days for a chimeric mAb (Raper et al., 1992). However in both these studies, expression in bulk populations were monitored and comparisons of producing and nonproducing populations were 69 Chapter 4 - Discussion not performed. The bimodal population in the GP63WT(M) CHO cultures was similar to those reported in hybridoma and transfectoma cultures (Merritt and Palsson, 1993; Bae et al., 1995; Couture and Heath, 1995). Loss of GP63WT(M) expression conferred a growth advantage (from 12 to 20% higher growth rates). Loss of antibody productivity in long-term hybridoma and transfectoma cultures has also been attributed to the appearance of nonproducers (Frame and Hu, 1990; Bae et al., 1995) with reported growth rate increases of 8% (Kromenaker and Srienc, 1994) and 22%> (Chuck and Palsson, 1992). A model was developed for loss of GP63WT(M) expression to estimate the rates of conversion based on the relative growth rates of producers and nonproducers. The close fit of the model to the slope of the decline in percent producers (Fig. 15) indicates that the growth rates measured at the start and end of the experiments were representative. The conversion rates (3.2 x 10"6 h"1 and 1.0 x 10-6 h"1) of GP63 producers to nonproducers in CHO cells were 100 to 1000-fold lower than those reported for myeloma and hybridoma cell lines (Cotton et al., 1973; Morrison et al., 1973; Gardner et al., 1985; Kromenaker and Srienc, 1994). This probably reflects the inherent instability of hybridoma cell lines, which are the product of cell fusion and thus multiploid (Castillo et al., 1993). In an attempt to increase the stability of GP63 protein production, expression of an active site mutant (GP63E265D) was investigated. A similar approach had been used to increase production of plasminogen (Busby et al., 1991). For GP63 to fulfill its role as a vaccine candidate it is not necessary for the protein to be proteolytically active. Expression of GP63E265D(M) was stable in the presence of M T X for 138 days (Fig. 12 B). The growth characteristics of these cells did not change after this extended time in culture. To produce recombinant proteins for therapeutics and vaccines it is necessary to grow cell lines for extended 70 Chapter 4 - Discussion periods in the absence of MTX. The expression of GP63E265D(M) was stable for 30 to 40 PD, but between 41 and 170 PD the distribution of expression per cell of GP63E265D(M) gradually declined. The cells expressing GP63E265D(M) had not been amplified (<10gene copies per cell) but had been selected and maintained in M T X prior to the experiment. Recloning the cells in the absence of M T X may result in cell lines with increased stability in the absence of MTX. In other studies where the bulk population production rates of amplified cells were monitored in the absence of M T X , cell lines were reported to show stable expression for up to only 30 to 38 PD (Kaufman et al., 1985). In this report the cells expressing GP63E265D(M) showed stable expression out of M T X for up to 40 PD after which expression levels gradually declined. Thus many reportedly "stable" cell lines may not be stable indefinitely. The differences observed in the stability of GP63WT(M) compared to GP63E265D(M) suggest that a correlation exists between proteinase activity and instability of GP63 protein expression. Even in the absence of selection, stability of GP63E265D(M) expression was enhanced compared to GP63WT(M) expression. After over 80 PD out of M T X selection (day 84 Fig. 12 A) cells expressing GP63E265D(M) were still 94% positive where as, by 80 PD out of M T X wild type clones had either completely lost expression or were less than 30% positive (Fig. 15). There are a number of reasons why expression of a broad-spectrum active proteinase could be deleterious to cells: (1) activity within the cell could result in proteolysis of the protein translocation machinery of the endoplasmic reticulum or proteins involved in transport to the Golgi, (2) activity at the cell surface or within compartments in transit to the cell surface could result in proteolysis of other proximal cell surface molecules such as receptors, and (3) growth factors in the medium could be degraded. 71 Chapter 4 - Discussion The molecular mechanisms that resulted in loss of protein expression in GP63WT(M) and GP63E265D(M) expressing clones appear to be different. In wild type clones, loss of protein expression correlated with an apparent loss of both GP63 mRNA and GP63 genes. Gene loss has also been reported in hybridomas (Hengartner et al., 1978; Wilde and Milstein, 1980; Westerwoudt et al., 1984). There was an apparent preferential loss of GP63 genes over DHFR genes, divergence in the levels of DHFR and recombinant protein genes have been reported previously (Cossons et al., 1991; Sinacore et al., 1994). Loss of GP63E265D(M) expression in the absence of M T X was not accompanied by a measurable decrease in either GP63 or DHFR gene copy number and was reversed in M T X containing medium. These observations suggest that gene inactivation (e.g. by methylation) (Busslinger et al., 1983; Antequera et al., 1990) may have occurred in the absence of MTX. This inactivation may have occurred in a two stage process, hence the appearance of an intermediate lower expressing population (Fig. 12 A). Resuming selection in M T X may then select for cells in which the genes were no longer methylated. Expression of recombinant GP63 on the surface of CHO cells enabled a quantitative approach to evaluate the stability of expression of an active proteinase and an active site mutant of the proteinase during long-term culture. Comparison of producing and nonproducing populations has given insight into the mechanisms involved in the loss of recombinant protein expression. 72 Chapter 5 - Results Chapter 5 Comparison of Membrane Expression versus Secretion Production Rates A process termed "controlled release" was developed (Kennard et al., 1993) for harvesting recombinant GPI-anchored proteins from the cell surface using PI-PLC. This approach has many advantages for downstream processing, however a direct comparison of the production levels of the same protein expressed on the cell surface versus secreted was not performed. In this study GP63 was expressed on the cell surface and as a secreted protein. Initial experiments indicated that levels of cell surface expression of GP63 were much lower than the levels of secretion. This may be due to post-translational limitations in membrane expression e.g. a limitation in the amount of protein expressed on the cell surface or a limitation in the transport of membrane bound protein to the surface. To address this possibility the production levels of membrane expressed and secreted GP63 were investigated. To compare the cell specific production (ng/106 cells/h) of membrane expression to secretion rates a "continuous harvest" protocol was used for clones expressing GP63E265D(M) on the cell surface. Clones expressing GPI-linked GP63E265D(M) were grown in the presence of PI-PLC so that GP63 was cleaved from the cell surface and accumulated in the cell culture supernatant. Monitoring cell surface production in this manner minimized production losses due to membrane turnover. In order to compare cell surface and secreted production in independent clones the relative cell specific production to mRNA ratios were determined. Comparisons made using these ratios normalized any effects on GP63 expression due to differences in transcription between clones and mRNA turnover rates. Thus potential differences between membrane expressed and secreted production levels that occurred after translation could be identified. 73 Chapter 5 - Results 5.1 Continuous Harvest of Membrane Expressed GP63E265D(M) The cell specific production of membrane expressed GP63E265D(M) was determined for three independent clones (#29.8.6, #51.2.8 and #65.12.10) by continuous harvesting of GPI-anchored proteins from the cell surface using PI-PLC. Cells adapted for suspension, were grown in serum-free medium containing 3 mU/mL of PI-PLC and control cultures were incubated without PI-PLC (Fig. 18). For all three clones the growth characteristics, viability and glucose utilization of the cells grown in the presence of PI-PLC were very similar to the control cultures, indicating that the cell growth was not affected by the presence of PI-PLC (Figs. 19, 20 & 21, A & B). The relative cell surface expression levels of GP63E265D(M) were determined by flow cytometry using a GP63 specific antibody and are presented as relative mean fluorescence (Figs. 19, 20 & 21, C). A rapid decline in mean fluorescence was observed during the first 24 h of culture in the presence of PI-PLC for all three clones. This was followed by a more gradually decline and after approximately 72 h a steady level of expression was achieved where the mean fluorescence was not reduced further. These steady levels of expression in independent clones ranged between 10-25% of control culture expression. Further reduction in mean fluorescence was not achieved by using 10-fold increased concentrations of PI-PLC. The decrease in mean fluorescence was accompanied by a steady increase in GP63E265D accumulation in the culture medium as determined by ELISA. For control cells grown without PI-PLC an initial increase in mean fluorescence was observed for all clones, followed by a gradual decline in expression after approximately 48 h in culture. Some GP63E265D accumulation was observed in the medium. 74 Chapter 5 - Results Fluorescence Figure 18. Continuous harvest of GP63E265D(M) from the cell surface of clone 29.8.6. Flow cytometry profiles of cells in batch culture at times given in hours. Cells from culture containing 3 mU /mL PI-PLC, labeled with mAb 96 against GP63 and GAM-FITC (light line). Cells from control cultures (no PI-PLC) labeled with mAb 96 against GP63 and GAM-FITC (heavy line). Cells labeled with GAM-FITC only (dashed line). 75 Chapter 5 - Results Time (h) B C Time (h) Figure 19. Continuous harvest of GP63E265D(M) from the cell surface of clone 29.8.6. Results for duplicate cultures with PI-PLC are presented as averages and were within 5%. Cells were cultured with (square) or without (circle) 3 mU/mL PI-PLC. (A) Growth curves (closed symbol) and viability (open symbol). (B) Glucose depletion from the medium. (C) Accumulation of GP63E265D(M) in the supernatant (closed symbol) and mean fluorescence of cells labeled with mAb 96 against GP63 and with GAM-FITC, analysed by flow cytometry (open symbol). 76 Chapter 5 - Results B u o o U U o c o O o o ?100 '*80 << -60 CT; § -40 -20 20 40 60 80 100 120 140 Time (h) 60 80 Time (h) 350 % c o U m vo O 60 80 Time (h) Figure 20. Continuous harvest of GP63E265D(M) from the cell surface of clone 51.2.8. Results for duplicate cultures with PI-PLC are presented as averages and were within 5%. Cells were cultured with (square) or without (circle) 3 mU/mL PI-PLC. (A) Growth curves (closed symbol) and viability (open symbol). (B) Glucose depletion from the medium. (C) Accumulation of GP63E265D(M) in the supernatant (closed symbol) and mean fluorescence of cells labeled with mAb 96 against GP63E265D(M) and with GAM-FITC, analysed by flow cytometry (open symbol). 77 Chapter 5 - Results Time (h) Time (h) Figure 21. Continuous harvest of GP63E265D(M) from the cell surface of clone 65.12.10. Results for duplicate cultures with PI-PLC are presented as averages and were within 5%. Cells were cultured with (square) or without (circle) 3 mU/mL PI-PLC. (A) Growth curves (closed symbol) and viability (open symbol). (B) Glucose depletion from the medium. (C) Accumulation of GP63E265D(M) in the supernatant (closed symbol) and mean fluorescence of cells labeled with mAb 96 against GP63 and with GAM-FITC, analysed by flow cytometry (open symbol). 78 Chapter 5 - Results 5.2 Secretion of GP63E265D(S) The cell specific production of secreted GP63E265D(S) was determined for three independent clones (#39.10, #55.13 and #66.1). Cells were grown in serum-free suspension batch culture. All three clones showed similar growth characteristics and glucose utilization reaching maximal cell densities between 1.3-2.0 x 106 (Figs. 22, 23 & 24, A & B). A steady accumulation of GP63E265D(S) was detected in the cell culture supernatant of all three clones by ELISA (Figs. 22, 23 & 24, C). 5.3 Cell Specific Production of GP63E265D(M) versus GP63E265D(S) The cell specific production (ng/106 cells/h) was determined from the batch culture data obtained for clones that express GPI-anchored or secreted GP63 (Fig. 25 A & B). The average cell specific production was calculated for each clone during exponential growth (approximately 24-96 h) (Table 3). There was an order of magnitude lower cell surface expression than secretion of GP63E265D. The average cell specific production for clones expressing GP63E265D(M) ranged from 0.5-1.2 ng/106 cells/h while in clones secreting GP63E265D(S) it ranged from 17.5-25.0 ng/106 cells/h. 5.4 Southern Blot Analysis to Determine Gene Copy Numbers Southern blot analysis was used to determine the gene copy numbers of cells expressing GP63E265D(M) on the cell surface or secreting GP63E265D(S). Hybridisation of Pst 1 digested genomic DNA from all clones with a GP63 specific probe resulted in the expected 0.9 Kb band (Fig. 26 A lanes 1-12). The blots were rehybridised with an actin specific probe to identify variation in sample loading (Fig. 26 B). To determine the gene copy number for each clone, the 79 Chapter 5 - Results B U o U 13 U o U O o o 20 40 60 80 100 120 Time (h) 20 40 60 80 100 120 Time (h) 20 40 60 80 100 120 Time (h) Figure 22. Secretion of GP63E265D(S) by clone 39.10. Results are averages of duplicate cultures and were within 5%. (A) Growth curves (closed symbol) and viability (open symbol). (B) Glucose depletion from the medium. (C) GP63E265D(S) production. 80 Chapter 5 - Results 0 20 40 60 80 100 120 140 Time (h) 0 20 40 60 80 100 120 140 Time (h) Figure 23. Secretion of GP63E265D(S) by clone 55.13. Results are averages of duplicate cultures and were within 5%. (A) Growth curves (closed symbol) and viability (open symbol). (B) Glucose depletion from the medium. (C) GP63E265D(S) production. 81 Chapter 5 - Results Figure 24. Secretion of GP63E265D(S) by clone 66.1. Results are averages of duplicate cultures and were within 5%. (A) Growth curves (closed symbol) and viability (open symbol). (B) Glucose depletion from the medium. (C) GP63E265D(S) production. 82 Chapter 5 - Results A Time (h) B 40 n Time (h) Figure 25. Cell specific production of GP63E265D. Results for duplicate cultures are presented as averages and were within 5%.(A) Cell surface expression, clones: 29.8.6 (circle), 51.2.8 (square) & 65.12.10 (triangle). (B) Secretion, clones: 39.10 (triangle), 55.13 (circle) & 66.1 (square). 83 Chapter 5 - Results level of GP63 genomic DNA obtained by densitometry and adjusted for loading variation, was compared to a standard curves obtained from densitometry of titrated plasmid DNA (Fig. 26 C). Due to variation in the loading and the necessity to adjust the level of GP63 genomic DNA, the gene copy numbers obtained for clones are relative values compared to each other. The gene copy number of clones expressing GP63E265D(M) on the cell surface ranged between 3-8 copies per cell. While the gene copy number of clones secreting GP63E265D(S) were much higher, greater than 63 copies per cell (Fig. 26 D). 5.5 Northern Blot Analysis of GP63E265D mRNA Levels To compare the levels of protein production of cell surface expression and secretion in clones with varying gene copy numbers and potentially varying levels of mRNA, it was necessary to determine the relative cell specific production to mRNA ratios. Northern blot analysis was performed on mRNA isolated from clones expressing GP63E265D(M) and GP63E265D(S) during exponential growth. Hybridisation of mRNA from all clones with a GP63 specific probe resulted in the expected 2.1 Kb band (Fig. 27 A lanes 1-6). No band was detected in mRNA isolated from untransfected CHO cells (Fig. 27 A lane 7). For two GP63E265D(S) clones (#39.10 & #55.13) lanes were loaded with 10 \ig mRNA (Fig. 27 A lanes 4 & 5) compared to 20 |ig loaded for the other clones (Fig. 27 A lanes 1, 2, 3, 6 & 7). A second blot loaded with 20 lag of mRNA for all clones was hybridised with a probe specific for actin (Fig. 27 B). The expected 1.9 Kb band was detected in mRNA from all clones including the CHO control. From this data, a clear difference in the abundance of GP63 mRNA was observed among clones expressing GP63E265D(M) on the cell surface (Fig. 27 A lanes 1-3) and clones secreting GP63E265D(S) (Fig. 27 A lanes 5-6). To determine the amount of GP63 mRNA for 84 Chapter 5 - Results 1 2 3 4 5 6 7 8 9 10 11 12 13 D Lanes Clone Gene Copy Number 1,2 29.8.6 GP63E26513 (M) 8 3,4 51.2.8 GP63E265D (M) 3 5,6 65.10.12 GP63E265D (M) 3 7,8 39.10 GP63E265D (S) 106 9,10 55.13 GP63E265D (S) 82 11,12 66.1 GP63E265D (S) 63 Figure 26. Southern blot analysis and of clones that express GP63E265D(M) on the cell surface or secrete GP63E265D(S). Southern blots hybridised with (A) & (C) GP63 and (B) actin specific probes. (A) & (B) Lanes contained Pst I digested, genomic D N A isolated from clones: 29.8.6 GP63E265D(M) 10 p.g (lane 1) and 20 ug (lane 2); 51.2.8 GP63E265D(M) 20 tig (lane 3) and 30 ug (lane 4); 65.12.10 GP63E265D(M) 20 ng (lane 5) and 30 irg (lane 6); 39.10 GP63E265D(S) 10 ug (lane 7) and 20 ug (lane 8); 55.13 GP63E265D(S) 10 iag (lane 9) and 20 p.g (lane 10); 66.1 GP63E265D(S) 10 ug (lane 11) and 20 ug (lane 12); untransfected C H O cells 20 u,g (lane 13). (C) Standard controls to determine relative gene copy numbers. Lanes contained the specified amounts in pg of pNUT/GP63 plasmid D N A digested with Pst I. (D) Table to show the relative gene copy numbers o f clones as determined by densitometry of Southerns (A), (B) & (C). 85 Chapter 5 - Results Figure 27. Northern blot analysis of clones that express GP63E265D(M) on the cell surface or secrete GP63E265D(S). Five day exposure of Northern blots hybridised with A) GP63 and (B) actin specific probes. Lanes contained 20 ug mRNA with the exception of (A) lanes 4 & 5 which contained 10 |ig mRNA/lane. The mRNA was isolated from clones: 29.8.6 GP63E265D(M) (lane 1), 51.2.8 GP63E265D(M) (lane 2), 65.12.10 GP63E265D(M) (lane 3), 39.10 GP63E265D(S) (lane 4), 55.13 GP63E265D(S) (lane 5), 66.1 GP63E265D(S) (lane 6) and untransfected CHO cells (lane 7). Position and size (Kb) of markers is indicated on left. 86 Chapter 5 - Results membrane expressing and secreting clones, densitometry was performed on autoradiographs after a shorter 2 day exposure. Shorter exposures were used to minimize the underestimation of GP63 mRNA levels in secreted clones. These results were normalised for variation in loading using the 1.9 Kb band in the actin controls. The GP63 mRNA level for membrane expressing clones was in the range of 0.2-1.2 and for secreting clones 6.2-45.7 (Table 3). The relative GP63E265D productivity to mRNA ratios varied between 1.0 - 4.5 for membrane expression and 0.5 - 2.8 for secretion (Table 3). These results indicate that there is no measurable difference in the relative GP63E265D productivity to mRNA ratios between clones expressing GP63E265D(M) on the cell surface and clones secreting GP63E265D(S). 5.6 Discussion Continuous harvest of GP63E265D(M) from the cell surface provided a meaningful way to compare the cell specific productivity of cell surface expression and secretion. Complete cleavage of GP63 from the cell surface was not obtained. Similar results were observed with a single time point harvest of GP63 (Chapter 3, Section 3.4) and also for the continuous harvesting of recombinant melanotransferrin from CHO cells (Sunderji, 1994). PI-PLC has been shown to be stable at 37°C for over 14 days (Sunderji, 1994) and increasing the initial concentration of PI-PLC 10-fold did not enhance the degree of cleavage obtained. The 10-25 % residual uncleaved GP63 may have been located in a position that was inaccessible to the PI-PLC. Inaccessibility to PI-PLC was previously reported for alkaline phosphatase expressed in two human cervical epithelial carcinoma cell lines (HeLa and SKG3a) (Wong and Low, 1992). Alternatively the uncleaved GP63 may represent a population of GPI-linked GP63 that was resistance to cleavage by PI-PLC. Partial or complete resistance to PI-PLC has previously been reported for GPI 87 Chapter 5 - Results Table 3. Cell specific production and mRNA levels of clones expressing GP63E265D(M) and GP63E265D(S). GP63 cell specific production rates were determined for cells during exponential growth. GP63 mRNA levels were determined by densitometry of Northern blots of mRNA isolated from exponentially growing cells and hybridised with GP63 and actin specific probes. Clone GP63 Production Rate (ng/106cells/h) GP63 Normalised mRNA levels GP63 Production Rate/ GP63 mRNA Levels 29.8.6 (M) 1.2 1.2 1.0 51.2.8 (M) 0.9 0.2 4.5 65.10.12 (M) 0.5 0.4 1.3 39.1(S) 25.0 45.7 0.5 55.13(S) 22.5 18.4 1.2 66.10(S) 17.5 6.2 2.8 88 Chapter 5 - Discussion anchors expressed in a variety of cell lines (Wong and Low, 1992). The degree of resistance was found to be cell line dependent. In most cases resistance to PI-PLC results from acylation by palmitate or other fatty acids (reviewed by Ferguson and Williams, 1988). Consistent with previous reports (Kennard et al., 1993) incubation of cells in the presence of PI-PLC and resulting cleavage of GPI-linked proteins on the surface of CHO cells did not appear to effect cell growth and viability. Some accumulation of GP63 in the cell culture supernatant was observed in control cultures that did not contain PI-PLC (Fig. 19, 20 & 21 C). Such shedding of GPI-anchored proteins in small membrane vesicles has previously been reported in mammalian cells (Test et al., 1991). The release of GPI-anchored DAF from the cell surface of HeLa cells under physiological conditions by cell associated PI-PLD has also been reported (Metz et al., 1994). The cell specific productivity of secreted GP63E265D(S) was approximately 25-fold higher than the cell specific productivity of membrane expressed GP63E265D(M). As a result of incomplete cleavage of GP63 with PI-PLC the values obtained for the cell specific productivity of cell surface expressed GP63E265D(M) would be slightly underestimated, however such small differences would not effect the overall result. A comparison of the relative GP63 productivity to mRNA ratios for independent clones expressing GP63E265D(M) on the cell surface or secreting GP63E265D(S) showed no differences in the range of values obtained. The results indicate that the reduced cell specific productivity of GPI-anchored protein was not due to a post-translational limitation. The increased productivity of secreted GP63 appeared to be directly related to elevated levels of GP63 mRNA and gene copy number. The relative gene copy number of independent clones secreting GP63E265D(S) were much higher (greater than 63) than in clones with cell surface expression (less than 10). These results were obtained even though transfections 89 Chapter 5 - Discussion of CHO cells with constructs for secretion and cell surface expression were carried out on the same day using cells from the same batch cultures and identical conditions for transfection and selection. Thus it appears that cells with high numbers of GP63E265D(M) gene copies were not obtained possibly due to selection against high level production of GPI-anchored protein. It is possible that post-translational limitations may be encountered for other recombinant proteins in cell lines with much higher levels of cell surface expression than the levels obtained for GP63E265D. 90 Chapter 6 - Results Chapter 6 Comparisons of the Post-translational Modifications of Membrane Expressed and Secreted GP63 The effect on the post-translational modifications of membrane versus secreted production of recombinant proteins has not been well documented. Post-translational modification of importance in the production of GP63 include processing of the 61 amino acid pro-region that results in activation of the proteinase and N-linked glycosylation. GP63 expressed on the cell surface of COS cells, was shown to be proteolytically active (Macdonald et al., 1995), whereas in both COS cells and the baculovirus expression system recombinant GP63 was secreted in a latent form that retained the pro-region (Button et al., 1993). Autocatalytic processing and activation of latent secreted GP63 by the addition of HgCl 2 occurred by a mechanism analogous to the "cysteine switch" (Button et al., 1993; Macdonald et al., 1995) previously described for members of the family of matrix metalloproteinases (Springman et al., 1990; Van Wart and Birkedal-Hansen, 1990). In this study, processing and activation of membrane expressed and secreted GP63 proteinase in CHO cells was investigated. The glycosylation of relatively few recombinant membrane proteins (Guan et al., 1988; Konig et al., 1989) and numerous secreted proteins (Takeuchi et al., 1988; Leonard et al., 1990; Hironaka et al., 1992; Bergwerff et al., 1993; Orita et al., 1994; James et al., 1995) has been analysed in great detail. However little is known about differences in the glycosylation pathways of secreted and membrane expressed proteins. Fukuda et al. (1988) appear to be one of the few groups to report a comparison of the glycosylation of a secreted and cell surface expressed protein produced in the same cell line. 91 Chapter 6 - Results GP63 has three potential sites for N-linked glycosylation. Western blot analysis of membrane expressed and secreted GP63 indicated potential differences in the glycosylation of these two forms of the protein. Fast atom bombardment mass spectrometry (FAB-MS) was used to analyse the structural differences in the glycans of membrane expressed and secreted GP63. Differences in the glycosylation may be due to differences in the dynamics of the secretion pathways and thus residence times of membrane expressed and secreted GP63 were determined by pulse chase experiments. 6.1 Processing of Pro-region and Activation of Cell Surface Expressed GP63WT(M) The processing of GP63WT(S), GP63WT(M) and GP63E265D(M) was investigated by Western blot analysis. GP63WT(M) and GP63E265D(M) were harvested from the cell surface using PI-PLC. GP63WT(S) was isolated from cell culture supernatants. Equal amounts of GP63WT(S), GP63WT(M) and GP63E265D(M), as determined by ELISA, were analysed. The controls used included PI-PLC harvested protein and cell culture supernatant from CHO cells transfected with the pNUT vector. Western blots were developed with a GP63 specific monoclonal antibody (Button et al., 1991) and the results confirmed that sample loadings were equal (Fig. 28 A lanes 1, 2 & 3). A second blot was developed with an affinity purified polyclonal antibody specific for the pro-region of L. major GP63 (Macdonald, 1995) and the results demonstrated that GP63WT(M) expressed on the cell surface did not contain the pro-region (Fig. 28 B lane 1), indicating that the pro-region was processed. In contrast, expression of GP63WT(S) as a secreted protein resulted in the expression of a latent form of the proteinase that still retained the pro-region (Fig. 28 B lane 2). To determine whether processing was due to the presence of a CHO processing enzyme that acted preferentially on the membrane bound protein or to self-processing 92 Chapter 6 - Results 106 71 4 4 B 106 71 4 4 1 2 3 4 5 1 2 3 4 5 Figure 28. Processing of GP63WT(M), GP63E265D(M) and GP63WT(S). Western blots probed with (A) mAb 139 against GP63 (B) rabbit polyclonal antibody specific for the pro-region of GP63. Lanes were loaded with (A) 15 ng /lane & (B) 30 ng /lane of: PI-PLC harvested GP63WT(M) (lanel), PI-PLC harvested GP63E265D(M) (lane 2), GP63WT(S) (lane 3). Controls: 30 uL PI-PLC harvested material (lane 4) and 20 uL of supernatant concentrated 5 times (lane 5) from CHO cells transfected with pNUT vector. The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. 93 Chapter 6 - Results of membrane bound GP63WT(M), the processing of GP63E265D(M) was analysed. Probing with the antibody specific to the pro-region showed that GP63E265D(M) expressed on the cell surface also retained the pro-region (Fig. 28 B lane 3). To confirm that the processing of the pro-region of GP63WT(M) resulted in activation of the proteinase, the activities of membrane expressed GP63WT(M) and GP63E265D(M) were investigated by zymogram analysis (Fig. 29). Equivalent loadings of PI-PLC harvested membrane expressed GP63WT(M) and GP63E265D(M) were analysed with controls including cell lysate of L. major promastigotes (Fig. 29 A & B lane 3) and PI-PLC harvested protein from CHO cells transfected with the pNUT vector (Fig. 29 B lane 0). A band of clearing indicating proteinase activity was detected for GP63WT(M) (Fig. 29 A lane 1) at a corresponding position to GP63 proteinase activity in L. major cell extracts (Fig. 29 A lane 3). No detectable proteinase activity was observed for GP63E265D(M) (Fig. 29 A lane 2). Western blot analysis confirmed that the loading of GP63WT(M) and GP63E265D(M) were similar (Fig. 29 B lane 1 & 2). 6.2 Comparison of GP63WT(S) and GP63E265D(S) Proteinase Activation GP63 was demonstrated to be secreted in a latent form that retained the pro-region (Fig. 28). Activation of secreted GP63WT(S) was investigated by a soluble succinylated casein proteinase activity assay and by Western blot analysis. The activation of GP63WT(S) was compared to activation of secreted active site mutant GP63E265D(S). Purified GP63WT(S) and GP63E265D(S) were incubated at 4°C or at 37°C in the presence and absence of 6.4 uM HgCl 2 (20-fold molar excess). Western blot analysis of samples using a GP63 specific antibody confirmed that sample loading were equal (Fig. 30 A). Using the antibody specific to the pro-region of GP63 (Fig. 30 B) it was shown that treatment of GP63WT(S) with HgCl 2 at 37°C 94 Chapter 6 - Results A ' 2 3 Figure 29. Proteinase activity of GP63WT(M) and GP63E265D(M) purified from the cell surface of CHO cells. (A) Zymogram (B) Western blot analysis using mAb 139 against GP63. Lanes were loaded with (A) 30 ng/lane & (B) 15ng/lane of: GP63WT(M) (lane 1) and GP63E265D(M) (lane 2). Controls: cell lysate of L. major promastigotes (lane 3) and PI-PLC harvested supernatant from CHO cells transfected with pNUT vector (lane 0 (B) only). The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. The position of L. major GP63 is indicated by arrows in (A) and (B). 95 Chapter 6 - Results resulted in a shift in molecular weight and at least partial removal of the pro-region indicated by the reduced intensity of the band (Fig. 30 B lane 1 & 3). Incubation at 37°C without HgCl 2 also resulted in a slight decrease in molecular weight without significantly diminishing the intensity of the band (Fig. 30 B lane 2). The decrease in molecular weight without complete loss of antibody binding is consistent with the known step-wise removal of the pro-region that occurs upon activation (Macdonald et al., 1995). Amino-terminal sequencing showed that activated secreted GP63WT(S) from CHO cells had the same mature NH2-terminus (VRVDN) observed for GP63 isolated from L. major promastigotes (Button and McMaster 1988). Incubating GP63E265D(S) at 37°C with HgCl 2 also resulted in a decrease in molecular weight and a slight reduction in band intensity (Fig. 30 B lanes 5 & 6), again indicating that the pro-region is at least partially processed in the presence of HgCl 2 at 37°C. Analysis of cyanogen bromide fragments of GP63E265D(S) by electrospray mass-spectrometry identified a fragment that was consistent with the mass of a peptide containing the intact pro-region (data not shown). This result confirms that GP63E265D(S) was secreted with the attached pro-region. The proteinase activity of GP63WT(S) and GP63E265D(S) was determined before and after activation using a soluble succinylated casein assay (Fig. 30 C). Incubation of GP63E265D(S) at 37°C resulted in 1.6-fold and 1.5-fold enhancements of proteinase activity with and without HgCl 2 , respectively. In contrast to secreted GP63WT(S) obtained from the baculovirus expression system (Macdonald et al., 1995), incubating GP63WT(S) from CHO cells at 37°C was sufficient to activate the proteinase. Also the low level of enhancement suggests that GP63WT(S) incubated at 4°C may have been partially activated during the assay which was incubated at 37°C for 2 h. The activity of GP63E265D(S) was considerably reduced (Fig. 30 C). It was necessary to incubate GP63E265D(S) with HgCl 2 at 37°C to achieve significant activation. 96 Chapter 6 - Results Figure 30. Processing and proteinase activity of purified secreted GP63WT(S) and GP63E265D(S). Western blots probed with (A) mAb 139 against GP63 (B) rabbit polyclonal antibody specific for the pro-region of GP63. (A) & (B) loaded with 50 ng /lane of: GP63WT(S) incubated for 24 h at 4°C (lane 2), 37°C (lane 3), 37°C with 6.4 um HgCl2 (lane 4) and GP63E265D(S) incubated for 24 h at 37°C (lane 5), 37°C with 6.4 um HgCl2 (lane 6). Control: 30 uL of supernatant from CHO cells transfected with pNUT vector (lane 1). The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. (C) Soluble succinylated casein proteinase activity assay comparing the activity of GP63WT(S) (closed symbols) and GP63E265D(S) (open symbols) incubated for 24 h at 4°C (square) 37°C (circle) and 37°C with 6.4 um HgCl2 (triangle). 97 Chapter 6 - Results 125.6-79.6-47.7 — 1 2 3 4 5 6 125.6 79.6 47.7 1 2 3 4 5 6 0.0 100 200 300 400 G P 6 3 (ng) 500 600 98 Chapter 6 - Results A 6-fold increase in activity compared to GP63E265D(S) incubated at 4°C was observed. However, GP63E265D(S) incubated at 37°C with HgCl 2 had only 24% of the activity of GP63WT(S) incubated under the same conditions. While GP63E265D(S) incubated at 4°C and 37°C without HgCl 2 had 10% activity of GP63WTTS) incubated under the same conditions. 6.3 Role of the Pro-region of GP63 in Secretion To determine whether it was possible to obtain a "mature" form of secreted GP63 that lacked the 61 amino acid pro-region, a construct referred to as matGP63WT(S) was expressed for secretion in CHO cells. The expression of matGP63WT(S) and GP63WT(S) were compared by pulse chase analysis with 3 5S methionine. In cells expressing GP63WT(S) bands corresponding to GP63 were detected in cell extracts immunoprecipitated with a polyclonal antibody specific to GP63 at the start of the chase (Fig. 31 A lane 1 b). A gradual decline in intracellular GP63 was observed with increasing chase time until by 480 min intracellular GP63 was no longer detected (Fig. 31 A lane 6 b). GP63 was detected in cell culture supernatants by 20 min and accumulated with increasing chase time (Fig. 31 B). In cells expressing matGP63WT(S) bands corresponding to GP63 were also detected in the cell extracts at the start of the chase (Fig. 32 A lane 1 b). Again a gradual decline in intracellular GP63 was observed with increasing chase time (Fig. 32 A). However no matGP63WT was detected in cell culture supernatants (Fig. 32 B). 6.4 Purification of Recombinant GP63E265D for FAB-MS analysis of Glycans GP63 for FAB-MS analysis was isolated from cells expressing the active site mutant form of GP63 either as a membrane expressed or secreted protein. To determine whether glycosylation patterns changed during exponential growth, GP63E265D(S) was isolated from culture 99 Chapter 6 - Results Figure 31. Pulse chase to follow expression of GP63WT(S) in clone 49.3. (A) Cell extracts and (B) supernatants at chase times in min: (1) 0, (2) 30, (3) 60, (4) 120, (5) 180 & (6) 480. Samples were immunoprecipitated with (a) control rabbit polyclonal antibody and (b) GP63 specific rabbit polyclonal antibody. The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. 100 Chapter 6 - Results Figure 32. Pulse chase to follow expression of matGP63WT(S) in clone 1.4.6.1. (A) Cell extracts and (B) supernatants at chase times in min: (1)0, (2) 30, (3) 60, (4) 120, (5) 180 & (6) 480. Samples were immunoprecipitated with (a) control rabbit polyclonal antibody and (b) GP63 specific rabbit polyclonal antibody. The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. 101 Chapter 6 - Results supernatants of cells, in mid and late-log phase growth (0.6 x 106 and 1.2 x l O 6 cells/mL, respectively) when cells were still approximately 90% viable (Fig. 33 A). Cells expressing GP63E265D(M) on the cell surface had a 25-fold lower cell specific productivity (Chapter 5 Table 3). To maximize the yield, GP63E265D(M) was harvested from the cell surface using PI-PLC in late-log phase cultures when the cells were still greater than 90% viable (Fig. 33 B). Using a monoclonal antibody affinity column GP63 was purified to homogeneity as assessed by silver stained SDS-PAGE (Fig. 34 lane 2, 3 & 5). The identity of the protein was confirmed by Western blot analysis (data not shown). 6.5 FAB-MS Analysis of Glycans of Membrane Expressed and Secreted GP63E265D N-linked glycans released from membrane expressed and secreted GP63E265D were analyzed by FAB-MS after permethylation (Fig. 35). Samples were analyzed in the mass range up to 4400 Da and assignments were made on the basis of m/z ratio of molecular and fragment ions (Table 4). In FAB-MS ions with single charges are obtained. The assignments were verified by linkage analysis (data not shown). The glycan structures of both secreted and membrane expressed GP63E265D were predominantly complex biantennary and triantennary types with lesser amounts of tetraantennary structures. The A-type cleavage ions observed for both membrane expressed and secreted GP63E265D suggest that, when present, fucose is attached to the innermost core N-acetylhexosamine (Table 4). However, other features of secreted and membrane sample spectra were dramatically different. The major peaks observed in membrane samples (at m/z 2244.7, 2606.1 and 2431.7) corresponded to the sodium forms of asialo and monosialo fucosylated biantennary and monosialo non-fucosylated biantennary structures respectively (Fig. 36 A). In secreted GP63E265D(S) isolated from late-log phase cultures, the 102 Chapter 6 - Results A Time (h) Figure 33. Production of secreted and membrane expressed GP63E265D. Growth curves of clones (A) 55.13 GP63E265D(S) and (B) 29.8.6 GP63E265D(M) showing cell concentration (square symbol) and viability (circle symbol). Harvest times of mid and late-log phase cultures indicated by arrows. 103 Chapter 6 - Results 1 2 3 4 5 Figure 34. Purification of GP63E265D(S) and GP63E265D(M). Silver stained SDS-PAGE gel, lanes loaded with 5 uL cell supernatant (lane 1), 170 ng of purified GP63E265D(S) from late-log phase cultures (lane 2), 270 ng of purified GP63E265D(S) from mid-log phase cultures (lane 3), 5 uL PI-PLC harvest from the cell surface (lane 4) and 140 ng of purified GP63E265D(M) (lane 5). The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. 104 Chapter 6 - Results N H , t t T t 1 1 1 s - - -s 1 ] C O O H Reduction & Carboxy methylation N-Glycans ^ O-Glycans N H , t f T f T 1 1— s-cm s -cm s-cm 3 C O O H s-cm Specific Protease Digestion i 1 1 PNGase Digestion Sep-Pak f M f r ^ i I Fractionation • Dy m m 1 1 • in • 0% 2 0 % 30% Permethylation and FAB-MS Reductive Elimination 1 FAB-MS or| ES-MS Mapping Figure 35. Scheme for analysis of N and O-linked glycan structures by fast atom bombardment-mass spectrometry (FAB-MS). This figure is adapted from Dell et al., 1993. 105 Chapter 6 - Results predominant peaks observed (at m/z 2945.0 and 2771.0) corresponded to fully sialylated fucosylated and non-fucosylated biantennary structures (Fig. 36 C). Similarly the predominant species of triantennary structures in late-log phase secreted GP63E265D(S) were fully sialylated with or without fucosylation (m/z 3778.6 and 3605.2) (Fig. 37 C), while higher structures from membrane expressed GP63E265D(M) had variable degrees of sialylation (Fig. 37 A). Thus, in membrane expressed GP63E265D(M) the degree of sialylation of glycans was greatly reduced compared to glycans isolated from secreted GP63E265D(S). Another difference between membrane and secreted glycans was indicated by the presence of A-type fragment ions in membrane samples at m/z 913.5 and 1274.7 (Table 4). The composition of these ions Hex 2HexNAc 2 + and NeuAcHex 2HexNAc 2 + are indicative of the presence of poly-N-acetyllactosamine repeats in the outer arms of complex glycan structures. Some of the higher structures observed for glycans isolated from membrane expressed GP63E265D(M) may therefore represent biantennary or triantennary structures containing up to three poly-N-acetyllactosamine repeats. The spectra of glycans isolated from GP63E265D(S) from mid and late-log phase cultures were found to be very similar. The predominant structures were identical (Figs. 36 & 37 B & C). One minor difference observed was the presence of a molecular ion at m/z 1579.7 in secreted GP63E265D(S) from late-log phase which corresponded to the sodium form of Hex5HexNAc2, an unsubstituted mannose structure (Table 4). GP63E265D(S) from late-log phase cultures was chemically treated by reductive elimination to remove potential O-linked glycans. O-linked glycans were not detected by FAB-MS analysis (data not shown). 106 Chapter 6 - Results B 100S, 95. 90. 85. 80J 75. 70. 65-1 60. 55. 50. 45J 40. 35. 30. 25. 20J| 15. 10. 5 0, 2244.7 2222.5 2071.0 2431.7 2 6 P 6 1 '2'o'o'o' V l W '^ 'o'o' 'ii'oO Wo'o' '25'dd '2V00' ^Vo'o' 2'8'o'o' 'zVo'o' VoW Vl'o'o' '^ 'o'o' '3'3'o'o' m/z 2771.4 2944.9 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 m/z 2018.2 2771.0 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 m/z Figure 36. FAB mass spectra of permethylated N-glycans from GP63 in the molecular ion region (mass range 1950-3400). (A) Cell surface expressed GP63E265D(M) (B) secreted GP63E265D(S) from mid-log phase cultures and (C) secreted GP63E265D(S) from late-log phase cultures. 107 Chapter 6 - Results Figure 37. FAB mass spectra of permethylated N-glycans from GP63 in the molecular ion region (mass range 2550-4400). (A) Cell surface expressed GP63E265D(M) (B) secreted GP63E265D(S) from mid-log phase cultures and (C) secreted GP63E265D(S) from late-log phase cultures. 108 Chapter 6 - Results Table 4. Assignments of FAB-MS peaks observed for the molecular and fragment ions of the permethylated N-linked glycans released from membrane expressed and secreted GP63E265D. Calculated masses are based on monoisotopic mass. Observed Mass (AMU) Calculated Membrane Late Log Mid Log Mass Assignment Expressed Phase Phase (AMU) GP63 Secreted Secreted GP63 GP63 376.2 N e u A c + 376.2 464.2 HexHexNAc + 464.3 825.4 NeuAcHexHexNAc + 825.5 825.3 825.5 913.6 Hex2HexNAc2 + 913.5 1171.6 Hex3HexNAc2 + N a + 1171.6 1171.5 1274.6 NeuAcHex2HexNAc2 + 1274.7 1345.6 Hex3HexNAc2Fuc + N a + 1345.6 1416.6 Hex3HexNAc3 + N a + 1416.9 1557.8/1579.7 Hex 5 HexNAc2 + H+/Na + 1558.0/1579.6 1639.7/1661.7 Hex3HexNAc4 + H + /Na+ 1662.0 1641.7 1770.8 Hex5HexNAc3 + 1771.1 1884.8 Hex3HexNAc5 + H + 1886.1 2018.2 Hex4HexNAc4Fuc + H + 2018.2 2069.9 Hex5HexNAc4 + N a + 2071.0 2070.3 2070.3 2222.0/2244.0 Hex5HexNAc4Fuc + Na 2222.5/2244.7 2222.4/2244.5 2409.1/2431.1 NeuAcHex5HexNAc4 + H + /Na+ 2431.7 2409.7 2409.5 2493.2 NeuAc2Hex5HexNAc3 + 2493.4 2519.3 HexgHexNAc5 + N a + 2520.0 2583 2/2605.2 NeuAcHex5HexNAc4Fuc + H+/Na+ 2584.2/2606.1 2770.3/2792.4 NeuAc2Hex5HexNAc4 + H + / N a + 2792.8 2770.7/2793.5 2771.4/2793.3 2880.3 NeuAcHexgHexNAcs + H + 2880.8 2944.6/2966.4 NeuAc2Hex5HexNAc4Fuc + H + /Na+ 2968.5 2945.0/2967.3 2944.9/2967.3 3032.4/3054.4 NeuAcHex6HexNAc5Fuc + H + / N a + 3056.2 3033.5 3073.5 NeuAcHexsHexNAcgFuc + H + 3074.1 3219.5/3241.5 NeuAc2Hex6HexNAc5 + N a + 3242.1 -3220.2 3602.7 NeuAc3HexgHexNAc5 + N a + 3603.8 3605.1 3603.8 3690.7 NeuAc2Hex7HexNAc6 + N a + 3691.7 3776.8 NeuAc3HexgHexNAc5Fuc + N a + 3778.6 3778.2 4051.9 NeuAc3Hex7HexNAc6 + N a + 4054.5 4226.0 NeuAc3Hex7HexNAcgFuc + N a + 4228.2 109 Chapter 6 - Results 6.6 FAB-MS Analysis of Neuraminidase Treated N-linked Glycans Removal of sialic acid residues by neuraminidase treatment of glycans highlighted other structural differences between membrane expressed and secreted GP63E265D glycans (Fig. 38, Table 5). There was a striking difference in the proportions of fucosylated and non-fucosylated glycan structures. In membrane GP63E265D(M) there were approximately equal proportions of fucosylated and non-fucosylated biantennary structures (m/z 2070.5 and 2245.1) while higher structures Hex6HexNAc5 and Hex7HexNAc6 were predominantly unfucosylated (m/z 2520.0 and 2969.4) with only small amounts of the fucosylated form (m/z 2694.7 and 3143.4). The presence of the structure Hex8HexNAc7 (m/z 3419.2) was further evidence for poly-N-acetyllactosamine repeats in glycans isolated from GP63E265D(M) (Fig. 38 A). Glycans isolated from GP63E265D(S) had different proportions of fucosylated and non-fucosylated structures. There was approximately 2-fold more non-fucosylated biantennary (m/z 2070.5) than fucosylated biantennary structures (m/z 2245.1). However there was approximately equal proportions of fucosylated and non-fucosylated triantennary and tetraantennary structures (m/z 2520.3 & 2694.3 and 2969.8 & 3143.7) (Fig. 38 C). The spectra of glycans isolated from secreted GP63E265D(S) from mid and late-log phase were very similar (Fig. 38 B & C). 6.7 Pulse chase of Clones Expressing GP63E265D(M) or GP63E265D(S) Pulse chase experiments with 3 5S methionine were performed to determine whether differences in glycosylation of membrane expressed and secreted GP63E265D were reflected by differences in the residence times of these alternate forms of the protein within the secretory pathway. Resistance of GP63 to Endo H digestion was used as a marker for processing of glycans in the medial compartment of the Golgi (Komfeld and Komfeld, 1985). 110 Chapter 6 - Results B 2000 100&, 95. 90. 85J 80. 75J 70 J 65. 60 J 55. 50. 45. 40. 35. 30. 25. 20J 15. 10. 2200 2400 2070.4 2600 2800 3000 3200 3400 m/z 2245.1 2520.3 2694.8 2970 .2^44^ 100% 9 5 4 90. 85_| 80 j 75, 70. 65 60 j 55 j 50. 45. 40. 35. 30j 25J 20J 15. 10. 5. 0. 2000 2200 12070.5 2600 3200 3400 m/z 2245.1 2520.3 2694.3 2969.8 3143.7 '20'00 ' ' 22'00 '24'00 ' ' 26'oO 3400 m/z Figure 38. FAB mass spectra of neuraminidase treated permethylated N-glycans from GP63 in the molecular ion region (mass range 1950-3500). (A) Cell surface expressed GP63E265D(M) (B) secreted GP63E265D(S) from mid-log phase cultures and (C) secreted GP63E265D(S) from late-log phase cultures. Ill Chapter 6 - Results •3 N •a V J J> P 8 •a 00 . - H ^ 03 0) <u «3 1) Q 4> ID «! _ VO 03 O co O B Q , so s-3 S C/2 C E a .SP SI cn IT) 0 s CN I T ) o x CN cn o ffi 0> ffi o c-o CN o x cn CN o x CN o x cn 03 z o 3 o <o ffi x ffi •t CN CN o 1-o x Z + <L> ffi CD ffi o CN CN o x o x Os + 0 s -cn CS3 + o 3 Ml u < U ffi <D ffi T OS SO CN O X CN CN o x C--a Z + so O ffi x ffi OS SO OS CN o x CN © x O s ° o x CN ox O + s ° o x CN CN z o 3 O < ffi x~ CD ffi z + O < to ffi oo X <o ffi CN OS 112 Chapter 6 - Results In cells expressing GP63E265D(M) on the cell surface, bands corresponding to GP63 were immunoprecipitated that remained sensitive to Endo H for at least 40 min as indicated by a shift in molecular weight following treatment with the enzyme (Fig. 39 A lanes 1,2, 3 b & c). Bands corresponding to GP63 were observed within 60 min that did not decrease in size after Endo H digestion (Fig. 39 A lane 4 c). The proportion of Endo H resistant GP63 increased with chase time (Fig. 39 A) and by 360 min virtually all the labeled GP63 was Endo H resistant (Fig. 39 A lanes 8 b & c). Pulse chase of cells incubated in the presence of PI-PLC confirmed that GP63 was cleaved from the cell surface and detected in the culture supernatant by 60 min (Fig. 39 B lane 4 b). GP63 detected in the supernatant was not sensitive to Endo H (Fig. 39 B lanes 6 c & d). GP63 was not detected in the cell culture supernatant that did not contain PI-PLC (data not shown). In GP63E265D(S) expressing cells, the protein was also detected in the supernatant by 60 min (Fig. 40 B lane 4 b). Again GP63 detected in the supernatant was not sensitive to Endo H (Fig. 41 B lanes 8 b & c). A band of Endo H sensitive GP63 persisted within the cell even after 360 min (Fig. 40 A lanes 8 b & c). Incubating GP63E265D(S) expressing cells in the presence of PI-PLC did not effect secretion, GP63 was still detected by 60 min in cell culture supernatants (data not shown). For cells expressing GPI-anchored and secreted GP63E265D, Endo H resistant protein was detected at the cell surface or in the culture supernatant by 60 min. These results indicate that the dynamics of the secretion pathway and residence times for membrane expressed and secreted GP63E265D were very similar. 113 Chapter 6 - Results Figure 39. Pulse chase to follow expression of GP63E265D(M) in clone 29.8.6. (A) Cell extracts and (B) supernatants from cells grown in the presence of 500 mU/mL PI-PLC, at chase times in min: (1)0, (2)20, (3)40, (4)60, (5)90, (6) 120, (7) 180 & (8) 360. Cell extracts and supernatants were immunoprecipitated with (a) control rabbit polyclonal antibody or (b) (c) & (d) GP63 specific rabbit polyclonal antibody. Cell extracts immunoprecipitated with GP63 specific antibody were either (b) mock digested or (c) digested with Endo H. Supernatant from 120 min (6) was either (c) mock digested or (d) digested with Endo H. The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. 114 Chapter 6 - Results 1_ 2 3 4 a b c a b c a b c a b c 5 6 7 8 J 2 3 4_ _5_ 6 a b a b a b a b a b a b e d 115 Chapter 6 - Results Figure 40. Pulse chase to follow expression of GP63E265D(S) in clone 55.13. (A) Cell extracts and (B) supernatants at chase times in min: (1) 0, (2) 20, (3) 40, (4) 60, (5) 90, (6) 120, (7) 180 & (8) 360. Cell extracts and supernatants were immunoprecipitated with (a) control rabbit polyclonal antibody or (b) & (c) GP63 specific rabbit polyclonal antibody. Cell extracts from all time points and supernatant from 360 min (8) that were immunoprecipitated with GP63 specific antibody, were either (b) mock digested or (c) digested with Endo H. The position and reported molecular mass (kDa) of molecular weight markers are indicated on the left. 116 Chapter 6 - Results Wl Chapter 6 - Discussion 6.8 Discussion Previously expression of GP63 on the cell surface of COS cells as a GPI-linked or as a transmembrane protein was shown to result in proteinase activation, while GP63 was secreted in a latent form (Macdonald et al., 1995). However, transient expression in COS cells did not yield sufficient amounts of protein to examine the processing further and the involvement of a mammalian processing enzyme could not be discounted. Expression of GP63WT(M) on the cell surface of CHO cells also resulted in processing of the pro-region and activation of the proteinase (Fig. 28 & 29). However the pro-region was not processed when the active site mutant was expressed on the cell surface. These results suggest that processing of GP63 is an autocatalytic event that does not appear to involve a CHO processing enzyme. In vitro, activation of secreted GP63 was shown to occur via an intramolecular rather than intermolecular processing event (Macdonald et al., 1995). In CHO cells, expression of GP63WT(M) in a membrane bound form was required for self-processing to occur since secreted GP63WT(S) was unprocessed. This may be due to an alteration in the conformation of membrane bound protein that renders the protein more susceptible to self processing compared to the soluble form. Alternatively, if processing in vivo was occurring via an intermolecular event, membrane expression may be required for increased proximity of GP63 molecules. Expression of the active site mutant and proteolytically active GP63 as secreted proteins generated sufficient protein to enable the use of a more quantitative soluble assay for proteinase activity. In contrast to GP63 secreted in the baculovirus expression system, GP63WT(S) from CHO cells was much more susceptible to activation. This may be due to slight denaturation of the protein during purification from CHO culture supernatant which involved an elution step at high pH. Incubation of GP63WT(S) at 37°C appeared to be sufficient to activate the proteinase. 118 Chapter 6 - Discussion The active site mutant of GP63 had greatly reduced proteinase activity and the addition of HgCl 2 was required to achieve significant activation. After activation at 37°C by the addition of HgCl 2 using this assay GP63E265D(S) had 24% of the activity of GP63WT(S) with 3-fold lower proteinase activity than GP63WT(S) incubated at 4°C. This activity was not detectable by zymogram analysis (Fig. 8 B). The relative proteinase activity of in vitro activated GP63E265D(S) to GP63WT(S) measured by the succinylated casein assay was higher than results published of other zinc metalloproteinases with the same Glu to Asp mutation in the active site. For example, pro-region processing by the active site mutant of gelatinase A was 10-fold slower than for wild type enzyme and kinetic assays revealed a reduction in proteinase activity to 1%> of the activity of wild type enzyme (Crabbe et al., 1994). Interestingly a Glu to Asp active site mutant of neutral endopeptidase expressed on the surface of COS cells had 400-fold lower proteinase activity than wild type enzyme (Devault et al., 1988). To further characterise the active site mutant of GP63, kinetic assays with a chromogenic substrate could be performed. GP63, like other zinc metalloproteinases (Corbeil et al., 1993; Crabbe et al., 1994), was secreted in a latent form that retained the pro-region. The pro-region appeared to be required for secretion since the expression of a "mature" form of GP63 that lacked the pro-region was not secreted but accumulated intracellularly (Fig. 32). The pro-region has been shown to be required for secretion of a number of proteinases in mammalian cells, including gelatinase A expressed in HeLa cells and a mouse myeloma cell line (Fridman et al., 1992; Crabbe et al., 1994) and renin expressed in CHO cells (Mathews et al., 1996). Lack of secretion of matGP63WT(S) may be due to misfolding of the protein in the absence of the pro-region and resulting retention of the protein in the ER. The pro-regions of a number of proteins have been shown to facilitate correct folding 119 Chapter 6 - Discussion (Silen and Agard 1989; Gray et al., 1990; Suter et al., 1991; Inouye, 1991). Expression of the pro-region in trans was sufficient for proteins such as TGF-pi and activin A, to be secreted from a human cell line (Gray and Mason, 1990). Alternatively, lack of secretion may be due to deleterious effects on the cells caused by expression of an active form of this broad spectrum proteinase, similar to the effect observed when expressing wild type GP63 on the cell surface of CHO cells (Chapter 4). FAB-MS analysis of N-linked glycan structures revealed that modification of a membrane expressed protein for secretion had clear effects on the profile of glycan structures. The sialylation of membrane expressed GP63E265D(M) was greatly reduced compared to secreted GP63E265D(S). This may be due to reduced activity of sialyltransferase or increased sialidase activity on the membrane bound GP63E265D(M). Reduced activity of sialyltransferase at specific glycosylation sites due to steric hindrance has previously been reported for IFN-y (Gu et al., 1997) and for erythropoeitin (Sasaki et al., 1988). Sialidase activity in culture supernatants of CHO cells has also been well documented (Gramer and Goochee, 1993; Gramer et al., 1995) and attributed to sialidase released by cell lysis during extended cultures (Munzert et al., 1996). In this study, membrane expressed GP63E265D(M) was harvested within the exponential growth phase when cells were >90% viable. Also, while sialylation of membrane expressed GP63 was reduced, secreted GP63E265D(S) isolated from the same culture phase contained predominantly fully sialylated glycans. Sialidases have been identified on the plasma membrane and in lysosomal compartments of mammalian cells (Miyagi et al., 1993) and turnover of sialic acid residues on membrane expressed proteins has been reported (Varki and Freeze 1994). Reduced sialylation of membrane expressed GP63E265D(M) may therefore be a result of localized sialidase activity associated with membrane turnover. This lack or reduction of sialylation could 120 Chapter 6 - Discussion have profound effects on the on the in vivo clearance rates of proteins destined for therapeutic use (Ashwell and Hartford, 1982). Neuraminidase treatment of membrane expressed and secreted GP63E265D revealed differences in the fucosylation pattern of glycans. In GP63E265D(M), the smaller biantennary structures without poly-N-acetyllactosamine repeats had the highest degree of fucosylation. Only a small proportion of higher structures were fucosylated. In GP63E265D(S) the degree of fucosylation of biantennary structures was reduced while fucosylated and non-fucosylated triantennary and tetraantennary structures were present in equal proportions. Fucosidase activity has been detected in CHO culture supernatants (Gramer et al., 1994) however cleavage of fucose from intact glycoproteins was not demonstrated (Gramer et al., 1995). Thus, differences in fucosylation were more likely due to altered accessibility to fucosyltransferase between membrane bound and soluble GP63E265D. Site specific differences in fucosylation due to local protein conformation and steric hindrance of fucosyltransferase has been reported (James et al., 1995). GP63E265D(M) was found to contain poly-N-acetyllactosamine repeats not observed in GP63E265D(S). Poly-N-acetyllactosamine repeats have been reported previously in glycans from CHO cells (Li et al., 1980; Sasaki et al., 1988; Takeuchi et al., 1988; Orita et al., 1994). A number of secreted proteins that contain one or two repeats have been reported (Takeuchi et al., 1988; Orita et al., 1994; Dell et al., 1995) while greater numbers of repeats (lactosaminoglycan structures) are generally associated with membrane proteins (Fukuda et al., 1985; Taguchi et al., 1994). Interestingly, when a secreted protein, human chorionic gonadotrophin-cc chain (hCG-a), was expressed as a transmembrane protein in COS cells it contained polylactosaminoglycans not detected when the protein was secreted (Fukuda et al., 1988). 121 Chapter 6 - Discussion Major differences in glycosylation were not observed between secreted GP63E265D(S) from mid and late-log phase cultures. This result was consistent with other reports in which changes in glycosylation patterns were observed only after extended time in culture when cells were no longer in exponential growth (Robinson et al., 1994; Hooker et al., 1995). The results that harvesting the cells in late-log phase increased the yield of recombinant protein 5-fold (data not shown) without affecting the product consistency with respect to glycosylation (Table 3 & 4) has important implications for production. The high degree of consistency of these independent samples, even taken from different culture times within the log phase, adds increased significance to the differences observed between membrane and secreted samples. A number of factors may account for the structural differences between glycans isolated from membrane expressed and secreted GP63E265D. These include (i) differences in residence times within the secretion pathway of membrane bound and soluble proteins, (ii) altered protein conformation due to GPI-linked membrane attachment and (iii) altered accessibility of soluble and membrane bound proteins to membrane bound glycosyltransferases and/or glycosidases. Similar factors were postulated for differences observed in glycosylation of transmembrane and secreted hCG-cc chain (Fukuda et al., 1988). To address the first point pulse chase experiments were performed. The results showed that the dynamics of the secretion pathway of membrane bound and soluble GP63E265D were very similar with no major differences in residence times. Both proteins acquired resistance to Endo H between 40-60 min and were detected extracellularly by 60 min. These secretion times are consistent with those reported for other recombinant proteins expressed in mammalian cells (Howell et al., 1994; Chung et al., 1995). It would seem therefore that differences in glycosylation of membrane bound and soluble protein 122 Chapter 6 - Discussion may arise due to changes in protein conformation and accessibility to specific glycosyltransferases and/or glycosidases. This study demonstrated that modifying a protein for secretion altered the glycosylation profile of the protein. Previously the importance of the careful selection of expression system due to cell type-specific glycosylation had been recognized. The present findings show that within a given host cell whether a protein is expressed anchored to the membrane or secreted can also have a profound effect on the protein glycosylation. 123 Chapter 7 - General Discussion Chapter 7 General Discussion GP63 was successfully expressed in CHO cells both as a GPI-anchored protein and as a secreted protein. In contrast to the expression of GPI-anchored proteins from other protozoan species (Moran and Caras, 1994), the Leishmania GPI signal sequence was recognized and cleaved efficiently in mammalian cells as indicated by the lack of intracellular accumulation of GP63E265D(M) determined by pulse chase experiments. In contrast to GP63WT(M), when the active site mutant GP63E265D(M) was expressed on the cell surface the pro-region was retained. Proteinase activity of both membrane expressed and secreted GP63E265D was not detectable by zymogram analysis. However using a soluble assay, and after forcing the activation of secreted GP63E265D(S) by incubation with HgCl 2 , some proteinase activity was observed for the mutant. To accurately quantitate the reduction in proteinase activity of GP63E265D compared to GP63WT a suitable chromogenic substrate would need to be identified and kinetic enzyme assays performed. Expression of recombinant GP63 on the surface of CHO cells enabled a quantitative approach to evaluate the stability of expression of the active proteinase and the active site mutant during long-term culture. Enhanced stability was observed for the expression of GP63E265D(M), even in the absence of selective pressure. The differential stability of GP63WT(M) and GP63E265D(M) suggested a correlation between proteinase activity and instability of GP63 expression, indicating that expression of the active broad spectrum proteinase was deleterious to the cell. Comparison of producing and nonproducing populations demonstrated that the molecular mechanisms involved in loss of GP63WT(M) expression and 124 Chapter 7 - General Discussion the loss of GP63E265D(M) in the absence of M T X were different. This study showed that instability problems in GP63 production could be avoided by the expression of GP63E265D and by limiting the length of production runs to approximately 40 population doublings when cells are grown out of M T X . These results are directly applicable to the production of other recombinant proteins and in particular to the production of other broad substrate proteinases such as the matrix metalloproteinases. Continuous harvesting of GP63E265D(M) from the cell surface by PI-PLC was used to compare the expression levels of membrane expressed and secreted protein. The cell specific production of membrane expressed GP63E265D was approximately 25-fold lower than secreted production. These differences could not be attributed to a post-translational limitation of membrane expression. It would be interesting to compare membrane and secreted production of a more highly expressed recombinant protein to confirm the results obtained in this study and to determine whether post-translational limitations are reached at higher expression levels. Analysis of the post-translational modifications of membrane expressed and secreted protein revealed significant differences. Differential processing of the pro-region of GP63 was observed. A comparison of the processing of GP63WT(M) and GP63E265D(M) indicated that GP63WT(M) underwent autocatalytic processing when expressed on the cell surface. This suggests that expression as a membrane bound protein may have altered the conformation compared to the soluble protein, thus rendering the enzyme more susceptible to self-processing. As reported for many other proteinases that are secreted as precursor proteins, the presence of the pro-region of GP63WT(S) was necessary for secretion. 125 Chapter 7 - General Discussion The glycosylation patterns of membrane bound and soluble GP63E265D were clearly different. Of particular importance was the reduction in sialylation of membrane bound protein. This would be a significant disadvantage in the production of recombinant proteins for therapeutic use, such as receptor molecules, as rapid clearing of asialylated proteins is known to occur in vivo (Ashwell and Hartford, 1982). Differences were also observed in the patterns of core fucosylation of glycans from secreted and membrane expressed protein and poly-N-acetyllactosamine repeats were detected only in membrane expressed GP63E265D(M). These differences could not be attributed to major differences in the dynamics of the secretion pathway of membrane bound and soluble protein. These results suggest that the accessibility to glycosyltransferases may have been altered in the membrane bound and soluble proteins. Reduced sialylation of glycans from membrane bound protein may alternatively occur as a result of increased sialidase activity at the cell surface. To extend these studies, it would be interesting to analyse the glycans present at individual sites to determine whether site specific steric hindrance is occurring. Radioactive precursors of sialic acid such as N-acetylmannosamine (Gu and Wang, 1997) could be used to determine whether sialic acids were present in glycan structures on the cell surface and if subsequent removal by sialidases was occurring. Studying the glycan structure of GP63 expressed as a transmembrane protein would determine whether differences in glycosylation are specifically associated with GPI expression or whether they are associated with membrane expression in general. These studies have shown that for GP63 production in CHO cells, secretion of soluble protein was more effective than cell surface expression. Clones secreting GP63 were consistently more stable and had higher productivity. More generally these studies have shown that expression of a membrane protein for secreted production can cause dramatic changes in the 126 Chapter 7 - General Discussion post-translational processing even if the same host cell line is used. Modifications such as the processing and activation of a membrane expressed proteinase, were shown to have profound effects on the stability of protein expression. While other differences in post-translational modifications such as the reduced sialylation of membrane expressed protein, would have serious consequences on the efficacy of recombinant therapeutic proteins. These results therefore represent important factors to be considered when expressing recombinant membrane proteins. 127 Nomenclature Nomenclature A M U Atomic Mass Unit BCIP 5-bromo-4-chloro-3-indoylphosphate-p-toluidine salt BHK baby hamster kidney BSA bovine serum albumin cDNA complimentary deoxyribonucleic acid CHO Chinese hamster ovary DAF decay accelerating factor DEPC diethyl pyrocarbonate DHFR dihydrofolate reductase D M E M Dulbecco's modified essential medium DMSO dimethylsulphoxide DNA deoxyribonucleic acid DNAse I deoxyribonuclease I dNTP deoxynucleotidetriphosphate DTT dithiothreitol EDTA ethyenediaminetetraacetic acid ELISA enzyme linked immunosorbent assay EndoH endoglycosidase H EPO erythropoeitin ER endoplasmic reticulum FAB-MS fast atom bombardment-mass spectrometry 128 Nomenclature Fuc fucose GAM-FITC goat anti-mouse IgG fluorescein isothiocyanate conjugated antibody Glc glucose GlcN glucosamine GlcNAc N-acetylglucosamine GPI glycosylphosphatidylinositol H C M V human cytomegalovirus Hex hexose HexNAc N-acetylhexosamine IFN-B interferon-B IFN-y interferon-y IL-2 interleukin-2 Kb kilobase kDa kilodalton mAb monoclonal antibody Man mannose MOPS 3-(N-morpholino)-propanesulphonic acid mRNA messenger ribonucleic acid MS mass spectrometry M T X methotrexate NBT nitroblue tetrazolium chloride NeuAc N-acetylneuraminic acid or sialic acid NPD number of population doublings 129 Nomenclature PBS phosphate buffered saline PCR polymerase chain reaction PD population doublings PI phosphatidylinositol PI-PLC phosphatidylinositol-phospholipase C PI-PLD phosphatidylinositol-phospholipase D PMSF phenylmethylsulphonyl fluoride PNGase F peptide N-glycosidase F P-P-Doli dolichol pyrophosphate RNA ribonucleic acid RT room temperature RT-PCR reverse transcription- polymerase chain reaction SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis TBS Tris buffered saline TGF-p 1 transforming growth factor-p 1 t-PA tissue plasminogen activator V S G variant surface glycoproteins 130 References References Andersen, D. 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