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Construction, expression and characterization of CD45-immunoglobulin fusion proteins Awrey, Shannon June 1996

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C O N S T R U C T I O N , EXPRESSION A N D C H A R A C T E R I Z A T I O N OF C D 4 5 - I M M U N O G L O B U L I N F U S I O N PROTEINS by S H A N N O N J U N E A W R E Y B.Sc, The University of British Columbia, 1993 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF M A S T E R OF SCIENCE i n T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Microbiology and Immunology We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A March 1996 © Shannon June Awrey, 1996 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 rtlttoKlOLO^ ^-^m^VtQOL O^j The University of British Columbia Vancouver, Canada Date Z-l/Cfio : DE-6 (2/88) A B S T R A C T The aim of this work was to create, express, and characterize fusion proteins consisting of different alternatively spliced exons of murine CD45, a protein tyrosine phosphatase, l inked to the heavy chain constant regions of murine immunoglobul in G. CD45-immunoglobul in fusion proteins were secreted as dimers in a relatively pure form using serum free media at an approximate yield of 1.5-4.5 u g / m l , depending on the isoform of CD45 and the cell l ine in which it was expressed. Fusion proteins secreted by Cos 7 cells had a higher apparent molecular weight by approximately 5-10 kDa than those expressed by X63-Ag8.653 or T28 cells. The interaction of CD45 wi th putative ligands may be mediated by specific carbohydrate residues on CD45, therefore, the carbohydrate residues expressed on CD45-immunoglobul in fusion proteins were characterized. O-glycosidase digestion and lectin analysis revealed that all fusion proteins were extensively O-glycosylated in a cell-specific manner. Neuraminidase digestion and analysis of subsequent Peanut agglutinin reactivity suggested that fusion proteins secreted by Cos 7 cells expressed more sialic acid when compared to that secreted by X63-Ag8.653 or T28 cells. Thrombin cleavage and PNGase F digestion revealed that the immunoglobul in portion was 34 kDa and the only site of N-l inked carbohydrate addition. A l l fusion proteins reacted with anti-CD45 exon-specific antibodies as predicted with the exception of RA3 6B2, a B220-specific antibody that reacted with C D 4 5 R A B C -Ig expressed by Cos 7 cells but not with that expressed by X63-Ag8.653 or T28 cells. RA3 6B2 reacted with fusion proteins containing exons A , B, and C inclusive in addition to fusion proteins containing only exon A . R A 3 6B2 binding was not affected by neuraminidase treatment, but d id correlate to the binding of wheat germ agglutinin. Once expressed and purif ied, CD45-immunoglobulin fusion proteins can be used as diagnostic tools in immunoadherence and adhesion assays in an attempt to further our understanding of T lymphocyte signalling via the identification an isoform-specific ligand(s) for murine CD45. ii T A B L E OF C O N T E N T S Page A B S T R A C T ii T A B L E O F C O N T E N T S i i i LIST OF TABLES vi LIST OF FIGURES ; v i i LIST OF ABBREVIAT IONS ix DEFINIT ION OF FUSION PROTEIN N O M E N C L A T U R E xi A C K N O W L E D G E M E N T xii D E D I C A T I O N xii i I N T R O D U C T I O N 1 CD45 1 Differential Expression of CD45 Isoforms 3 The Extracellular Domain 3 Role of CD45 in the Immune System 5 Potential Ligands 7 Summary of Goals 8 M A T E R I A L S A N D M E T H O D S 11 RESULTS 24 1.0 Generation of murine CD45Tmmunoglobul in Isoform-specific Fusion Constructs 24 1.0.1 Reconstruction of the Oncostatin M signal sequence by Polymerase Chain Reaction 27 i i i Page 1.0.2 Creation of the Modi f ied murine IgG2a Vector with the Oncostatin M signal sequence 28 1.0.3 Creation of Isoform-specific murine CD45 inserts by Polymerase Chain Reaction 30 2.0 Expression of murine CD45-Immunoglobul in Isoform-specific Constructs in Cos 7 cells 36 2.0.1 Transient expression 36 2.0.2 Stable expression 40 3.0 Expression of murine CD45-Immunoglobul in Isoform-specific Constructs in Myelo id and Lymphoid cells 47 3.0.1 Stable Expression in the X63-Ag8.653 murine myeloma 47 3.0.2 Transient Expression in T28 murine T lymphoma 49 4.0 Characterization of Expressed murine CD45Tmmunoglobul in Fusion Proteins 52 4.0.1 Reactivity with anti-CD45 Exon-specific Antibodies 52 4.0.2 Analysis of N-Linked Glycosylation 57 4.0.3 Analysis of O-Linked Glycosylation 61 4.0.4 Characterization of Carbohydrate residues by Lectin Binding 63 4.0.5 Determination of Peanut Lectin Reactivity in the Absence of Sialic Ac id 67 4.0.6 Apparent Molecular Weight in Non-reducing S D S - P A G E Conditions 69 iv Page DISCUSSION 71 Creation of murine CD45-Immunoglobul in Fusion Constructs 71 Expression of murine CD45-Immunoglobul in Fusion Proteins 73 Characterization of Expressed CD45-Immunoglobul in Fusion Proteins 77 R E F E R E N C E S 86 V LIST OF T A B L E S Page Table I Description of Exon-specific Antibodies 17 Table II Results of Polymerase Chain Reaction of murine CD45 isoform-specific Inserts 31 Table III Apparent Molecular Weight of CD45-Immunoglobul in Fusion Proteins Expressed in Three Cel l Lines 39 Table IV Results of Stable Transfection of CD45-Immunoglobul in Isoform Fusion Constructs into Three Cel l Lines 41 Table V Estimated Yield of CD45-Immunoglobul in Fusion Proteins Expressed in Two Cel l Lines 45 Table VI Reactivity of CD45-Immunoglobul in Fusion Proteins wi th Ant i -CD45 Exon-Specific Antibodies 54 Table VII Reactivity of CD45-Immunoglobul in Fusion Proteins wi th Various Lectins 66 vi LIST OF FIGURES Page Figure 1 Schematic Diagram of Mur ine CD45 Isoforms 2 Figure 2 Schematic Representation of C D 4 5 R A B C T m m u n o g l o b u l i n Fusion Protein 26 Figure 3 Creation of Oncostatin M / M u r i n e IgG2 a Vector 29 Figure 4 CD45 Isoform-specific P C R products 33 Figure 5 Subcloning of murine CD45 Isoform-specific Inserts into the murine IgG Vector '. 34 Figure 6 CD45- Immunoglobul in Fusion Constructs in Mammal ian Expression Vector pBCMGSneo 35 Figure 7 Transient Expression of murine CD45- Immunoglobul in Fusion Proteins in Cos 7 cells 38 Figure 8 Western Blot of CD45- Immunoglobul in Fusion Proteins Expressed in Cos 7 cells 42 Figure 9 Relative Yield and Puri ty of CD45- Immunoglobul in Fusion Proteins Expressed in Cos 7 cells 43 Figure 10 F A C S C A N of Cel l Lines Transfected with CD45- Immunoglobul in Isoform Constructs..... 48 Figure 11 Western Blots of CD45-Immunoglobul in Fusion Proteins Expressed in X63-Ag8.653 and T28 cells 50 Figure 12 Reactivity of CD45-Immunoglobul in Fusion Proteins Expressed in Cos 7 cells with Ant i-CD45 Isoform-specific Antibodies 55 Figure 13 Reactivity of CD45-Immunoglobul in Fusion Proteins Expressed in vi i Page X63-Ag8.653 cells with Ant i -CD45 Isoform-specific Antibodies 56 Figure 14 Reactivity of CD45-Immunoglobul in Fusion Proteins Expressed in T28 cells with Ant i -CD45 Isoform-specific Antibodies 58 Figure 15 PNGase F Digestion of CD45-Immunoglobul in Fusion Proteins 59 Figure 16 Thrombin Cleavage of CD45Tmmunoglobul in Fusion Proteins 60 Figure 17 O-Glycosidase Treatment of CD45-Immunoglobul in Fusion Proteins..62 Figure 18 Reactivity of CD45-Immunoglobul in Fusion Proteins wi th various Lectins 64 Figure 19 Peanut Agglut in in and RA3 6B2 Reactivity of Neuraminidase Treated CD45-Immunoglobulin Fusion Proteins 68 Figure 20 S D S - P A G E Analysis of CD45-Immunoglobul in Fusion Proteins in Reducing and Non-reducing Condit ions 70 vii i LIST OF A B B R E V I A T I O N S A T P Adenosine triphosphate B S A Bovine serum albumin bp Base pairs D M E M Dulbecco's Modi f ied Eagle Med ium d N T P Deoxynucleotides DTT Di th iothrei to l E D T A Ethylenediaminetetraacetic acid FCS Fetal calf serum gal Galactose g a l N A c N-acetylgalactosamine glc Glucose g l cNAc N-acetylglucosamine H B S Hepes buffered saline H R P Horseradish peroxidase H S Horse Serum h u H u m a n kb Kilobase pairs L A Lur ia + ampici l l in LB Lur ia Broth m a n Mannose M Mo la r Microgram Hi Micro l i t re u.M Mic romola r j imo l M ic romo le ix m g M i l l i g r a m m l M i l l i l i t re m M M i l l i m o l a r n e u N A c N-acetylneuraminic acid n g Nanogram n m Nanomet re OD Optical Density PBS Phosphate buffered saline P C R Polymerase Chain Reaction p m o l Picomole TBS Tris buffered saline term Termina l Tris Tris (hydroxymethyl) amino methane F A C S Fluorescence activated cell sorter FITC Fluorescein isothiocyanate PTPase Protein tyrosine phosphatase P V D F Polyvinyl idene dif luoride SH2 Src homology 2 T C R T cell receptor S D S - P A G E Sodium dodecyl sulfate-polyacrylamide gel electrophoresis X DEFINIT ION OF F U S I O N P R O T E I N N O M E N C L A T U R E C H 2 CH3 M u / m u M u l g G CD45 alternatively spliced exons muCD45R0:MuIgG m u C D 4 5 R A : M u ! g G muCD45RB:MuIgG m u C D 4 5 R C : M u ! g G muCD45RBC:MuIgG m u C D 4 5 R A B C : M u I g G Onco M Constant region heavy chain 2 Constant region heavy chain 3 M u r i n e Mur ine immunoglobul in G A , B, C =4,5,6 Fusion protein consisting of murine CD45 exons 3,7, and 8 l inked to the hinge, C H 2 and C H 3 regions of murine IgG2a heavy chain Fusion protein consisting of murine CD45 exons 3,4,7, and 8 l inked to the hinge, C H 2 and C H 3 regions of murine IgG2a heavy chain Fusion protein consisting of murine CD45 exons 3,5,7, and 8 l inked to the hinge, C H 2 and C H 3 regions of murine IgG2a heavy chain Fusion protein consisting of murine CD45 exons 3,6,7, and 8 l inked to the hinge, C H 2 and C H 3 regions of murine IgG2a heavy chain Fusion protein consisting of murine CD45 exons 3,5,6,7, and 8 l inked to the hinge, C H 2 and C H 3 regions of murine IgG2a heavy chain Fusion protein consisting of murine CD45 exons 3,4,5,6,7, and 8 l inked to the hinge, C H 2 and C H 3 regions of murine IgG2a heavy chain Oncostatin M XI A C K N O W L E D G M E N T I wou ld l ike to acknowledge the fo l lowing people for their contribution towards the attainment of my goal: Dr. Pauline Johnson for her scientific guidance and the opportunity to do my Master's degree in her laboratory, Dr. Julie Deans for her ideas and inspiration for without her this project wou ld not have been possible, Mojgan Jabali for technical laboratory support, and Arp i ta Mai t i and Dav id N g for stimulating scientific discussions and helping me wi th everyday lab problems. x i i D E D I C A T I O N I wou ld l ike to dedicate this body of work to my parents, Gary and June Awrey , whose continual love, guidance, and understanding have helped me to become the person I am today. A n d to Torsten, whose love and encouragement forced me to hold my head up and keep going, even when the tasks ahead seem insurmountable. x i i i I N T R O D U C T I O N CD45 CD45 (T200, B220, Ly-5, L -CA) is a protein tyrosine phosphatase (PTPase) that is expressed on all nucleated cells of hematopoietic l ineage, reviewed in [1, 2]. Members of the leukocyte common antigen (L-CA) family are abundant as they can account for up to 10% of the protein on the lymphocyte cell surface [3]. PTPases such as CD45 are involved in the reversible process of tyrosine dephosphorylation, a key regulatory mechanism for control l ing the growth and d iv is ion of eucaryotic cells. CD45 is a transmembrane protein containing a large, heavi ly glycosylated, variable, amino-terminal external domain, a single transmembrane region, and a large carboxy-terminal cytoplasmic domain (Figure 1). The h ighly conserved cytoplasmic domain consists of two tandem PTPase domains of approximately 240 amino acids separated by a short spacer region. Research has demonstrated that the cytoplasmic domain of CD45 possesses intrinsic enzymatic activity [4] independent of the transmembrane and extracellular domains [5, 6]. Mutat ion of a conserved cysteine residue at posit ion 817 of PTPase domain I resulted in a total loss of enzymatic activity whereas mutation of the comparable cysteine in domain II d id not signif icantly affect enzymatic activity, suggesting that PTPase domain II is inactive [6]. However, using an in vitro rabbit reticulocyte translation system, it was found that deletion of PTPase domain II resulted in a total loss of enzymatic activity, thus its presence is required in order for domain I to be active [7]. A l though the cytoplasmic function of CD45 has been studied extensively, the functional role of the external domain and the alternatively spliced exons remains largely unknown. The external domain of CD45 can undergo alternative spl icing resulting in the creation of different isoforms that are expressed on various cell types and at different stages of immune cell development. Mul t ip le isoforms of CD45 have been shown to bind 1 CD45 Isoforms N A B C TTT M P II }>>}))} SSSSSSS sssssss / / / / / / / / / / / • / / / / / / / / / / sssssss sssssss SSSSSSS / / / / / / / / / / / / / / SSSSSSS sssssss SSSSSSS SSSSSSS sssssss \ \ \ \ \ \ \ w w \ w \ \ N S \ S V w w w \ w w w v w w w \ w w w s \ \ \ s \ \ \ w w w v B c sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss • I U U U w w w - . w w w \ WWW". V. w w w w w w \ www sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss w w w \ \ \ \ \ \ \ \ \ \ \ \ \ s \ w w w \ \ \ \ \ \ \ \ w w w \ W W W \ w w w \ w w w \ B sa^h- IT sssssss, sssssss, ssssss, sssssss, sssssss, sssssss. sssssss, sssssss, sssssss, sssssss, sssssss'. sssssss. W W w w w ; \ w w v w w w w w w w w w , W WW' -WWW' . w w w ; ' w w w w w w w w w \ w w v c R A B C RBC R A RB sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss sssssss | \ w w w • w w w s w \ w w w w w s w w w s , w w w s • s s s s s s s • s s s s s s s l W \ W W s s s s s s s s s s s s s s s s s s s s s w w w m Ml Isssssss, \sssssss, \sssssss \sssssss, sssssss, sssssss, sssssss, sssssss, sssssss, sssssss. \SSSSSSS, sssssss, sssssss, sssssss, sssssss, sssssss. sssssss. w w w -w w w -w w w - -t". w w w * -s s s s s s - -w w w -s s s s s s * -w w w -w w w -RC RO Figure 1: Schematic Diagram of Mur ine CD45 Isoforms. Six different isoforms of murine CD45 containing different alternatively spliced exons are shown. N , amino terminus; A ,B ,C , alternatively spliced exons; M P , membrane proximal region; I, PTPase domain one; S, spacer region; II, PTPase domain two; C , carboxy terminus. 2 to a molecule expressed on B cells, CD22, via specific carbohydrate residues on CD45 [8, 9], but as yet, an isoform-specific l igand has not been identified. Differential Expression of Isoforms Different isoforms of CD45, ranging in molecular weight from 180 to 220 kDa, exist due to alternative splicing of at least three variable exons denoted A , B, and C (4, 5, and 6) located in the extracellular domain. The different isoforms vary not on ly in the exons expressed at the protein level but also in carbohydrate composition and antigenicity [3]. In addit ion, the expression of different isoforms varies wi th cell type, developmental stage and antigenic exposure. For example, B cells express predominantly the highest M r 220,000 isoform, immature T cells or thymocytes express predominantly the lowest M r 180,000 isoform and mature T cells can express mul t ip le isoforms. In general, the maturat ion of T lymphocytes involves the acquisi t ion of higher molecular weight forms of CD45 , whereas antigen activation of T cells results in a down regulation to the lowest M r 180,000 isoform. The Extracellular Domain The extracellular domain of CD45 can be div ided into two structural domains: the variable region and the cysteine r ich domain. Compar ison of human and murine CD45 c D N A sequences indicated that although a high degree of sequence identity exists between the cytoplasmic, transmembrane, and signal sequences, the external domain identity between the two species is only 39% [10]. It may be possible that just the isoform-specific regions of the extracellular domain are conserved, thus provid ing conserved l igand binding epitope(s). A t least eight different isoforms of CD45 can exist due to alternative splicing of three exons of approximately 50 amino 3 acids each (A, B, and C or 4, 5, and 6) located in the N-terminal region of the gene. The largest isoform of CD45, so called CD45RABC due to the presence of exons A , B, and C , has an extracellular domain of 542 amino acids. The smallest isoform of CD45, lacking exons A , B, and C, is denoted CD45R0, and has an extracellular domain of 404 amino acids. Of the 33 exons comprising the entire protein, exon 3 and exons 7-15 encode extracellular coding sequences common to all isoforms of CD45 [11]. Differential usage of the three alternatively spliced exons results not only in proteins of different length but also in isoforms wi th different carbohydrate content and antigenicity. The variable, N-terminal region is r ich in serine and threonine residues which are potential sites for extensive O- l inked glycosylation [12]. In addit ion, the variable region is highly negatively charged due to the addition of sialic acid residues [13, 14]. Furthermore, electron microscopy analysis by low-angle shadowing shows the external domain as an extended rod [15]. The cysteine-rich domain is approximately 360 amino acids long and is common to all isoforms of CD45. This region is thought to contain fibronectin type III modules similar to those found in tapeworm proteins and the fruit fly receptor protein tyrosine phosphatase DPTP[6, 16]. This region contains 15 potential N -l inked g lycosylat ion sites for the addi t ion of carbohydrate residues dur ing processing. Studies by Pul ido and Sanchez-Madrid indicated that inhibit ion of N -l inked glycosylat ion by treatment wi th tunicamycin resulted in decreased cell surface expression and decreased phosphatase act iv i ty of CD45 in K-562 erythroleukemic cells [17]. Hence, it appears that intact glycosylation is needed for correct transport, stability or expression of CD45 at the cell surface. Glycosy lat ion studies of CD45 expressed in K-562 erythroleukemic cells revealed the presence of complex, highly sialylated, O- and N- l inked carbohydrate chains [13]. A n independent structural study of the sugar chains on human CD45 on T cells was done by Sato et al . , reveal ing that CD45 contains complex type carbohydrate chains containing poly-N-acetyl- lactosamine and exclusively a-2,6 4 l inked sialic acid resides [18]. The fact that only certain isoforms of CD45 co-cap with certain T cell surface molecules suggests that isoforms-specif ic interactions are possible [19]. As different isoforms of CD45 express different alternatively spliced exons with distinct glycosylation sites, each isoform of CD45 has the potential for a differential glycosylation pattern. As alternate isoforms of CD45 are expressed on the surface of different cell types and at distinct stages of immune cell development, it is reasonable to hypothesize that the presence or absence of a certain isoform, hence the presence or absence of certain carbohydrate residues, at a particular time dur ing development wou ld have specific functional outcomes. Role of CD45 in the Immune System The role and funct ion of CD45 in immune cell interactions has been investigated by many groups, in particular by studying CD45 deficient cell l ines, reviewed in [2, 20, 21]. Evidence indicated that CD45 negative T cell clones reactive against pigeon cytochrome c proliferated in response to interleukin-2 but failed to proliferate in response to antigen. F low cytometric analysis d id not detect the absence of other molecules required for T cell activation and antigen responsiveness was restored in revertant cells that re-expressed CD45 [22]. This result was confirmed by another CD45 negative mutant of L3 CD8+ cytotoxic T cells, which was also unable to respond to antigenic st imulat ion whi le the expression of CD45 rescued the T cell response [23]. In addit ion, CD45 has been implicated in coupling of the T cell receptor (TCR) to the phosphatidylinositol second messenger pathway. Tyrosine phosphorylation and generation of soluble inositol phosphates was absent in spontaneous CD45 negative variants of a human T leukemic cell l ine, H P B - A L L . However , response to ant i-CD3 stimulation, as measured by intracellular calcium increase and generation of inositol phosphates, was restored upon transfection of murine CD45 , suggesting that expression of CD45 is required for efficient T C R 5 signall ing and linkage to downstream phosphorylat ion events [24]. Moreover, in CD45 negative mutants of Jurkat T cells, expression of CD45 was required for tyrosine phosphorylation induced by anti-TCR antibodies [25]. Furthermore, there is evidence that CD45 is required in an analogous fashion for signall ing via the B cell receptor [26,27]. A chimeric molecule consisting of the external and transmembrane domains of M H C class I and the cytoplasmic domain of CD45 expressed in CD45 deficient Jurkat cells restored the T C R induced tyrosine phosphorylat ion of phospholipase C y l [28]. S imi lar ly , the expression of a chimeric molecule consist ing of the extracellular and transmembrane domains of the epidermal growth factor receptor (EGFR) l inked to the CD45 cytoplasmic domain, rescued T cell responses to antigenic stimulation in CD45 deficient H P B - A L L cells [29]. Hence, both of these experiments suggest that the extracellular and transmembrane regions of CD45 are not required for T C R mediated signal l ing. However , these experiments do not rule out the possibil ity of regulation of CD45 function by l igand binding to the external domain of CD45. There is some evidence that specific isoforms of CD45 have differential effects on T cell signal l ing. In an experiment in which C D 4 5 R A B C and CD45R0 were expressed as transgenes in murine T cells regulated by the thymocyte-specific Ick promoter, the largest isoform, C D 4 5 R A B C , was observed to increase thymic CD4+ CD8- T cell proliferation in response to anti-TCR antibodies, resulting in an increase in phosphotyrosine levels and intracellular calcium concentration [30]. In the same experiment, expression of the nu l l isoform, CD45R0, resulted in a decrease in phosphotyrosine levels. This data is in contrast to other observations in which BW5147 C D 4 / T C R transfectants, expressing different isoforms of CD45, responded equally to stimulation via ant i -TCR antibodies whi le st imulat ion v ia addit ion of antigen presenting cells (APC) and conalbumin peptide resulted in the lower molecular weight isoforms having the highest level of T cell response [31]. Despite 6 the seemingly contradictory data, a unifying theme exists that different isoforms of CD45 can have differential effects on T cell signall ing events. Potential Ligands Binding of a l igand specific for an isoform of CD45 may result in a direct alteration in cellular activity or activation state. A t this time, a CD45 isoform-specific l igand has not been clearly identified, although two molecules, CD22 [32] and galectin-1 [33], have been shown to be invo lved in the extracellular domain interactions of CD45. CD22 is an adhesion molecule expressed exclusively on B cells that has been shown to bind to carbohydrate residues on CD45 expressed by T cells. A CD22-immunoglobul in fusion protein (CD22Rg) was reported to b ind to many different ligands on cells of the T, B, and myeloid lineages. One of the T cell l igands was a s ia ly lated g lycoprote in w i th enzymat ic act iv i ty ident i f ied as CD45R0 [32]. Subsequently, CD22 was shown to interact with multiple isoforms of CD45 [34] and that the interaction was dependent on the presence of N- l inked carbohydrates containing oc-2,6 l inked sialic acids on CD45 [8, 9]. Recent studies have shown that the engagement of CD45 by soluble CD22Rg can modulate early T cell signall ing events such as tyrosine phosphorylat ion of phospholipase C-y (PLC-y) resulting in the product ion of inositol tr iphosphate leading to an increase in intracellular calcium concentration [35]. Moreover, successive deletion of each of the seven extracellular immunoglobul in (Ig) domains of CD22 mapped the sialylated l igand-binding domain to Ig domains 1 and 2 of CD22 [36]. However, as CD22 binds to other molecules on cells of the hematopoietic lineage other than CD45, it is not defined as either a CD45-specific or an isoform-specific l igand. Another potential l igand for CD45 is a f3-galactoside-binding lectin molecule expressed on human thymic cells and stromal cells called galectin-1 [33]. Galectin-1 7 has been shown to b ind to core 2 O- l inked glycan structures on the surface of thymocytes and T lymphoblastoid cell lines. This interaction was inhibited not only by an antibody to galectin-1, but also by an antibody, 2B11, that recognizes a carbohydrate-dependent epitope of CD45, suggesting that carbohydrate residues on CD45 are mediat ing this interaction [37]. Interestingly, the degree of galectin-1 binding to thymocytes correlated with maturation stage wi th immature thymocytes b inding more galectin-1 than mature thymocytes. Whether the developmental ly regulated fashion of galectin-1 binding reflects the differential expression of CD45 isoforms dur ing T cell development remains to be elucidated. In addit ion, recent evidence indicates that although galectin-1 binds to both activated human T cells and resting T cells, apoptosis is induced only in CD45R0+, CD45RA- activated T cells and not in CD45RA+ resting T cells [38]. H P B - A L L cells expressing CD45 underwent apoptosis upon addit ion of galectin-1 whereas a CD45 negative variant of this cell l ine d id not, suggesting that CD45 is integral in this response. In addi t ion, inhib i t ion of N-g lycan processing by treatment wi th swainsonine as wel l as inhibit ion of O-glycan elongation by addit ion of benzyl -a-ga lNAc decreased binding of galectin-1 to T cells. Swainsonine treatment reduced galectin-1 mediated cell death whereas benzyl-oc-galNAc treatment increased apoptosis due to galectin-1 binding, suggesting that N- l inked glycans are involved in the apoptotic response whi le O- l inked glycans may have a regulatory function, perhaps acting to mask the effect of N-l inked glycan induced apoptosis. Summary of Goals The aim of this thesis was to further understand the role of the different isoforms of CD45 in immune cell function. As alternate isoforms of CD45 are expressed on the surface of different cell types and at distinct stages of immune cell development, it is reasonable to hypothesize that the presence or absence of a certain 8 isoform at a particular time dur ing development wou ld have specific functional outcomes. The identification of a l igand that could bind to specific isoforms of CD45 wou ld contribute greatly to our understanding of the role of CD45 in immune cell interactions. The creation of isoform-specific CD45:MuIgG fusion proteins and use as a diagnostic tool in immunoadherence and fluorescent b inding assays may prove instrumental in determining the identity of isoform-specific ligands for CD45. As the interaction of CD45 with a potential l igand may be mediated by unique carbohydrate residues located on the variable exons of CD45, it is important that CD45:MuIgG fusion proteins are correctly glycosylated. Therefore, the cell line used for expression of the fusion proteins is crit ical. The fusion proteins were first transiently expressed in Cos 7 cells, a non-hematopoietic cell line commonly used to express recombinant proteins. Once transient expression was observed and stable clones were obtained in Cos 7 cells, the focus of future experiments turned to expression of CD45:MuIgG fusion proteins in functionally relevant hematopoietic cell l ines, part icular ly those of the T and B cell l ineages. Stable clones were expressed in X63-Ag8.653 cells, a murine myeloma cell l ine frequently used as a fusion partner in the production of monoclonal antibodies. In addit ion, transient expression was observed in the T28 T lymphoma cell line. Once expressed, the CD45:MuIgG fusion proteins had to be ful ly characterized w i th respect to molecular weight, d imer izat ion in non-reduc ing S D S - P A G E conditions, and reactivity with anti-CD45 exon-specific antibodies, as wel l as to the carbohydrate content by reactivity wi th various sugar-specific lectins, PNGase F digestion, O-glycosidase treatment, thrombin cleavage and neuraminidase digestion. Fusion proteins expressed by different cell lines were analyzed in an attempt to determine if CD45:MuIgG fusion proteins were differentially glycosylated by each cell line. The amount and type of carbohydrate added was analyzed by treatment wi th enzymes that specifically remove N-l inked sugars or terminal sialic acids. It is important to ful ly characterize the expressed fusion proteins because if specific 9 binding is observed by the use of these fusion proteins in b inding assays then one can argue in favor of one of the fol lowing two options. If the binding observed is the same between differentially glycosylated CD45:MuIg fusion proteins then the importance of correct glycosylation becomes secondary to correct protein sequence and fo ld ing patterns. However , if b ind ing is different between fusion proteins expressed in different cell lines, thus having differential carbohydrate content, then correct carbohydrate addit ion becomes crucial in order for the interaction to occur wi th the putative l igand. Hence, the three goals of this work were as fol lows: 1) to create soluble, isoform-speci f ic , mur ine C D 4 5 : M u I g G fus ion proteins conta in ing different alternatively spliced exons, 2) to express these fusion proteins in lymphoid cell lines 3) to characterize the. expressed fusion proteins wi th respect to molecular weight, antigenicity, dimerization, and glycosylation and to compare and contrast isoforms of CD45 produced in different cell lines with respect to these characteristics. 10 M A T E R I A L A N D M E T H O D S PCR of Isoform-specific muCD45 inserts M u r i n e CD45 (muCD45) isoform-speci f ic inserts were created by the Polymerase Chain Reaction (PCR) using isoform-specific plasmid D N A as templates. A 5' primer corresponding to amino acid residues 1 to 6 of muCD45 exon 3 (UBC # 60: 5' G C G A G C A T G - > C A A A C A C C T A C A C C C A G T 3': Sph 1 site underl ined, where —> indicates the beginning of muCD45) and a 3' pr imer corresponding to amino acid residues 171-177 of murine exon 8 (UBC 63: 5' A C A A C G A A G C A A A C A < - G A T C T G GTT C C T C G T G G A T C C TCT G A T C A G G A G C C C 3": Bel 1 site underl ined, where <— indicates the 3' end of exon 8) were used to generate muCD45 PCR products. Primer U B C #63 was engineered to contain a thrombin cleavage site, 5' C T G GTT C C G C G T G G A T C C 3', which would be incorporated at the junction of CD45 and murine IgG (mulgG) to al low for removal of the IgG portion if desired. Once isoform-specific plasmid D N A had been l inearized by overnight digestion at 37 °C wi th 5 units of C la I (NEB: N e w England Biolabs, Mississauga, O N ) , P C R reactions were carried out using 1 ng of template, 10 ul of 10X Vent polymerase buffer (supplied by N e w England Biolabs), 2 ul of 10 m M d N T P (Pharmacia Biotech Inc., Piscataway, NJ), 50 pmol each primer and 2 Units of Vent D N A polymerase (New England Biolabs) in a final volume of 100 ul in an Ericomp Inc. Easy Cyc le r™ thermocycler. Samples were subjected to 30 cycles of 1 minute at 95 °C for denaturation, 1 minute at 55 °C for primer annealing, fol lowed by 1 minute at 72 °C for primer extension. P C R products were checked for correct size and purity on 2% agarose gels against 4 ul of 1 kb D N A ladder (Gibco BRL , Li fe Technologies, Burl ington, O N ) , digested wi th Sph 1 and Bel 1 (New England Biolabs), extracted from low melt agarose, and ethanol precipitated. Sph 1 does not cut at the end of D N A fragments with an acceptable efficiency so for each isoform, P C R products were 11 blunt end ligated using 400 Units of T4 D N A ligase (New England Biolabs) to create mult imers, fol lowed by Sph 1 and Bel 1 digestion to create the correct size insert wi th the required 5' and 3' overhangs to facilitate subcloning into the modif ied murine IgG plasmid vector. Creation of the Modified Oncostatin M/Murine JgGia Vector The murine IgG (mulgG) fusion protein vector was k indly provided by Dr. Peter Linsley [39]. A plasmid vector containing the murine IgG2 a hinge, C H 2 and C H 3 regions and the Oncostatin M (Onco M) signal sequence [40] was constructed using a human CD45R0 H i n d III/Bel 1 fragment containing exons 3, 7, and 8 as a starting point (provided by Dr. Julie Deans). Firstly, the CD45 signal sequence was replaced wi th the Onco M signal sequence by three successive rounds of P C R (conditions described previously) using over lapping ol igonucleotide 5' primers representing the Oncostatin M sequence (UBC #45, 46, or 47) and a 3' primer complementary to human CD45 exon 8 (UBC #18). A H i n d III restriction enzyme site was created at the 5' end of Oncostatin M . Primers used for the first round of P C R were U B C #45 and U B C #18, for the second round U B C #46 and U B C #18, and the third round U B C #47 and U B C #18. 5' H i n d III U B C 47 >3' 5- U B C 46——>3' 5> U B C 45 ->3' 5'—Sph 1—huCD45R0 Bel 1 3' / / 5' H ind III—huCD45 signal sequence / 3'<—UBC 18—5' The Onco M/huCD45R0 H ind III/Bcl 1 P C R fragment was ligated to Bel 1/Xba 1 digested murine IgG2 a D N A fragment and the H i n d I I I /Xba 1 digested plasmid vector pBluescript SK (+/-) (pBS) (Strategene C lon ing Systems, La Jol la, C A ) . Posit ive clones were grown up in GM48 dam", dem" Escherichia coli and digested 12 with Sph 1 and Bel 1 (New England Biolabs; Bel 1 restriction site is dam methylation sensit ive) to remove the human CD45 fragment, fo l l owed by subsequent purification and dephosphorylation of the Onco M / M u I g G / p B S fragment using 1 ul (10 units) of calf intestinal alkaline phosphatase (CIP; N e w England Biolabs). The murine IgG2a vector is hereafter referred to as the Ig vector. Care was taken to ensure the production of a pure preparation of the Ig vector absent of human CD45 contaminat ion. N-butanol Oligonucleotide Purification Oligonucleot ide primers were ordered from the Nucle ic Ac ids Processing Uni t (NAPS Unit , U B C , Vancouver, BC) and purif ied by the n-butanol method[41]. Primers were reconstituted in 100 jil of 30% ammonium hydroxide ( N H 4 O H ) and 1 ml of n-butanol (BDH Inc., Vancouver, BC) After vigorous vortexing, the sample was centrifuged at 12,000 x g fol lowed by removal of the single aqueous phase. The oligonucleotide pellet was resuspended in 100 pi of water and 1 ml of n-butanol and the above procedure was repeated. The resulting pellet was dried under vacuum and resuspended in 500 ul of dist i l led, deionized water. Opt ical Density (OD) readings at 260 nm and 280 nm were recorded and used to calculate the concentration of ol igonucleotide recovered by using the equation: | i m o l / m l of ol igonucleotide = O D 260/ext inct ion coefficient. The value for the extinction coefficient was calculated as 10 times the length of the ol igonucleotide. A l l oligonucleotides were heated to 80 °C and rapidly transferred to -20 °C to prevent self-hybridization. Preparation of Competent Bacteria for Transformation XL1 Blue and G M 4 8 Escherichia coli were rendered competent for 13 transformation by resuspending a 500 ml log phase culture (OD6oo= 0.4) in 200 ml of 30 m M potassium acetate, 100 m M rubid ium chloride, 10 m M calcium chloride, 50 m M manganese chlor ide, 15% glycerol p H 5.8 on ice for 5 minutes. Fo l lowing centrifugation, the pellet was resuspended in 20 ml of 10 m M 3-(N-Morphol ino-propanesulphonic acid (MOPS), 75 m M calcium chloride, 10 m M rubid ium chloride, 15% glycerol p H 6.5 on ice for 15 minutes. Prepared cells were frozen in a dry ice/ethanol bath and stored in 1 ml aliquots at -80 ?C until later use. Ligation and Transformation 20 ng of muCD45 isoform-specific P C R product digested wi th Sph 1 and Bel 1 was ligated to 20 ng of Ig vector using 1 ul of 10 X ligase buffer [50 m M Tr is -HCl p H 7.8, 10 m M magnesium chloride, 10 m M dithiothreitol (DTT), 1 m M adenosine triphosphate (ATP), 50 u g / m l bovine serum albumin (BSA)], and 1 ul (400 Units) of T4 D N A ligase (New England Biolabs) in a final volume of 10 ul for 2-3 hours at 15°C. Ligation reactions were diluted 1/4 with pyrogen reduced water fol lowed by addi t ion of 100 ul of competent X L l - B l u e E. coli and incubation on ice for 15 minutes. Samples were heated at 42 °C for 90 seconds, fol lowed by incubation on ice for 2 minutes. 100 ul of pre-warmed Lur ia Broth (LB) was added, and the mixture incubated at 37°C for 30 minutes to al low time for bacterial growth and acquisition of ampici l l in resistance. Ligation mixtures (240 ul) were spread on LB agar plates supplemented with 50 mg/1 ampici l l in and incubated at 37 °C overnight to al low for the formation of colonies. Colonies were randomly selected and placed in 2 m l of L A broth and incubated overnight at 37°C in preparation for alkaline lysis miniprep analysis. 1 ml of culture was pelleted and resuspended in 100 ul of cold TEG lysis buffer (25 m M Tris p H 8.0, 10 m M ethylenediaminetetraacetic acid (EDTA), 50 m M D-glucose) and 200 ul of 0.2 M N a O H / 1 % sod ium dodecyl sulfate (SDS) and left at room 14 temperature for 5 minutes. 150 ul of 3 M sodium acetate p H 5.2 was then added to the tube, mixed by inversion, and incubated on ice for 5 minutes to a l low for precipitation of bacterial chromosomal D N A . After centrifugation at 12 000 x g for 5 minutes, the chromosomal pellet was discarded while the plasmid D N A within the supernatant was kept for further analysis. Excess protein was removed from the supernatant by extraction w i th an equal vo lume of pheno l / ch lo ro fo rm • (1:1) fol lowed by precipitation of the plasmid D N A on ice for 5 minutes wi th 500 ul of isopropanol. The plasmid D N A pellet was washed once wi th 70% ethanol, dried briefly in a desiccator, and resuspended in 50 ul of TE buffer (10 m M Tr is -HCl p H 7.5, 1 m M EDTA) . 2 ul of miniprep D N A was digested with the appropriate restriction enzymes to determine if isoform-specific murine CD45 was present. Once ful ly sequenced[42], D N A from positive clones was digested wi th Xho 1 and Not 1, and the resulting Onco M / m u C D 4 5 / M u I g G piece was subcloned into the Xho 1/Not 1 digested mammalian expression vector pBCMGSneo. Nucleobond A X 2000 column preps (Macherey-Nagel G m b H & Co.) were performed according to manufacturers instructions to obtain large amounts of pure D N A for transfection into eucaryotic cells. Cell Culture A l l eucaryotic cell lines were maintained in 90% Dulbecco's Modi f ied Eagle Med ium (DMEM) , 10% fetal calf serum (FCS), 100 U n i t s / m l penici l l in G , 100 u g / m l streptomycin sulfate, and 0.25 u g / m l amphotericin B. Cos 7 adherent cells (American Type Culture Collection, Rockvi l le, Mary land; C R L #1651) were washed once wi th IX phosphate buffered saline (PBS; 154 m M N a C l , 2.7 m M KC1, 4.3 m M N a 2 H P 0 4 , 1.5 m M KH2PO4) and then removed from 5 m l N u n c tissue culture dishes (Gibco BRL , Life Technologies, Burl ington, ON) using 2X versene (0.7 m M E D T A , 0.14 M N a C l , 0.7 m M KC1, 8 m M N a 2 H P 0 4 , 1.3 m M KH2PO4 p H 7.3) and 15 gentle pipet action. X63-Ag8.653 murine myeloma cells were obtained from Dr. Hung-Sia Teh. T28 T lymphoma cells were obtained from Dr. Fumio Takei. A l l incubations were at 37°C and 5% CO2. Antibodies Exon-specific rat-anti-mouse CD45 antibodies used were the exon A-specif ic antibody 14.8 [43], the B220 isoform-specific antibody R A 3 6B2 [44], the exon B-specific antibodies 23 G2 and M B 4B4 [45], and the exon C-specific antibody, DNL1.9 (Ly 5-B220) [46] (Table I). A l l antibodies with the exception of DNL1.9 were used as tissue culture supernatants whereas DNL1.9 was obtained in a puri f ied form from Pharmingen (San Diego, C A ) . Goat-anti-mouse-Horseradish peroxidase ( G A M I g G -HRP) was obtained from Biorad Laboratories, Mississauga, O N and was used to detect the Fc port ion of CD45:MuIgG fusion proteins at a d i lu t ion of 1/5000. Secondary antibody goat-anti-rat-HRP (GARIgG-HRP) was obtained from Southern Biotechnology Associates, Inc., Birmingham, A L and used at a di lut ion of 1/10,000. Antibodies used for F A C S analysis included the pan specific rat-anti-mouse CD45 antibody, 13/2 [47] (gift from Dr. Ian Trowbridge), the anti-CD45 exon A-specif ic antibody R A 3 2C2 [48], fluorescein isothiocyanate (FITC) conjugated goat-anti-rat immunoglobul in (GARIgG-FITC) (Pierce, Rockford, IL), and anti CD45 exon-specific antibodies as indicated previously. Transient Transfection of Cos 7 cells with CD45-Immunoglobulin Constructs Transient expression of CD45:MuIgG isoform constructs was obtained by the DEAE-dextran method. Briefly, 30 ug of plasmid D N A was mixed wi th 68 ul of IX PBS, 20 JLXI of 50 m g / m l DEAE-dextran, and disti l led deionized H 2 O to a total volume of 100 ul. The D N A solution was then added to 1.6 ml of transfection media (10 ml 16 Table I Ant ibody Isotype Specificity Reference 13/2 I g G 2 b all isoforms [47] 14.8 IgG2b CD45 exon A [43] R A 3 2C2 IgM CD45 exon A [48] M B 4B4 I g G 2 a CD45 exon B [45] 23 G2 IgG2a CD45 exon B [45] DNL1.9 IgG2a CD45 exon C [46] R A 3 6B2 IgG2a B220 isoform [44] Table I: Description of Exon-specific Antibodies The isotype and specificity of each anti-CD45 exon-specific antibody used in the characterization of expressed CD45:MuIgG fusion proteins is indicated. 17 of D M E M plus 10 ul of 0.1 M chloroquine), mixed by pipett ing, and then added dropwise to a confluent 60 mm plate of adherent Cos 7 cells (1 x 10^ cells). Plates were rocked gently to ensure complete coverage of cells wi th the D N A / m e d i a solution and then incubated at 37°C, 5% CO2 for 4 hours. After 4 hours, the media was removed and the cells were gently washed in 3 m l of 90% PBS, 10% dimethylsulfoxide (DMSO) for 2-3 minutes, fol lowed by one wash in 4 m l of D M E M and subsequent incubation in 3 ml of D M E M , 10% FCS for 72-96 hours at 37*C. After 3 days, the culture supernatant was harvested and tested for the presence of secreted CD45:MuIgG isoform-specific fusion proteins. In addit ion, the transfected cells were removed from the tissue culture dish using 3 ml of 2X versene, centrifuged and the cell pellet lysed in 0.5 ml of 1% T r i t on /TNE lysis buffer (25 m M Tris p H 7.5, 150 m M N a C l , 2 m M E D T A , 1% Triton-X-100) for 20 minutes on ice. Lysates were centrifuged at 12,000 x g for 20 minutes and the supernatant removed and kept for further analysis. Stable Transfection of Cos 7 cells with CD»45-Immunoglobulin Constructs Stable expression of CD45 :MuIgG isoform constructs was obtained by the calcium phosphate method [49]. 30 ug of each plasmid D N A fusion construct was ethanol precipitated and resuspended in 220 ul of disti l led, deionized H2O and 250 |il of 2X Hepes buffered saline (HBS; 280 m M N a C l , 10 m M KC1, 1.5 m M Na2HPC>4/ 10 m M dextrose) in a 1.7 ml microcentrifuge tube (Island Scientific, Bainbridge Island, WA) . 30 ul of 2 M CaCl2 was added slowly, 5 ul at a time, over 30 seconds, and left at room temperature for 15 minutes to al low the D N A to precipitate. The precipitated D N A solution was added to a 50% confluent monolayer of Cos 7 cells in a 60 mm N u n c tissue culture dish containing 5 ml of D M E M / 1 0 % FCS. The cells were incubated at 37 °C, 5% CO2 for 3 to 4 hours at which time the D N A containing media was removed and replaced wi th fresh D M E M / 1 0 % FCS. A t 48 hours after the 18 addit ion of D N A , selection of neomycin resistant clones was achieved by removing cells from the dish using 4 ml of 2X versene and placing 1 ml into each of four 60 mm tissue culture dishes and adding 4 ml of D M E M / 1 0 % FCS and 350 u g / m l of active G418 (Gibco BRL Life Technologies, Burl ington, ON) . The four dishes were then incubated at 37°C, 5% CO2 for 13 days until small colonies started to appear on the surface of the dish. Single colonies were removed from the plate using 2 ul of 2X versene and transferred to one wel l of a 96 wel l plate containing 200 ul of D M E M / 1 0 % FCS and al lowed to grow to confluency. A t that time, the culture supernatant was removed for analysis and detection of secreted isoform-specific CD45:MuIgG fusion proteins. Transfection of X63-Ag8.653 and T28 cells by Electroporation Stable C D 4 5 R A B C : M u I g G , C D 4 5 R B : M u I g G , and CD45R0 :Mu IgG fusion protein producing clones were obtained by the fol lowing method. Briefly, 1 X 10^ X63-Ag8.653 cells or 5 X 10 6 T28 cells were harvested and resuspended in 800 ul of D M E M with 20 ug of ethanol precipitated plasmid D N A in a Biorad electroporation cuvette. Samples were electroporated at 250 volts and 960 uF using a Biorad Gene Pulser Electroporator (Biorad Laboratories, Mississauga ON) . After electroporation, the cells were placed on ice for 10 minutes, then removed from the cuvette and placed in a 100 mm tissue culture dish (Fisher Scientific, Vancouver, BC) wi th 10 ml of D M E M / 1 0 % FCS and incubated at 37°C, 5% CO2 for 48 hours. A t this time, cells containing the transfected gene for neomycin resistance were selected by the addit ion of 500 u g / m l (X63-Ag8.653) or 1 m g / m l (T28) of active G418. Culture supernatants were harvested at 48 hours and tested for transient expression of CD45:MuIgG fusion protein whi le the transfected cells were resuspended in 20 ml of D M E M / 1 0 % FCS, 500 u g / m l to 1 m g / m l of active G418 and cloned into 96 wel l plates and incubated at 37°C for 2-3 weeks until discrete colonies appeared. A t that 19 time, 150 ul of supernatant was removed from each wel l containing an indiv idual colony and tested for the presence of secreted CD45:MuIgG fusion protein. Cells that tested positive for stable fusion protein expression were grown to confluency and aliquots were frozen down in l iquid nitrogen.. Precipitation CD45:MuIgG fusion proteins were precipitated from 100-500 ul of tissue culture supernatant (after a 3 day incubation with 1 X 10^ cells) using 20 ul of a 10% solution of protein A produced by Staphylococcus aureus for 2 hours at 4°C on a rotator. Protein A producing Pansorbin cells were obtained from Calbiochem (San Diego, C A ) and were washed once in 1% Tr i t on /TNE lysis buffer prior to use. After 2 hours, the precipitate was washed once with lysis buffer, boiled for 5 minutes at 100°C in 20 ul of IX reducing SDS sample buffer containing 0.125 M Tr is -HCl p H 7.5, 10% glycerol, 100 m M dithiothreitol (DTT), 2% SDS and 0.2% bromophenol blue. The supernatant was loaded onto 7.5% SDS-polyacrylamide (SDS-PAGE) minigels and electrophoresed in running buffer (25 m M Tris p H 7.5, 192 m M glycine, 0.1% SDS) for 45-50 minutes at 200 volts. The resulting S D S - P A G E gel was transferred to polyvinyl idene dif luoride (PVDF) membrane (Mil l ipore, Bedford, M A ) in 0.3 M Tris p H 7.5, 0.23 M Glycine, 20% methanol for 1 hour at 100 volts. Western Blots Western blots were performed by wett ing P V D F membranes in methanol, r insing once in Tris buffered saline consisting of 25 m M Tris p H 7.5, 150 m M N a C l (TBS) fol lowed by a blocking step using either 5% bovine serum albumin fraction V (BSA) (Gibco BRL , Life Technologies) or 5% skim mi lk powder in TBS for 1 hour. Ant i -CD45 tissue culture supernatants were di luted 1/10 for 14.8 and 1/20 for M B 20 4B4, 23 G2, R A 3 6B2 in TBS plus 0.1% Tween-20 (TTBS) and incubated wi th the membranes for 1.5 hours. Membranes were then washed three times in TTBS, fo l lowed by subsequent incubation wi th 1/10,000 G A R I g G - H R P in TTBS for 45 minutes. A l l incubations were at room temperature on a rotary shaker. After washing membranes for 35 minutes wi th mult iple changes of TTBS, blots were developed by incubat ion w i th E C L reagents (Enhanced Chemi luminescence, Amersham Life Science, Oakvi l le, ON) according to manufacturers instructions and exposure to f i lm (Eastman Kodak Biomax M R , Rochester, N e w York). Flow Cytometry Cel l lines used for transfection of CD45:MuIgG isoform fusion constructs were tested for the expression of different isoforms of murine CD45. Briefly, 2 X 10 5 cells were harvested in log phase growth, washed once in IX PBS, resuspended in 200 ul IX PBS/2 .5% horse serum (HS) and placed in one wel l of a 96 wel l plate. The plate was centrifuged at 1000 rpm for 2 minutes to pellet the cells. The supernatant was removed by sharply invert ing the plate and blott ing on paper to remove excess l iqu id. After vortexing to resuspend the cell pellet, 100 ul of pr imary anti-CD45 antibody was added (undi luted tissue culture supernatant) and the plate was incubated on ice for 20 minutes. Fol lowing a wash step using 100 ul of IX PBS/2 .5% HS, lOOul of a 1/100 dilution of GARIgG-FITC was added and incubated in the same manner. The cells were washed once in IX PBS/2 .5% HS, fol lowed by transfer to a #2052 Falcon tube (Fisher Scientific) containing 150 pi of IX PBS/2 .5% H S and 10 u g / m l p rop id ium iodide (PI). Immunocytometry was performed using a Becton Dickinson F A C S C A N . Analysis of data was carried out using Lysis II software . 21 Lectin Analysis CD45:Mu IgG fusion proteins were precipitated as described previously. Membranes were blocked for one hour using 5% BSA fraction V in TBS, washed three times, 5 minutes each, in TTBS, fol lowed by incubation wi th 0.05 to 0.2 u g / m l of biotinylated lectin in 5% B S A / T T B S for one hour. The secondary antibody used was st reptavid in-HRP (Pierce, Rockford, IL) in 5% B S A / T T B S di luted 1/10,000. F ina l ly , membranes were washed 6 times in TTBS, 5 minutes each, and then developed by E C L as previously described. Lectins used are as fol lows: R C A , Ricinus communis 120; P N A , Arachis hypogaea (Peanut agglut inin); V V A , Vicia villosa; Con A , Concanavalin A ; W G A , Triticum vulgaris (Wheat germ agglutinin); S N A ; Sambucus nigra; M A A , Maackia amurensis. The S N A [50] and M A A [51] lectins were obtained from Oxford GlycoSystems, Rosedale, N Y . A l l other lectins were purchased from the Sigma Chemical Company, St. Louis, M O . PNGase F Digestion and Thrombin Cleavage Precipitated CD45:MuIgG fusion proteins were digested wi th PNGase F (New England Biolabs) as follows. Immunoprecipitates were resuspended in 10 ul of 1% T r i t o n / T N E lysis buffer and 1 ul of 10X denaturation buffer (supplied by N E B ; 5% SDS, 10% B-mercaptoethanol) and boiled for 10 minutes at 100°C. 10X reaction buffer (supplied by N E B ; 0.5 M N a 2 P 0 4 p H 7.5) and 10% Nonidet P-40 (NP-40) were then added to final concentrations of 50 m M and 1% respectively, fo l lowed by addit ion of 1000 units of PNGase F and incubation at 37°C for 90 minutes. In addit ion, in separate experiments, CD45:MuIgG fusion proteins were digested with 12.5 cleavage units of thrombin (Sigma Chemical Company, St. Louis, M O ) for two hours at room temperature. A l l samples were then electrophoresed under reducing conditions on SDS-PAGE gels as previously outlined. 22 Neuraminidase Digestion Precipitated CD45:MuIgG fusion proteins were digested wi th 2 mUnits of neuraminidase (sialidase) isolated from Vibrio cholerae (Boehringer M a n n h e i m G m b H , Lava l , Quebec) at 37°C for one hour in 50 m M sodium acetate, 4 m M calcium chloride p H 7.8, fo l lowed by S D S - P A G E and western blot analysis as previously described. O-Glycosidase Digestion Precipitated CD45:MuIgG fusion proteins were digested wi th 0.5 mUnits of BSA-f ree O-glycosidase isolated f rom Diplococcus pneumoniae ( B o e h r i n g e r Mannheim G m b H , Laval , Quebec) for 16-18 hours at 37°C in 20 ul of 20 m M sodium cacodylate, 20 m M sodium dihydrogen phosphate p H 6.5, fol lowed by S D S - P A G E and western blot analysis as previously described. To optimize the removal of O-l inked sugars, samples were treated wi th neuraminidase as previously described prior to digestion with O-glycosidase. 23 R E S U L T S 1.0 Generat ion of M u r i n e CD45- Immunog lobu l in Isoform-speci f ic Fus ion Constructs The objective of this work was to generate recombinant, secreted CD45-Immunoglobul in (CD45:MuIgG) fusion proteins containing isoform-specific regions of murine CD45. Previous attempts at expression of human CD45:MuIgG fusion constructs containing the original CD45 signal sequence were unsuccessful (Dr. Julie Deans, unpublished). Therefore, in an attempt to increase the expression of fusion proteins to a level that could be detected by precipitat ion of secreted culture supernatant wi th protein A , S D S - P A G E analysis and western blott ing wi th anti-murine immunoglobul in G (anti-muIgG), a new expression strategy was devised. The plasmid vector previously used for expression of human CD45:Ig constructs, p C D M 8 , was replaced in favor of an alternate mammal ian expression vector, pBCMGSneo [52]. As wel l , the CD45 signal sequence was removed and replaced with that of the growth factor, oncostatin M (Onco M) [40]. Transient expression of CD28Ig and B7Ig fusion constructs (containing the Onco M signal sequence) into Cos 7 cells by the DEAE-dext ran method resulted in the secretion of Ig fusion proteins that were easi ly pur i f ied f rom culture supernatants by affinity chromatography on immobi l ized protein A columns to concentrations of 1.5-4.5 mg/1 [39]. Puri f ied fusion proteins are a soluble form of recombinant protein that can be used in subsequent applications such as adhesion and binding assays. In the past, Ig fusion proteins have been instrumental in the identification of molecules that bind to CD44, [53] CD22, [8, 9] C T L A - 4 , [54] and CD28 [39]. In this body of work, transient expression of modif ied human CD45:MuIgG constructs in p C D M 8 resulted in a detectable level of expression by precipitation of culture supernatant wi th protein A and subsequent western blott ing wi th anti-24 mulgG (data not shown). As the inclusion of the Onco M signal sequence increased the levels of expression of human CD45 fusion constructs, it was reasonable to hypothesize that the expression of murine CD45:MuIgG fusion proteins wou ld also be favorably affected by this modification. Different isoforms of CD45 exist due to alternative spl icing of at least three exons located in the variable region of the extracellular domain. CD45 isoform-specific Ig fusion proteins were designed to include extracellular exon 3 (the first exon encoding mature protein), exon 7, and the majority of exon 8 as wel l as one or more of the variable, alternatively spliced exons 4, 5, and /o r 6. Exons 1 and 2 of the CD45 gene encode 5' untranslated regions and the CD45 signal sequence, hence they were not included in fusion protein constructs. The exons encoding the remainder of the extracellular domain of CD45 were omitted from fusion protein constructs, thus a l lowing the focus to remain on the b ind ing of the alternatively spl iced, isoform-specif ic exons. A s wel l , inclusion of addit ional invariable exons could result in potentially confusing interactions occurring wi th the invariable region of CD45. Moreover , as the invariable region contains 16 cysteine residues, the inclusion of this domain could cause incorrect disulf ide linkages to occur dur ing expression that may cause the fusion protein to fold incorrectly. The cysteine residue in exon 8 at amino acid position 178 was not included for the same reason. A schematic diagram of a secreted CD45Ig fusion protein is shown in Figure 2. Expressed fusion proteins are predicted to mimic the structure of an antibody heavy chain, except wi th the conventional variable, antigen b inding region replaced by isoform-specific regions of CD45. In addit ion, Ig fusion protein constructs contain the Onco M signal sequence as well as the hinge, C H 2 (constant region heavy chain 2), and C H 3 (constant region heavy chain 3) regions of murine IgG2a- The inclusion of the C H 2 and C H 3 domains of the Ig heavy chain allows for rapid purification of Ig fusion proteins by b inding to protein A-sepharose. In order to investigate all possible isoform-specific interactions, six plasmid D N A constructs were created, each 25 CD45RABC hinge Fc of MuIgG2a Figure 2: Schematic Representation of CD45RABC- Immunog lobu l in Fusion frotein. CD45RABC:MuIgG Fusion Protein is shown in dimerized form. Numbers indicate the exon of murine CD45 incorporated. Letters indicate alternatively Spliced exons of murine CD45. C H 2 and C H 3 denote constant (Fc) regions two and three of murine immunoglobulin G heavy chain. 26 containing a different alternatively spliced isoform of murine CD45: C D 4 5 R A B C , CD45RBC, CD45RA, CD45RB, CD45RC and CD45R0. 1.0.1 Reconstruction of the Oncostatin M signal sequence by Polymerase Cha in Reaction The Onco M signal sequence was reconstructed at the 5' end of the H i n d III/Bel 1 fragment containing exons 3, 7 and 8 of human CD45R0. This was achieved by three successive rounds of P C R us ing ove r lapp ing o l igonuc leot ides corresponding to the Onco M signal sequence. The first round used two oligonucleotide primers: at the 5' end, U B C #45, which corresponds to the 3' 33 nucleotides of the Onco M signal sequence and the 5' 18 nucleotides of human CD45 exon 3, and at the 3' end, antisense primer U B C #18, which anneals to the 3' end of human CD45 exon 8. The expected D N A fragment size for the first round of P C R was 180 base pairs (bp): the 3' 33 nucleotides of the Onco M signal sequence in addit ion to 145 nucleotides of human CD45 exons 3, 7, and 8. Analys is of P C R products from the first round by agarose gel electrophoresis revealed a 180 bp band at an approximate concentration of 20 ng /u l as wel l as a minor contaminating band of lower concentration at approximately 300 bp (data not shown). As the total volume of the P C R reaction was 100 ul, approximately 2 ug of the 180 bp product was created from 1 ng of template. The second round of PCR, using the 180 bp product from the first round as the template, the 5' primer U B C #46 which annealed to the sequences created in round one by U B C #45, and the same 3' primer as in round one, yielded a D N A fragment of approximately 207 bp, consistent wi th the addi t ion of an addit ional 27 nucleotides of the Onco M signal sequence to the 5' end of the D N A strand. The third round of P C R , using the 207 bp product from round two as the template, the 5' primer U B C #47, and the same 3' primer as in previous rounds, resulted in a 234 bp band, consistent with the addit ion of the final 15 nucleotides of 27 the Onco M signal sequence and a further 12 nucleotides creating a H i n d III restriction enzyme site at the 5' end of the D N A strand. The 234 bp product, representing the complete Onco M signal sequence l inked at the 5' end to human CD45 exons 3, 7 and 8, was digested wi th H i n d III and Bel 1 and pur i f ied to a concentration of 20 n g / u l (approximately 1.2 ug recovered) in preparation for 3-way ligation to the Bel 1 /Xba 1 digested murine heavy chain sequences (700 bp) and the H ind II I /Xba 1 digested plasmid vector pBluescript (pBS; 3 kb). The resulting 3.9 kb plasmid was expressed in the E. coli strain XL1 Blue, fully sequenced [42] and named clone #30. 1.0.2 Creation of the Modi f ied Mur ine IgG2 a Vector with the Oncostatin M signal sequence The m o d i f i e d Ig vec tor was c rea ted by u s i n g the O n c o M / h u C D 4 5 R 0 / M u I g G / p B S plasmid (#30) created previously as a starting point (Figure 3). This plasmid was digested separately wi th Sph 1 or Bel 1 to y ie ld a linearized 3.9 kb band upon agarose gel analysis. In addit ion, bands at approximately 6 kb and 7 kb were observed after 3 hours of digestion, indicating uncut plasmid D N A was present. Therefore, in order to al low for complete digestion by both enzymes at the same time, it was necessary to cleave the 3.9 kb plasmid #30 with Bel 1 overnight fol lowed by addition of an aliquot of Sph 1 every hour for 5 hours. The resulting fragments were the desired 3.75 kb Ig vector and the 150 bp fragment corresponding to huCD45 exons 3, 7, and 8. Whereas the huCD45 port ion was discarded, the resulting vector containing the murine IgG C H 2 and C H 3 domains as wel l as the Onco M signal sequence was dephosphorylated to remove the 5' phosphate to prevent self re-l igation, fo l lowed by puri f icat ion to 60 n g / u l by extraction from low melt agarose and ethanol precipitation. Approximately 1.2 ug of digested, dephosphorylated, and purif ied Ig vector was recovered. The Ig vector 28 huCD45R0 Sph Onco M Hind III pBluescript digest with 1 Sphl and Bel 1 Sph 1 Onco M Hind III Bel 1 pBluescript Figure 3: Creation of the Oncostatin M / Mur ine IgG2 a Vector The 3.9 kb pBluescript plasmid construct containing huCD45R0/Onco M /MuIgG2a (#30) was digested overnight with Bel 1 followed by 5 hours digestion with Sph 1 to remove huCD45. The 3.75 kb Ig vector portion was subsequently purif ied by low melt agarose extraction in preparation for cloning in of murine CD45 inserts. 29 was then ligated to muCD45 isoform-specific P C R inserts, created as described in the next section, to create muCD45:MuIgG fusion constructs. 1.0.3 Creat ion of Isoform-specif ic mur ine CD45 inserts by Polymerase Cha in Reaction The predicted size of each isoform-specific P C R product is shown in Table II. Primers were designed to incorporate muCD45 exon 3, exon 7, and the majority of exon 8 (3' primer was designed to omit the cysteine residue at position 178) into all six isoform-specific P C R products. In addit ion, one or more of the alternatively spliced exons, 4, 5, and 6, was incorporated into the final product depending on the isoform-specific template used. Upon using the oligonucleotide primers indicated in Table II, P C R products of the predicted size for each of the six isoforms were observed upon agarose gel analysis (Figure 4, Panel A). For CD45R0, in addition to the band at 159 bp, a band was observed at roughly 400 bp. However, the 400 bp band was present at a much lower concentration than the desired 159 bp band. For CD45RBC, a 200 bp band was observed in addition to the desired 447 bp product, but again this non-specific band was present at a much lower concentration. In addition to non-specific pr iming, some smearing and degradation was observed for CD45RA and CD45RB P C R products. A l l isoform-specific P C R products were digested with Sph 1 and Bel 1 and then purif ied to a single, sharp band by extraction from low melt agarose and ethanol precipitation (Figure 4, Panel B). Approximately 800 ng of each purif ied isoform-specific P C R fragment was available for l igation to the Ig vector. Successive attempts at ligation of the Sph 1/Bcl 1 digested P C R products to the Ig vector containing the Onco M and murine IgG2 a sequences were not immediately successful. Further analysis revealed that Sph 1 cuts at the ends of linear strands of D N A with a very low efficiency, approximately 25% in 20 hours when there are 8 nt 5' to the restriction site. Us ing the 5' oligonucleotide primer U B C #60 for P C R 30 Table II CD45 Exons 5' Pr imer 3' Primer Predicted Size Isoform Incorporated Used Used in base pairs CD45R0 3,7,8 #60 #63 159 CD45RA 3,4,7,8 #60 #63 280 CD45RB 3,5,7,8 #60 #63 300 CD34RC 3,6,7,8 #60 #63 300 CD45RBC 3,5,6,7,8 #60 . #63 447 C D 4 5 R A B C 3,4,5,6,7,8 #60 #63 547 Table II: Results of Polymerase Cha in Reaction of murine CD45 isoform-specific Inserts. The exons of the variable, extracellular domain incorporated by P C R using isoform-specific plasmid D N A templates and the indicated primers is shown. The predicted size of each isoform-specific product is also shown. 31 reactions yielded products that had only 5 nucleotides 5' to the Sph 1 restriction site. In order to create an isoform-specific muCD45 insert that Sph 1 could digest with an acceptable efficiency, each P C R product was blunt end l igated to itself to create mult imers. A s the Sph 1 site was no longer at the end of the fragment in a multimer, digestion was able to occur to create an isoform-specific product with the required sites for ligation to the Ig vector. Once the muCD45 isoform-specific inserts contained the appropriate cloning sites, l igation to the Ig vector was successful (Figure 5). A l l six fusion protein constructs were sequenced [42] to confirm accurate amplif ication of isoform-specific templates by PCR. A l l isoform-specific constructs were 100% confirmed wi th the exception of CD45RC, which was only 97% confirmed. A 100 ml min i plasmid prep of one positive clone for each isoform of CD45 was prepared in order to produce sufficient amounts of D N A for subcloning. The muCD45:MuIgG portion was cut out of the pBluescript p lasmid vector using restriction enzymes Xho 1 and Not 1 and the pur i f ied fragment subcloned into the Xho 1 /No t 1 digested and dephosphory lated mammal ian expression vector, p B C M G S n e o (Figure 6), in preparation for transient and stable expression in eucaryotic cells. 32 Figure 4: CD45 Isoform-speci f ic P C R products Isoform-specific inserts were created by PCR using isoform-specific plasmid D N A templates, 5' primer UBC#60 and 3' primer UBC#63. PCR products of the correct size were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining followed by purification by extraction from low melt agarose and ethanol precipitation. A ; unpurified PCR products, B; purified PCR products. Isoforms of CD45 are as indicated. Markers indicate size in base pairs. 33 3 A B C 7 8 pBluescript I cut out C D 4 5 : M u I g G 2 a Y Xho 1/ Not 1 and subclone pBCMGSneo Figure 5: Subcloning of murine CD45 Isoform-specific Inserts into the murine Ig Vector. Isoform-specific muCD45 inserts were created by PCR using isoform-specific plasmid D N A templates, an upstream 5' primer corresponding to muCD45 exon 3, and a downstream 3' primer corresponding to muCD45 exon 8. Restriction enzyme sites (Sph 1 upstream and Bel 1 downstream) are present to allow for cloning into the Ig vector. The thrombin cleavage site is shown. 3,7,8, muCD45 exons incorporated;A,B,C, alternatively spliced exons incorporated; Onco M , Oncostatin M signal sequence. 34 12 1.6 1.0 ^Vv ^ *° ^  Figure 6: C D 4 5 : M u I g G Fus ion Constructs i n M a m m a l i a n Express ion Vector p B C M G S n e o Fusion protein constructs in pBCMGSneo were digested with Xho 1 and Not 1 followed by 2% agarose gel electrophoresis and ethidium bromide staining to determine if isoform-specific muCD45 :MuIgG was present. Isoforms of CD45 are as indicated. Markers indicate size is kilobase pairs. t 35 R E S U L T S 2.0 Expression of murine CD45-Immunoglobul in Isoform-specific Constructs in Cos 7 cells The goal of this series of experiments was to determine if CD45:MuIgG fusion proteins could be expressed by Cos 7 cells, a non-hematopoietic monkey kidney cell l ine that has been used successful ly in the past for transient expression of recombinant proteins [9]. Cos 7 cells were used as an efficient transient expression system to rapidly establish if isoform-specific fusion proteins could be expressed and secreted into the culture supernatant. If isoform-specif ic fusion proteins are expressed by Cos 7 cells, it is reasonable to think that expression in more functionally relevant myelo id and lymphoid cell lines wou ld also be successful. Attempts were made to transiently express six murine CD45 isoform-specific fusion protein constructs in Cos 7 cells by the DEAE-dext ran transfection method. In theory, if an isoform-specific l igand interacts wi th the fusion protein consisting of the largest isoform of muCD45, C D 4 5 R A B C : M u I g G , v ia the product of the three alternatively spliced exons, the same l igand w i l l not b ind to the smallest isoform of muCD45, CD45R0:MuIgG, due to the absence of exons A , B, and C. In addit ion, the putative l igand may or may not bind to a fusion protein containing only one or two of the alternatively spliced exons. Furthermore, CD45RB is a isoform commonly expressed by murine cell types, thus it is l ikely that this one-exon isoform wi l l have a physio logical l igand on murine cells. Therefore, in order to maximize the possibility of determining the identity of an isoform-specific l igand, stable clones of three isoform-specific fusion constructs, C D 4 5 R A B C : M u I g G , CD45RB:MuIgG, and CD45R0:MuIgG were obtained by calcium phosphate transfection. 2.0.1 Transient Expression 36 Transient expression of five isoform-specific CD45:MuIgG fusion proteins was obtained by the DEAE-dext ran method. Culture supernatants from 1 X 10 6 Cos 7 cells were harvested 3 days post-transfection and analyzed by precipitation with protein A followed by SDS-PAGE and western blotting with G A M I g G - H R P . Secreted fusion proteins were v isual ized as one distinct band (Figure 7, Panel A) . The predicted molecular weight of unglycosylated fusion proteins and the apparent molecular weight as determined by precipitation and S D S - P A G E analysis of each isoform-specific fusion protein is shown in Table III. C D 4 5 R A B C : M u I g G had an apparent molecular weight of 125 k D a , C D 4 5 R B : M u I g G was 70 k D a , and CD45R0:MuIgG was 55 kDa. In addit ion, fusion proteins containing two other isoforms of CD45, CD45RA:Mu IgG, and CD45RBC:MuIgG, were expressed at the transient level and were observed as one band at 70 kDa and 85 kDa respectively. A l though mult ip le attempts were made, transient expression of CD45RC:Mu IgG was not observed. A s the CD45RC P C R product was only 97% confirmed by D N A sequencing, this sequence should be reconfirmed before subsequent attempts at transient expression are made. Analysis of Cos 7 cell lysates for all expressed isoforms after a l lowing three days for secretion of fusion proteins revealed the existence of one or two bands at lower molecular weights than observed for the mature, glycosylated, secreted fusion protein. Lower molecular weight protein bands at approximately 60 kDa were observed in lysates of Cos 7 cells expressing CD45RA:MuIgG and CD45RB:MuIgG, at 73 kDa for CD45RBC:MuIgG, and at 75 kDa for CD45RABC:MuIgG. In addition, a band at approximately 35-40 kDa was observed in lysates of CD45RB:MuIgG and CD45RBC:MuIgG, indicating possible cleavage of proteins at the thrombin site. At least two independent clones of each isoform were analyzed by transient transfection and comparable results were obtained wi th respect to apparent molecular weight, purity, and protein bands in lysates. Figure 7, Panel B 37 S L S L S L S L S L 175 — 83 — 63 — 4 8 — •A S L S L S L S L S L 175 • 83" 63-48" Figure 7: Transient Expression of murine CD45-Immunoglobul in Fusion Proteins in Cos 7 cells. 1 X 106 Cos 7 cells were transfected 30 ug of plasmid D N A by the DEAE-dextran method. Transfected cells were incubated in 3 ml of DMEM/10% FCS for three days prior to precipitation of 100 ul of supernatant (S) or 100 ul of lysate (L) with 20 ul of a 10% solution of protein A producing Pansorbin cells. Each sample was subjected to 7.5% SDS-PAGE and western blotting with: A , 1/5000 GAMIgG-HRP; B, 1/10 RA3 6B2. Cos 7 cells were lysed in 0.5 ml of 1% Triton/TNE lysis buffer for 20 minutes on ice. 38 Table III Fusion Protein Predicted Apparent Molecular Weight Unglycosylated in each cell l ine Molecular Weight Cos 7 X63-Ag8.653 T28 CD45R0:MuIgG 34 55 50 50 CD45RA:MuIgG 39 75 nd nd CD45RB:Mti IgG 40 70 65 70 CD45RC:MuIgG 40 no no no CD45RBC:MuIgG 46 90 nd nd CD45RABC:MuIgG 50 . 125 110 120 Table III: Apparent Molecular Weight of CD45-Immunoglobul in Isoform Fusion Proteins Expressed in Three Ce l l Lines. The apparent molecular weight as determined by precipitation of 100 ul of cell culture supernatant with 20 ul of a 10% solution of protein A producing Pansorbin cells. The entire sample was subjected to 7.5% S D S - P A G E analysis against N E B unstained molecular weight standards and western blotting with 1/5000 G A M l g G -H R P . Values reflect an average of four experiments, nd; not determined, no; not obtained. 39 indicates that R A 3 6B2, a B220 isoform-specif ic ant ibody, reacted wi th both C D 4 5 R A B C : M u I g G and C D 4 5 R A : M u I g G . Fus ion proteins conta in ing no alternatively spliced exons or exons B and /o r C d id not react wi th R A 3 6B2 as predicted. 2.0.2 Stable Expression As attempts at stable expression of CD45:MuIgG fusion proteins by the D E A E -dextran method were unsuccessful, stable expression of three isoform fusion constructs, CD45R0:MuIgG, CD45RB:MuIgG, and C D 4 5 R A B C : M u I g G , was obtained by the calc ium phosphate method. Approx imate ly 5% of neomycin resistant colonies tested posit ive for secretion of isoform-specific fusion proteins into the culture supernatant (Table IV). As with transient expression experiments, fusion proteins expressed in culture supernatants were observed as a single, distinct band upon precipitation with protein A , S D S - P A G E analysis, and western blotting with G A M I g G - H R P (Figure 8). In addit ion, comparison of apparent molecular weight between transient and stable expression systems revealed similar results (Figure 7 versus Figure 8). However, slight clonal variation in the apparent molecular weight of CD45RABC:MuIgG, and to some extent of CD45RB:MuIgG was observed. CD45:MuIgG fusion proteins were produced in relatively pure amounts using serum free hybridoma media (Figure 9). Incubation of fusion protein-secreting cells in D M E M / 1 0 % FCS followed by precipitation of culture supernatant with protein A , S D S - P A G E analysis, and Coomassie bril l iant blue staining resulted in the presence of contaminating proteins at approximate molecular weights of 55 kDa, 65 kDa and > 175 kDa. In contrast, incubation of fusion protein-secreting cells in serum free hybr idoma media resulted in the appearance of only one protein band at the molecular weight previously observed to be that of either CD45R0:MuIgG or CD45RABC:Mu IgG. Fusion protein yield appeared to be less in serum free media. 40 Table IV Ce l l CD45 Total Number Number of neo r Number of Line Isoform neo r colonies CD45:MuIgG colonies analyzed secreting colonies Cos 7 R A B C 96 54 2 R B 100 26 2 R0 110 26 3 X63-Ag8.653 R A B C 20 12 4 R B 6 6 3 R0 30 16 13 T28 R A B C 80 24 3 R B 83 23 19 R0 90 4 4 Table IV: Results of Stable Transfection of CD45-Immunoglobul in Isoform Fusion Constructs into Three Ce l l Lines. Colonies were screened by precipitation of 150 ul of culture supernatant with 20 ul of a 10% solution of protein A producing Pansorbin cells fol lowed by 7.5 % SDS-P A G E analysis of the entire sample and western blotting with 1/5000 G A M I g G - H R P for 1 hour in 5 % B S A / T B S . 41 RABC RB RO Figure 8: Western Blot of CD45-Immunoglobul in Fusion Proteins Expressed in Cos 7 cells. 100 ul of cell culture supernatant was precipitated with 20 ul of a 10% solution of protein A producing Pansorbin cells followed by 7.5% SDS-PAGE analysis of the entire sample and western blotting with 1/5000 GAMIgG-HRP. Duplicate lanes represent samples from different clones. 42 Isoform <P ^ ^ Serum + + Figure 9: Relative Y ie ld and Puri ty of CD45-Immunoglobul in Fusion Proteins Expressed in Cos 7 cells. 1.8 ml of cell culture supernatant was precipitated with 150 ul of a 10% solution of protein A producing Pansorbin cells followed by 7.5% SDS-PAGE and staining with Coomassie brilliant blue-R250. Serum +, supernatant from 1 X 106 cells grown in 10 ml of DMEM/10% FCS for three days; Serum -, supernatant from 1 X 106 cells grown in 10 ml of Hybridoma serum free media for three days. 43 Scanning densitometry and comparison to standards of known amount established that 1 X 106 Cos 7 cells secreted approximately 1.5-2.0 ug/ml CD45RABC:MuIgG or 3.0-4.0 ug/ml CD45R0:MuIgG over a three day period in 10 ml of serum free media (Table V). IgG standards of 1 ug, 5ug, and 8ug corresponded to OD readings of 4.78, 22.8, and 32.0 respectively. Therefore, upon calculation of the standard curve, the OD readings of 12.3 for CD45RRABC:MuIgG and 30.5 for CD45R0:MuIgG corresponded to 2.7 ug and 7.4 ug of protein respectively (per 1.8 ml of supernatant precipitated, therefore, 1.5 ug/ml CD45RABC:MuIgG and 4.0 ug/ml CD45R0;MuIgG). Limitations of this method include the following: protein A precipitated fusion proteins may not bind coomassie blue with the same affinity as unprecipitated murine IgG standards, or alternatively, the presence of carbohydrate residues may differentially affect the binding of coomassie blue. The results obtained using Cos 7 cells as a model expression system serve to establish four important points. Firstly, it is clear from both transient and stable expression experiments that CD45:MuIgG fusion proteins are secreted into the culture supernatant. In addition, expressed fusion proteins are observed to have an apparent molecular weight significantly higher than the predicted molecular weight of unglycosylated proteins, suggesting that fusion proteins are extensively glycosylated before secretion. Thirdly, it has been established that by using the expression vector, pBCMGSneo, fusion proteins are secreted at sufficient levels to be detected in culture supernatants by precipitation with protein A followed by SDS-PAGE and Coomassie brilliant blue staining or western blotting with an anti-murine IgG antibody. Finally, in addition to transient expression, stable fusion protein secreting clones can be obtained by calcium phosphate transfection. It has now been determined that this strategy for expression of murine CD45:MuIgG fusion proteins results in the secretion of detectable amounts of protein by the methods described. Thus, it is now important to express these fusion proteins in functionally relevant myeloid and lymphoid cells. If the interaction of 44 Table V Ce l l L ine CD45RABC:MuIgG CD45RO:MuIgG Cos 7 1.5-2.0 u g / m l 3.0-4.0 u g / m l X63-Ag8.653 1.5-2.5 Ug/ml 3.5-4.2 u g / m l Table V : Estimated Y ie ld of CD45-Immunoglobvi l in Isoform Fusion Proteins Expressed in Two Cel l Lines. Est imated y ie ld of secreted CD45:MuIgG fusion proteins was determined by precipitation of 1.8 ml of cell culture supernatant wi th 150 (al of a 10% solution of protein A producing Pansorbin cells followed by 7.5 % S D S - P A G E and staining with Coomassie br i l l iant blue-R250. Band intensity was determined by scanning densitometry. Values reflect the concentration of fusion protein produced by 1 X 10 6 cells in 10 ml of media over a three day period. Results are a range over three experiments. 45 CD45 with a putative ligand is indeed mediated by specific carbohydrate residues on CD45, it is crucial to express CD45:MuIgG fusion proteins in a cell l ine that w i l l mimic endogenous CD45 glycosylation. If glycosylat ion of CD45:MuIgG fusion proteins is not comparable to that of endogenous CD45, a l igand that binds to endogenous CD45 would not bind to a CD45:MuIgG fusion protein or vice versa. As CD45 is expressed on the surface of all nucleated cells of hematopoietic lineage, the use of myeloid and lymphoid cells in place of Cos 7 cells to express CD45:MuIgG fusion proteins wou ld most l ikely result in a closer, if not identical, glycosylation pattern to that of endogenous CD45. Characterization of carbohydrate residues on endogenous CD45 as wel l as on CD45 :MuIgG fusion proteins is necessary to determine if secreted fusion proteins are mimicking endogenous CD45. 46 R E S U L T S 3.0 Expression of murine CD45-Immunoglobul in Isoform-specific Constructs in Myelo id and Lympho id Cel l Lines As a detectable level of expression of muCD45:MuIgG fusion proteins was observed in Cos 7 cells using the methods previously described, the focus of the next series of experiments was to obtain stable, fusion protein-secreting clones in functionally relevant cells, specifically myeloid and lymphoid cell lines. The cell lines transfected with fusion construct D N A were X63-Ag8.653, a murine myeloma, and T28, a murine T cell lymphoma. As with stable clones expressed by Cos 7 cells, three isoforms of muCD45 l inked to heavy chain constant regions 2 and 3 of mulgG were expressed: C D 4 5 R A B C : M u I g G , CD45RB:MuIgG, and CD45R0:MuIgG. The expression of endogenous CD45 by X63-Ag8.653 myeloma and T28 lymphoma may reflect the ability of these cell types to express CD45:Ig fusion proteins in a manner that would mimic endogenous CD45 expression. F A C S analysis indicates that X63-Ag8.653 cells do not express any isoform of endogenous CD45 whereas T28 cells express the common epitope of CD45, as recognized by a pan-specific antibody 13/2, as wel l as one isoform of CD45, CD45RB, as recognized by exon B-specific antibodies M B 4B4 and 23 G2 (Figure 10). Al though the goat-anti-rat IgG negative control for X63-Ag8.653 cells appears to be slightly positive, indicating possible heavy chain Fc receptor binding, the binding of subsequent exon-specific antibodies d id not increase significantly when compared to the negative control. 3.0.1 Stable Expression in the X63-Ag8.653 Mur ine Myeloma Cel l Line The results of transfection of muCD45 :MuIgG fusion proteins into X63-Ag8.653 mur ine mye loma cells by electroporat ion is shown in Table IV. 47 Figure 10: F A C S C A N of Ce l l Lines Transfected wi th CD45-Immunoglobul in Isoform Constructs. Expression of muCD45 isoforms as determined by Flow Cytometry. 2 X ItF cells were incubated with 100 ul of exon-specific tissue culture supernatant and 1/100 Goat-anti-rat-FITC. A , T28 cells; B, X63-Ag8.653 cells. Exon-specific antibodies: RA3 6B2, B220 isoform; 14.8 and RA3 2C2, CD45 exon A; 23 G2 and MB 4B4, CD45 exon B; DNL1.9, CD45 exon C, 13/2, pan-specific anti-CD45. 48 Approximately 30% of neomycin resistant clones, as determined by precipitation with protein A followed by western blot analysis using an anti-muIgG antibody, tested positive for secretion of CD45RABC:MuIgG whereas 50% and 80% of clones tested positive for secretion of CD45B:MuIgG and CD45R0:MuIgG respectively. The apparent molecular weight of CD45:MuIgG fusion proteins expressed by X63-Ag8.653 cells as determined by precipitation with protein A and western blot analysis with anti-muIgG antibody is shown in Figure 11 and summarized in Table III. CD45RABC:MuIgG secreted by X63-Ag8.653 cells had an apparent molecular weight of 110 kDa, CD45RB:MuIgG was 65 kDa and CD45R0:MuIgG was 50 kDa. In general, the apparent molecular weight of fusion proteins expressed by X63-Ag8.653 cells was 5-10 kDa lower than that of the equivalent fusion protein expressed by Cos 7 cells. In addition, as with fusion proteins secreted by Cos 7 cells, the apparent molecular weight of fusion proteins expressed by X63-Ag8.653 cells was significantly higher than the predicted molecular weight of unglycosylated fusion protein. Scanning densitometry established that 1 X 106 X63-Ag8.653 cells in 10 ml of DMEM/10% FCS secreted 1.5-2.5 ug/ml of CD45RABC:MuIgG and 3.5-4.2 ug/ml of CD45R0:MuIgG over a three day period (Table V). IgG standards of lug, 5ug, and 8ug corresponded to OD readings of 8.0, 23.1, and 30.0 respectively. Upon calculation of a standard curve, the OD reading of 20.1 for CD45RABC:MuIgG corresponded to 4.5 ug of protein (in 1.8 ml of supernatant precipitated, therefore, 2.5 ug/ml). Likewise, calculation of a standard curve for CD45R0:MuIgG revealed that the OD reading of 33.2 corresponded to 7.6 ug of protein (in 1.8 ml, therefore, 4.2 ug/ml). These values are comparable to those obtained using Cos 7 cells. 3.0.2 Transient Expression in the T28 Murine T lymphoma Cell Line Unfortunately, stable fusion protein secreting clones were not obtained by electroporation of T28 cells. As a result, transient expression of muCD45:MuIgG 49 Figure 11: Western Blots of CD45-Immunoglobul in Fusion Proteins Expressed by X63-Ag8.653 and T28 cells. 100-400 ul of cell culture supernatant was precipitated with 20 ul of a 10% solution of Pansorbin cells followed by 7.5% SDS-PAGE analysis of the entire sample and western blotting with 1/5000 GAMIgG-HRP. A, fusion proteins expressed by X63-Ag8.653 cells; B, fusion proteins expressed by T28 cells. 50 fusion proteins by T28 cells was observed before levels of fusion protein expression decreased below the l imit of detection possible by precipitation wi th protein A and western blotting wi th anti-muIgG antibody. The results of transfection of fusion construct D N A into T28 cells are shown in Table IV. C D 4 5 R A B C : M u I g G had an apparent molecular weight of 120 k D a , C D 4 5 R B : M u I g G was 70 k D a and CD45R0:MuIgG was 50 kDa (Figure 11, Table III). As wi th CD45:MuIgG fusion proteins expressed by Cos 7 and X63-Ag8.653 cells, the apparent molecular weight of fusion proteins expressed by T28 cells was significantly higher than the predicted molecular weight of unglycosylated fusion protein. In addit ion, apparent molecular weight of fusion proteins expressed in T28 cells was in the same range as that observed wi th Cos 7 and X63-Ag8.653 cells (Figure 8 versus Figure 11, Table III). Initially, expression of fusion proteins by T28 cells was comparable to that observed for Cos 7 and X63-Ag8.653 cells, but within 3 days, T28 expression levels decreased to below detectable l imits by S D S - P A G E and Coomassie bri l l iant blue staining or western blot. 51 R E S U L T S 4.0 Character iza t ion of Expressed mur ine CD45- Immunog lobu l i n Fus ion Proteins Once expressed, muCD45:MuIgG fusion proteins were ful ly characterized not only as to apparent molecular weight, dimerizat ion in non-reducing S D S - P A G E conditions, and reactivity with anti-CD45 exon-specific antibodies, but also as to re -l inked and O-l inked carbohydrate content by reactivity wi th carbohydrate residue-specific lectins, PNGase F digestion, O-glycosidase treatment, thrombin cleavage, and neuraminidase digestion. As the interaction w i th a putat ive l igand may be mediated by specific carbohydrate residues on CD45 , it is important to ful ly characterize the amount and type of carbohydrate added by each cell line. 4.0.1 Reactivity with anti-CD45 Exon-specific Antibodies Transient expression in Cos 7 cells of five isoforms of CD45 l inked to the heavy chain constant regions of murine IgG is shown in Figure 7. A l l secreted fusion proteins reacted strongly with an antibody that recognizes the constant region of murine IgG2a upon precipitation with protein A and western blot analysis. In addit ion, the anti-muIgG antibody reacted with lower molecular weight proteins in the lysates of Cos 7 cells transfected with fusion construct D N A (Figure 7, Panel A) . R A 3 6B2, an antibody specific for the B220 isoform of CD45, reacted not only with C D 4 5 R A B C : M u I g G but also w i th the fus ion prote in conta in ing on ly one alternatively spliced exon (4,A), CD45RA:MuIgG (Figure 7, Panel B). This is the first demonstration that R A 3 6B2 reacts with an epitope dependent on the expression of exon A . A l l transiently expressed fusion proteins reacted wi th anti-CD45 exon-specific antibodies M B 4B4, 23 G2 (exon B dependent), 14.8 (exon A dependent), and 52 DNL1.9 (exon C dependent) as predicted depending on the presence of alternatively spliced exons A , B, and/or C (data not shown). In addit ion, anti-CD45 exon-specific antibodies d id not react with the proteins in Cos 7 cell lysates (Figure 7) wi th the exception of the exon C-specific antibody, DNL1.9 (data not shown). In addition to transient Cos 7 clones, stable clones produced by Cos 7 and X63-Ag8.653 cells secreting either CD45RABC:MuIgG, CD45RB:MuIgG, or CD45R0:MuIgG were analyzed for reactivi ty w i th ant i -muIgG and ant i -CD45 exon-specif ic antibodies. The results are summarized in Table VI. Moreover, antibody reactivity analysis was conducted on fusion proteins expressed transiently by T28 cells. Ant ibody reactivity analysis of CD45:MuIgG expressed in a stable fashion by Cos 7 cells is shown in Figure 12. A l l isoform-specific fusion proteins reacted strongly with the anti-muIgG antibody. In addit ion, all fusion proteins reacted with exon-specific antibodies as expected depending on the presence of exons A , B, and/or C. CD45RABC:Mu IgG reacted faintly wi th exon A-specif ic 14.8 and strongly with B220 isoform-specif ic RA3-6B2, al though var iat ion in RA3-6B2 reactivity was observed between the two clones obtained. Exon B-specific M B 4B4 reacted very strongly wi th CD45RABC:Mu IgG and CD45RB:MuIgG but not wi th CD45R0:MuIgG as expected. A n alternate exon B-specific antibody, 23 G2, reacted wi th all fusion proteins containing exon B, but to a lesser extent and lower intensity than that observed w i th M B 4B4. Var ia t ion in 23 G2 react iv i ty was observed for CD45RB:MuIgG in that one clone reacted with a significantly higher intensity than the other. Exon C-specif ic DNL1 .9 reacted wi th C D 4 5 R A B C : M u I g G and not CD45RB:MuIgG and CD45R0:MuIgG as expected. Ant ibody reactivity analysis of CD45:MuIgG fusion proteins expressed by X63-Ag8.653 murine myeloma cells is shown in Figure 13. Four independent fusion protein secreting clones were analyzed for C D 4 5 R A B C and five independent clones were analyzed for CD45R0. A l l fusion proteins reacted with anti-muIgG as expected. A s observed w i th fus ion prote ins expressed by Cos 7 ce l ls , fus ion 53 Table VI C D 4 5 R A B C CD45R0 CD45RB Cos 7 X63-Ag8.653 Cos X63-Ag8.653 Cos 7 X63-Ag8.653 A n t i l g + + + + + + R A 3 6B2 + - - -M B 4B4 + + - + + 23G2 . + + - - + + 14.8 + + - - - -DNL1.9 + + - -Table V I : Reactivity of CD45-Immunoglobul in Fusion Proteins wi th An t i -CD45 Exon-Specific Antibodies. CD45:MuIgG Fusion proteins were precipitated from 100 ul of cell culture supernatant using 20 ul of a 10% solution of protein A producing Pansorbin cells. The entire sample was subjected to 7.5 % SDS-PAGE analysis and subsequent western blotting using anti-murine CD45 exon-specific antibody tissue culture supernatants. Ant ibody specificity is as follows: Ant i Ig, heavy and light chains of mouse IgG2 a ; R A 3 6B2, B220 isoform; M B 4B4 and 23 G2, CD45 exon B; 14.8, CD45 exon A ; DNL1.9, CD45 exon C. +, positive reaction; - , negative reaction. 54 A n t i m u l g G RABC RB RO C 175 — 83 — 63 — 48 — Exon A. RABC RB RO C RABC RB RO 175— 1 7 5 -83 - " 8 3 -63— 6 3 -4 8 -Exon B RABC RB RQ C RABC RB RQ C 1 7 5 ~ 175-83— * * 83 -6 3 - 6 3 - • 4 8 - 4 8 -Exon C RABC RB RQ C 175 — 83— * " 63 — 48 — Figure 12: Reactivity of CD45-Immunoglobul in Fusion Proteins Expressed in Cos 7 cells wi th Ant i -CD45 Exon-specific Antibodies. Precipitation of 100 ul of cell culture supernatant with 20 ul of a 10% solution of protein A producing Pansorbin cells followed by 7.5% SDS-PAGE and western blotting with the antibody indicated. From left to right top to bottom: Anti mulgG, GAMIgG-HRP; Exon A, 14.8 and RA3 6B2; Exon B, MB 4B4 and 23 G2; Exon C, DNL1.9. C, negative control. 55 A n t i M u l g G RABC RO 175-8 3 -6 3 -48-Exon A RABC RO COS-7 AG8 653 C 175-8 3 -63 -4 8 -175-83-6 3 -48-Exon B RABC RO C RABC RO 175- 1 7 5 -8 3 - 8 3 -63- 6 3 -48- 4 8 -Exon C RABC RO 175- , 83 -63-48-Figure 13: Reactivity of CD45-Immunoglobul in Fusion Proteins Expressed in X63-Ag8.653 cells wi th Ant i -CD45 Exon-specific Ant ibodies. Precipitation of 100 ul of cell culture supernatant with 20 ul of a 10% solution of protein A producing Pansorbin cells followed by 7.5% SDS-PAGE and western blotting with the antibody indicated. From left to right, top to bottom: Anti mulgG, GAMIgG-HRP; Exon A, 14.8 and RA3 6B2 (CD45RABC:MuIgG only); Exon B, MB 4B4 and 23 G2; Exon C, DNL1.9. C, negative control. 56 proteins expressed by X63-Ag8.653 cells reacted wi th exon A-specif ic 14.8, exon B-specific M B 4B4 and 23 G2, and exon C-specific DNL1.9 as expected depending on the presence of the appropriate alternatively spliced exon (Figure 12 versus Figure 13). In contrast to C D 4 5 R A B C : M u I g G expressed by Cos 7 cells, C D 4 5 R A B C : M u I g G expressed by X63-Ag8.653 cells did not react with B220 isoform-specific RA3-6B2. Figure 14 shows antibody reactivity of CD45:MuIgG expressed transiently by T28 cells. Results were similar to that obtained by using fusion proteins expressed by X63-Ag8.653 cells (Figure 13 versus Figure 14). Each of two independent clones for C D 4 5 R A B C , CD45RB, and CD45R0 reacted with anti-muIgG and the exon-specific antibodies previously outlined as expected. In agreement wi th the results obtained using X63-Ag8.653 cells but in contrast to results obtained using Cos 7 cells, fusion proteins secreted by T28 cells d id not react with the B220 isoform-specific antibody R A 3 6B2. 4.0.2 Analysis of N- l inked Glycosylation The presence of N-l inked carbohydrate was detected and analyzed by digestion wi th PNGase F (Figure 15). Treatment of protein A-precipitated CD45RABC:MuIgG and CD45R0:MuIgG with PNGase F resulted in a slight decrease in apparent molecular weight of approximately 5 kDa for both isoforms of CD45. As a positive control, endogenous CD45 expressed by T28 cells was precipitated wi th the 1 3 / 2 antibody conjugated to sepharose beads, and then digested wi th PNGase F, resulting in a decrease in apparent molecular weight as visualized by S D S - P A G E and western blotting with CD45 exon B-specific M B 4B4. CD45:MuIgG fusion proteins can be cut into two pieces at the engineered thrombin cleavage site between CD45 and murine IgG (see Figure 5). This treatment allows for determination of the the apparent molecular weight of each portion of the fusion protein. The results of thrombin cleavage are shown in Figure 16. 57 Anti MulgG RB RO RABC C 175-m 8 3 --* mm 6 3 -4 8 - m m Exon .A. RB RO RABC C RB RO RABC C 175- — 175-8 3 - 8 3 -6 3 - 6 3 -4 8 - 4 8 -Exon B RB RO RABC RB RO RABC 175. 83. 63. 48' 175. 83-63-48> Exon C RB RO RABC C 175-8 3 -6 3 -4 8 -Figure 14: Reactivity of CD45-Immunoglobul in Fusion Proteins Expressed in T28 cells wi th Ant i -CD45 Exon-specific Antibodies. Precipitation of 100 ul of cell culture supernatant with 20 ul of a 10% solution of protein A producing Pansorbin cells followed by 7.5% SDS-PAGE and western blotting with the antibody indicated. From left to right, top to bottom: Anti mulgG, GAMIgG-HRP; Exon A, 14.8 and RA3 6B2; Exon B, MB 4B4 and 23 G2; Exon C, DNL1.9. C, positive control, CD45RABC:MuIgG expressed by Cos 7 cells precipitated in the same manner. 58 CD45R0 CD45RABC C Figure 15: PNGase F Digest ion of CD45-Immunoglobul in Fusion Proteins . 100-600 ul of cell culture supernatant was precipitated with 20 ul of a 10% solution of protein A producing Pansorbin cells followed by treatment with 1000 Units of PNGase F (New England Biolabs) for one hour at 37°C. The entire sample was subjected to 7.5% SDS-PAGE and western blotting with 1/5000 GAMIgG-HRP. +, treated samples; -, untreated samples. From left to right: isoform-specific fusion proteins secreted by Cos 7 cells, isoform-specific fusion proteins secreted by X63-Ag8.653 cells. C, PNGase F treated CD45 precipitated from 3 X 106 T28 cells with 20 ul of I 3/2 conjugated sepharose beads. 59 CD45R0 CD45RABC Figure 16: Thrombin Cleavage of CD45-Immunoglobulin Fusion Proteins. 100-600 ul of cell culture supernatants was precipitated with 20 ul of a 10% solution of protein A producing Pansorbin cells followed by treatment with 12.5 cleavage units of thrombin (Sigma) for two hours at room temperature. The entire sample was subjected to 7.5% SDS-PAGE and western blotting with 1/5000 GAMIgG-HRP. +, treated samples; untreated samples. From left to right: isoform-specific fusion proteins secreted by Cos 7 cells, isoform-specific fusion proteins secreted by X63-Ag8.653 cells. 60 Digestion of protein A-precipitated C D 4 5 R A B C : M u I g G and CD45R0 :Mu lgG with thrombin resulted in the appearance of a 34 kDa band upon western blot analysis wi th anti-muIgG. As two bands are observed in the lanes treated wi th thrombin, one at the apparent molecular weight previously observed for the isoform-specific fusion protein and the second band at 34 kDa, thrombin cleavage was not 100% complete. Removal of the immunoglobu l in port ion may be necessary dur ing l igand identif ication. For example, macrophages express Fc receptors which can non-specif icial ly b ind to the Ig portion of fusion proteins, thus a l lowing cellular interactions to occur that are not specific to CD45. 4.0.3 Analysis of O-l inked Glycosylation The addit ion of O-l inked sugars to CD45:MuIgG was analyzed by digestion wi th O-glycosidase (Figure 17). Prior to treatment wi th Diplococcus pneumoniae O-glycosidase, terminal sialic acid residues were removed by digestion wi th Vibrio cholerae neuraminidase, thus al lowing for optimal cleavage of O- l inked sugars by O-glycosidase. Upon digestion with O-glycosidase, a decrease in apparent molecular weight of 10-12 kDa was observed for CD45R0:MuIgG expressed by both Cos 7 and X63-Ag8.653 cells, as visualized by precipitation with protein A and western blotting w i th an t i -muIgG. The pred ic ted molecu la r we ight of ung lycosy la ted CD45R0:MuIgG is 34 kDa. In X63-Ag8.653 cells, the observed 50 kDa fusion protein is comprised of 5 kDa N- l inked carbohydrate (as determined by PNGase F digestion) and 11 kDa O-l inked carbohydrate (as determined by O-glycosidase digestion) presumably attached to the predicted 34 kDa protein backbone. The predicted molecular weight of unglycosylated CD45RABC:MuIgG is 50 kDa. Upon comparing untreated and O-glycosidase treated samples, a decrease in apparent molecular weight of 30 kDa was observed for C D 4 5 R A B C : M u I g G in X63-Ag8.653 cells. A sma l le r decrease in apparent mo lecu la r we igh t of 14 k D a was 61 CD45R0 CD45RABC O-glycosidase: — +— + — + — + 175 — Figure 17: O-Glycosidase Treatment of CD45-Immunoglobul in Fusion Proteins. 50-200 ul of cell culture supernatant was precipitated with 20 ul of a 10% solution of Pansorbin cells. The sample was digested with 1 mUnit of neuraminidase for 4 hours at 37°C followed by digestion with 1 mUnit of O-glycosidase for 20 hours at 37°C / subjected to 7.5% SDS-PAGE and western blotting with 1/5000 G A M I g G - H R P . +, treated samples; -, untreated samples. Leftmost two lanes, isoform-specific fusion proteins secreted by Cos 7 cells; rightmost 2 lanes, isoform-specific fusion proteins secreted by X63-Ag8.653 cells. 62 observed for CD45RABC:MuIgG secreted by Cos 7 cells upon O-glycosidase digestion. 4.0.4 Characterization of Carbohydrate residues by Lectin Binding The identity of specific carbohydrate residues on CD45RABC:MuIgG and CD45R0:MuIgG expressed by both Cos 7 and X63-Ag8.653 cells was determined by precipitation of culture supernatant with protein A followed by blotting membranes with various biotinylated sugar residue-specific lectins and detection with streptavidin-HRP (Figure 18). A summary of observed lectin reactivity of CD45:MuIgG is compiled in Table VII. Two isoforms of CD45 were analyzed: the highest molecular weight form containing alternatively spliced exons A , B, and C, and the lowest molecular weight isoform that does not contain the alternatively spliced exons. As exons A , B, and C contain multiple sites for addition of O-linked sugars, the two isoforms indicated are predicted to have differing lectin reactivity. In addition, lectin reactivity may be different between fusion proteins expressed by different cell lines. If ligand binding is dependent on specific carbohydrate residues on CD45, differing lectin reactivity could provide insight into the identity of an isoform-specific ligand. The strong reaction of both fusion proteins with anti-muIgG serves as a control ensuring that relatively equal amounts of fusion protein are present for subsequent lectin analysis. Wheat Germ Agglutinin (WGA) reacted with CD45RABC:MuIgG expressed by Cos 7 cells but not with the same fusion protein expressed by X63-Ag8.653 cells. Vicia villosa (VVA) lectin did not react with either fusion protein expressed by either cell line. Ricinus communis 120 (RCA) reacted strongly with CD45RABC:MuIgG expressed by both Cos 7 and X63-Ag8.653 cells. In addition, CD45R0:MuIgG expressed by X63-Ag8.653 cells reacted moderately with RCA. Concanavilin A (Con A) reacted faintly with CD45RABC:MuIgG expressed by X63-Ag8.653 cells but did not react with other fusion proteins secreted by Cos 7 cells. 63 A n t i M u l g G RO R A B C Cos Ag8 Cos Ag8 C 175" 83. 62-47" W G A RO R A B C Cos Ag8 Cos Ag8 C 175" V V A R 0 R A B C Cos Ag8 Cos Ag8 c 175 — Figure 18: Reactivity of CD45 - Immunoglobul in Fusion Proteins wi th Var ious Lectins. CD45:MuIgG fusion proteins were precipitated from 100-500 ul of cell culture supernatant using 20 ul of a 10% solution of protein A producing Pansorbin cells followed by 7.5% SDS-PAGE, lectin blotting, and detection with Streptavidin-HRP. Page 1, top to bottom: Anti MulgG, 1/5000 GAMIgG-HRP; 0.5 ug WGA; 0.5 ug V V A . Page 2, top to bottom: 0.5 ug RCA, 0.5 ug Concanavalin A , 3 ug SNA, 0.5 ug M A A . C, positive control - 3 X 106 T28 cells lysed in 20 ul 1% Triton/TNE. 64 RCA 175— 83 — 62— 47— RO RABC Cos Ag8 Cos Ag8 C Con A SNA MAA RO RABC Cos Ag8 Cos Ag8 C 175— 83 — 62 — 47— RO RABC Cos Ag8 Cos Ag8 C 175— 83— 62 — 47— RO RABC C Cos Ag8 Cos Ag8 175 — 83 — 62— 47— 5 • 65 Table VII Lectin Specif icity Cos 7 X63-Ag8.653 C R O R A B C R O R A B C P N A (3 gal(l,3)galNAc (O linked) +/- ++ ++ ++++ R C A term (3 gal(l,4)glcNAc (N linked) +++ + ++ ++++ V V A Con A term a galNAc a man, a glc ++++ +/- ++++ W G A glcNAc a neuNAc +++ ++++ S N A M A A a(2,6) sialic acid (N linked, O linked) a(2,3) sialic acid (N linked) +/-+ ++++ ++ + ++++ Table VII : Reactivity of CD45Tmmunoglobu l in Isoform Fusion Proteins w i th Var ious Lectins. CD45:MuIgG fusion proteins were precipitated from 100-500 ul of culture supernatant using 20 ul of a 10% solution of protein A producing Pansorbin cells. The entire sample was subjected to 7.5% SDS-PAGE and transfer to Immobilon P membrane. The lectins are as follows: RCA, Ricinus communis 120; P N A , Arachis hypogaea (Peanut agglutinin); V V A , Vicia villosa; Con A, Concanavalin A; W G A , Triticum vulgaris (Wheat germ agglutinin); SNA, Sambucus nigra; M A A , Maackia amurensis.. Signal strength is as follows: C,++++, positive control; +++, very strong reaction; ++, strong reaction; +, moderate reaction; +/-, faint reaction; -, no reaction. Lectin specificity reflects carbohydrate residues commonly found on N -linked or O-linked chains as indicated. 66 Incubation with Sambucus nigra (SNA) resulted in a moderate reaction with CD45RABC:MuIgG expressed by X63-Ag8.653 cells although SNA did not react with CD45R0:MuIgG expressed by X63-Ag8.653 or either fusion protein expressed by Cos 7 cells. Finally, Maackia amurensis (MAA) reacted strongly with CD45R0:MulgG secreted by X63-Ag8.653 cells, moderately with CD45RABC:MuIgG secreted by X63-Ag8.653 cells, faintly with CD45RABC:MuIgG secreted by Cos 7 cells and not at all with CD45R0:MuIgG secreted by Cos 7 cells. These results indicate that the same isoform of CD45 can be glycosylated differently in different cell types, possibly due to cell-specific expression of certain glycotransferases. 4.0.5 Determination of Peanut Lectin Reactivity in the Absence of Sialic Acid The reactivity of Arachis hypogaea Peanut Lectin (PNA) with CD45:MuIgG in the presence and absence of sialic acid was determined (Figure 19). The presence of sialic acid on the end of a.carbohydrate chain prevents P N A from binding as this lectin binds to galactose residues beneath sialic acid on O-linked chains. Removal of sialic acid wil l therefore increase P N A reactivity, thus providing a method to quantitate the relative amount of sialic acid present. Panel A of Figure 19 shows reactivity of CD45R0:MuIgG and CD45RABC:MuIgG secreted by both Cos 7 and X63-Ag8.653 cells with anti-muIgG. This experiment demonstrated that equal amounts of fusion protein were present for subsequent experiments. The reactivity of CD45:MuIgG with P N A lectin is shown in Panel B of Figure 19. This experiment serves to show the initial level of P N A reactivity of fusion proteins before the removal of sialic acid by neuraminidase (Vibrio cholerae) digestion. Incubation of CD45R0:MuIgG expressed by either cell line with neuraminidase did not affect PNA reactivity (Figure 19, Panel C). In contrast, digestion of CD45RABC:MuIgG secreted by Cos 7 cells with neuraminidase significantly increased P N A reactivity, while similar incubation of CD45RABC:MuIgG expressed by X63-Ag8.653 cells increased 67 Cos Ag8 Cos Ag8 Neur: — + — + Neur: — + — + 47— Cos Ag8 Cos Ag8 Neur: — + — + — + — + 175 — 83 — 62-47-Figure 19: Peanut Agg lu t in in and RA3 6B2 Reactivity of Neuraminidase Treated CD45- Immunoglobul in Fusion Proteins. CD45:MuIgG fusion proteins were precipitated from 100-500 ul of cell culture supernatant using 20 ul of a 10% solution of protein A producing Pansorbin cells followed by treatment with 1 mUnit of Neuraminidase for one hour at 37°C and electrophoresis on 7.5% SDS-PAGE. A, CD45R0:MuIgG and CD45RABC:MuIgG expressed by Cos 7 and X63-Ag8.653 cells blotted with 1/5000 GAMIgG-HRP; B, 0.5 ug Arachis hypogaea (Peanut agglutinin); C, PNA reactivity of neuraminidase treated CD45R0:MuIgG; D, P N A reactivity of neuraminidase treated CD45RABC:MuIgG; E, RA3 6B2 reactivity of neuraminidase treated CD45R0:MuIgG and CD45RABC:MuIgG. 68 P N A reactivity, but to a much lesser extent (Figure 19, Panel D). Neuraminidase treatment of CD45:MuIgG did not affect subsequent RA3 6B2 reactivity of fusion proteins secreted by either cell line (Figure 19, Panel E). As sialic acid residues have been shown to be instrumental in the binding of CD22 to endogenous CD45, it is possible that sialic acid residues on CD45:Ig fusion proteins may function in a similar manner. 4.0.6 Apparent Molecular Weight in Non-reducing SDS-PAGE Conditions The apparent molecular weight of CD45:MuIgG was determined in both reducing and non-reducing SDS-PAGE conditions (Figure 20). In the presence of the reducing agent, DTT, protein A-precipitated fusion proteins migrated to the apparent molecular weights previously described (Figure 8, Figure 11, Table III). However, in the absence of DTT, fusion proteins expressed by both Cos 7 and X63-Ag8.653 cells were observed to have significantly higher apparent molecular weights of 120 kDa for CD45R0:MuIgG and approximately 240 kDa for CD45RABC:MuIgG, thus indicating dimer formation. In addition, a band at approximately 190 kDa was observed for CD45R0:MuIgG secreted by Cos 7 cells, indicated the possible formation of a multimer. 69 Figure 20: S D S - P A G E Analys is of CD45-Immunoglobul in Fusion Proteins in Reducing and Non-redticing Condit ions. 100-500 ul of cell culture supernatant was precipitated with 20 ul of a 10% solution of protein A producing Pansorbin cells. Samples were boiled in 1 X SDS-PAGE sample buffer in the presence or absence of 0.1 M Dithiothreitol (DTT) followed by electrophoresis on 7.5% SDS-PAGE and western blotting with 1/5000 GAMIgG-HRP Leftmost two lanes, isoform-specific fusion proteins secreted by Cos 7 cells; rightmost 2 lanes, isoform-specific fusion proteins secreted by X63-Ag8.653 cells. 70 DISCUSSION Creation of murine CD45-lmmunoglobulin Fusion Constructs At the start of this work, previous attempts at expression of human CD45-immunoglobulin constructs with the native CD45 signal sequence were unsuccessful. To try to improve expression levels, two things were changed: the signal sequence and the expression vector. The Onco M signal sequence has been used successfully by other groups to mediate the secretion of human IgG fusion proteins of the B lymphocyte activation antigen B7, its counter receptor CD28, on T lymphocytes, CD5 [39] as well as CTLA-4 [54]. The inclusion of the Onco M signal sequence in place of the native CD45 signal sequence increased the expression of human CD45-immunoglobulin fusion proteins by over 10 fold. As a result, the Onco M signal sequence was included in fusion constructs consisting of different isoforms of murine CD45 linked to the hinge, CH2 and CH3 regions of the murine IgG2a heavy chain. Polymerase Chain Reaction is an extremely useful method for the exponential amplification of small fragments of D N A ; By using oligonucleotide primers complementary to the ends of the desired sequence, a specific segment of D N A can be targeted and amplified for further analysis. In this body of work, the creation of murine CD45 isoform-specific inserts by PCR for subcloning into the modified Ig vector, 1 ng of plasmid D N A template was amplified to approximately 2 ug of final product - an efficient increase of over 2000 fold. The presence of non-specific bands upon agarose gel electrophoresis of PCR products was due to binding of oligonucleotide primers to non-complementary sequences, resulting in the extension and subsequent amplification of an alternate portion of the D N A strand. Annealing of oligonucleotide primers is optimal at the melting temperature ( T m ) of the primer. Tm can be calculated for oligonucleotide primers shorter than 18 71 nucleotides by allowing 2°C for each adenine or thymine and 4°C for each cytosine or guanine. For longer primers, an equation based on guanine/cytosine content and ionic strength can be used to get an more accurate value [55]. As primer annealing does not occur at temperatures above the calculated T m , hybridization reactions should be carried out under stringent conditions - typically 5-10°C below the calculated T m . To eliminate non-specific bands, the annealing temperature should be increased to the highest temperature possible without going above the calculated Tm-The occurrence of smearing and apparent degradation of some isoform-specific PCR products was due to two factors. Firstly, the 30 second extension time at 72°C using Vent D N A polymerase (New England Biolabs) may have been too long. To minimize smearing, manufacturers instructions suggest using an extension time correlating to the expected length of the final product: 1 minute for every 1000 base pairs of DNA. For CD45RA and CD45RB, the predicted size of the final products was 280 bp and 300 bp respectively. Therefore, the optimal extension time for CD45RA was 17 seconds and for CD45RB, 18 seconds. Alternatively, the smear extending down from the agarose gel well commonly indicates that a particular reagent or condition is in excess. Therefore, using less enzyme, primer, or template may have reduced smearing. Ligation of muCD45 isoform-specific inserts into the modified Ig vector containing the Onco M signal sequence required the correct restriction enzyme-generated overlapping ends. Digestion of PCR inserts with Sph 1 was inefficient for two reasons. Firstly, the half life of activity for Sph 1 is approximately one hour. Secondly, Sph 1 digests at the end of D N A strands with a very low efficiency: 25% in 20 hours when there are 8 nucleotides (nt) 5' to the cleavage site, 10% in 2 hours or 50% in 20 hours when there are 9 nt 5' to the cleavage site. Taken together, these statistics indicated that Sph 1 was not an efficient enzyme for creation of a restriction enzyme site at the end of a PCR fragment. Blunt end ligation, end to end, of PCR 72 products allowed for creation of multimers that could then be more efficiently digested with Sph 1 to create the required subcloning site. Expression of murine CD45-Immunoglobulin Fusion Proteins Cos 7 cells, a non-hematopoietic monkey kidney cell line, were used as an initial expression system to determine if murine CD45 isoform-specific fusion proteins could be secreted into the culture supernatant at levels high enough to be 1) detected by precipitation with protein A and western blot analysis and 2) purified in larger amounts by protein A affinity chromatography. If muCD45:MuIgG fusion proteins were expressed by Cos 7 cells, it would be reasonable to hypothesize that expression in more functionally relevant myeloid and lymphoid cell lines would also be successful. Transient expression of five isoforms of CD45 linked to murine heavy chain constant regions was obtained in Cos 7 cells by the DEAE-dextran method. In addition, stable expression in Cos 7 cells of three isoform-specific constructs was obtained by calcium phosphate transfection. Both DEAE-dextran and calcium phosphate transfection are highly efficient and widely used methods for the introduction of plasmid D N A into eucaryotic cells [55]. Although the exact mechanism remains obscure, the D N A is thought to enter the cell by endocytosis followed by transport to the nucleus. While DEAE-dextran transfection is used only for transient expression, the calcium phosphate method can be used for both transient expression and stable integration of D N A into the eucaryotic genome. Although repeated attempts were made, transient and stable expression of one isoform of muCD45, CD45RC, was not observed. While all other isoform constructs in pBluescript were 100% confirmed by D N A sequencing prior to subcloning into the mammalian expression vector, pBCMGSneo, 8/300 nt of CD45RC could not be confirmed due to compression of guanine/cytosine rich 73 sequences resulting in unreadable results with respect to the nitrogenous base present at that position of the D N A sequence. Hence, the D N A sequence of CD45RC was only 97% confirmed. It is plausible that one or more of the unconfirmed nucleotides was incorrect, resulting in the substitution of a different amino acid residue that then acted to prevent subsequent expression of the fusion protein. In addition, it is possible that the unconfirmed sequence contained the incorrect number of nucleotides, resulting in the downstream protein being out of frame and subsequently being translated into an entirely different protein that could not be detected by the methods employed. The apparent molecular weight of CD45:MuIgG fusion proteins secreted by Cos 7 cells as determined by precipitation with protein A and western blotting with anti-muIgG was significantly higher than the predicted molecular weight of unglycosylated protein (Table III). In Cos 7 cells, the difference between predicted and apparent molecular weight is 21 kDa for CD45R0:MuIgG and 75 kDa for CD45RABC:MuIgG. As each alternatively spliced exon consists of approximately 50 amino acids, the addition of all three exons would increase the apparent molecular weight by 18 kDa. Thus, the remaining 57 kDa observed for CD45RABC:MuIgG can be explained by the fact that exons A, B, and C contain multiple sites for O-linked carbohydrate addition, which upon addition, would result in a further increase in apparent molecular weight. The appearance of lower molecular weight bands in lysates of Cos 7 cells can be readily explained. The observed bands react with the anti-muIgG antibody, suggesting they contain the heavy chain constant regions of murine IgG. Hence, the lower molecular weight proteins may be CD45:MuIgG fusion proteins that are not yet fully processed by addition of carbohydrate in the endoplasmic reticulum and golgi apparatus. The lack of complete glycosylation accounted for the lower apparent molecular weight and inability to be secreted into the culture supernatant. Although the lower molecular weight proteins did not react with anti-CD45 exon A-74 specific 14.8 and exon B-specific MB 4B4 and 23 G2, purified exon C-specific DNL1.9 did react with proteins in the lysates, implying that exon A and B-specific antibodies may recognize a combination of protein sequence and carbohydrate residues whereas DNL1.9 may recognize protein sequence only. Related evidence from isoforms of human CD45 expressed in Escherichia coli as unglycosylated glutathione-S-transferase (GST) fusion proteins also suggested that antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide sequence but influenced by glycosylation[56]. For example, in this study, the monoclonal antibody MRC OX22, which recognizes the product of exon C in the rat, was shown to have significantly higher binding affinity to glycosylated CD45 from spleen than to unglycosylated CD45-GST fusion proteins produced in E. coli. Expression of CD45:MuIgG by Cos 7 cells incubated in D M E M / 1 0 % FCS resulted in the appearance of three anti-IgG antibody unreactive protein bands at 55 kDa, 65 kDa and > 175 kDa upon precipitation with protein A and coomassie brilliant blue staining. The contaminating proteins were components of fetal calf serum, as incubation of CD45R0:MuIgG and CD45RABC:MuIgG in serum free hybridoma media resulted in fusion proteins free of contaminating bands at the previously observed molecular weights. Scanning densitometry revealed that approximately 1.5-2.0 ug of CD45RABC:MuIgG and 3.0-4.0 ug CD45R0:MuIgG was secreted per ml of serum free media (1 X 106 cells in 10 ml media) over a three day period. The fusion protein containing the smallest molecular weight form of CD45, CD45R0, was secreted at a higher concentration possibly due to the decreased requirement for addition of carbohydrate residues during processing. As CD45R0:MuIgG lacks alternatively spliced exons A, B, and C, CD45R0:MuIgG fusion proteins contain only 18 potential sites for O-linked sugar addition and 1 site (on Ig portion) for N-linked sugar addition. Therefore, the time required for processing and secretion of CD45R0:MuIgG would be less than that required for CD45RABC:MuIgG, which contains 57 potential O-linked glycosylation sites and 3 75 potential N-linked glycosylation sites. The yield of 1.5-4.0 ug/ml is comparable to that observed by other groups performing similar experiments: B7Ig and CTLA-4Ig fusion proteins (both containing the Onco M signal sequence) were secreted by 1 X 10 6 Cos 7 cells at 1.5-4.5 ug/ml over a three day period [39, 54] whereas CD44Ig accumulated in Cos 7 cell supernatants at 0.5 ug/ml 7 days post-transfection [53]. Once it had been determined that CD45:MuIgG could be expressed at reasonable levels by Cos 7 cells, the focus of subsequent experiments was to express fusion proteins in the myeloma cell line, X63-Ag8.653, and the T lymphoma cell line, T28. As observed with Cos 7 cells, fusion proteins secreted by X63-Ag8.653 and T28 cells had apparent molecular weights significantly higher than the predicted protein size, suggesting extensive post-translational modifications via carbohydrate addition. The apparent molecular weight of CD45RABC:MuIgG, CD45RB:MuIgG, and CD45R0:MuIgG was generally 5-10 kDa lower than the equivalent fusion protein expressed in Cos 7 cells (Table III). One explanation for this result could be increased addition of carbohydrate residues by Cos 7 cells when compared to the sugars added by X63-Ag8.653 and T28 cells, resulting in the formation of a different glycosylation pattern. In addition, this result suggests that expression of the same protein by different cell lines can result in proteins with differing carbohydrate patterns which then may, in turn, affect subsequent ligand interactions. Therefore, the importance of expressing CD45:MuIgG fusion proteins in more than one cell line becomes apparent in order to identify all potential ligand interactions due to the presence or absence of a particular carbohydrate residue(s). The yield of CD45:MuIgG obtained in X63-Ag8.653 cells was not significantly different from that observed in Cos 7 cells and, as previously described, is in the range of concentration observed by other groups performing similar experiments. Transient expression of CD45:MuIgG was observed in T28 cells. The apparent molecular weights of fusion proteins were comparable to that obtained by expression in X63-Ag8.653 cells (Table III). Unfortunately, the level of fusion protein 76 expression by T28 cells decreased steadily over a period of three days, eventually leading to undetectable levels by precipitation with protein A and subsequent western blotting with anti-muIgG. The decrease in fusion protein expression may have occurred because fusion protein-secreting clones were contaminated with non-fusion protein-secreting cells. As untransfected T28 cells grow faster than transfected cells, the non-expressing cells eventually became the dominant cell type in the population. Possible ways to prevent this problem in future experiments may be to further increase the already high concentration of active G418 used for selection, thus allowing for complete destruction of cells that do not carry the gene for neomycin resistance. Alternatively, increasing the voltage and/or capacitance used for electroportation may result in a cell population in which neomycin sensitive cells are more stressed, thus more easily killed. As T28 cells appear to have a higher growth rate and faster recovery from electric shock than other T cell lines, electroportation of half the number of cells per sample (5 X 106 cells rather than 1 X 107) followed by selection of neomycin resistance by addition of G418 at 24 hours rather than 48 hours, may increase the percentage of fusion protein-secreting cells in the population and prevent neomycin sensitive cells from further propagating. Characterization of Expressed CD45-Immunoglobulin Fusion Proteins Although an isoform-specific ligand for CD45 has not yet been identified, one theory postulates that specific carbohydrate residues on CD45 mediate the interaction(s) with a putative ligand(s). Therefore, if carbohydrate residues are important, it is critical that CD45:MuIgG fusion proteins be correctly glycosylated so that they express the necessary sugars required for ligand binding. As endogenous CD45 is expressed on the surface of all nucleated hematopoietic cells, the glycosylation pattern of fusion proteins expressed by lymphoid cells would probably 77 be closer to that of endogenous CD45 than that of fusion proteins expressed by non-hematopoietic Cos 7 cells. The reactivity of secreted CD45:MuIgG fusion proteins with various murine CD45-specific antibodies was analyzed. A l l fusion proteins, expressed transiently or as stable clones by all three cell lines, reacted with an antibody recognizing the murine IgG heavy chain, suggesting that all fusion proteins contain the predicted heavy chain regions. At the transient expression level in Cos 7 cells, all isoform-specific fusion proteins reacted with anti-CD45 exon-specific antibodies as predicted with the exception of RA3 6B2, a B220 isoform-specific antibody observed to react not only with CD45RABC:MuIgG, but also with CD45RA:MuIgG (Figure 7, Panel B). This suggests that although RA3 6B2 can bind to the largest isoform of CD45 containing all three alternatively spliced exons, its reactivity is dependent on the presence of exon A. This is the first demonstration that RA3 6B2 reactivity is dependent on the expression of exon A. In addition, as RA3 6B2 did not react with lower molecular weight proteins in lysates of Cos 7 cells, the formation of the RA3 6B2 epitope may possibly depend on carbohydrate modifications of exon A. Interestingly, RA3 6B2 reacted with stable clones in Cos 7 cells secreting CD45RABC:MuIgG but did not react with stable CD45RABC:MuIgG clones in X63-Ag8.653 cells or transient clones in T28 cells. The difference in the binding of the B220-isoform-specific antibody may be due to differences in carbohydrate addition between Cos 7 versus X63-Ag8.653 and T28 cells. It is plausible that differential carbohydrate modification of fusion proteins expressed by different cell lines may contribute to the presence or absence of different epitopes for antibody binding. In addition, the fact that fusion proteins expressed by Cos 7 cells had a slightly higher apparent molecular weight than those secreted by X63-Ag8.653 or T28 cells, may correlate with additional glycosylation and the appearance of RA3 6B2 reactivity. Analysis of apparent molecular weight suggests that CD45:MuIgG fusion proteins were extensively glycosylated. Hence, it was important to characterize the 78 carbohydrate residues expressed by the fusion proteins secreted by different cell lines. Fusion proteins containing CD45RABC are more highly modified, presumably by addition of carbohydrate, than those containing CD45R0. Removal of asparagine (N) -linked glycan chains by PNGase F digestion followed by SDS-PAGE and western blot analysis with anti-muIgG (Figure 15) revealed a small reduction in apparent molecular weight of approximately 5 kDa. Interestingly, approximately equal decreases in apparent molecular weight upon PNGase F digestion for both CD45R0 and CD45RABC Ig fusion proteins suggests that the N-linked sugars present must be located on a common portion of the fusion proteins. This common region could be one of two parts of the fusion protein: exons 3, 7, and 8 of CD45, or the heavy chain regions of muIgG 2a- As exons 3, 7, and 8 of CD45 lack any potential N-linked glycosylation sites conforming to the required sequence motif - Asn-X-Ser/Thr where X is any amino acid except proline [57] - N-linked glycosylation wil l not occur in this region. Therefore, N-linked sugars present are likely located on the murine IgG portion of the fusion protein. Indeed, there is a conserved site between all isotypes of IgG molecules (Asn 297) for potential N-l inked sugar addition conforming to the required motif [58]. The murine IgG portion was shown to be 34 kDa by thrombin cleavage (unglycosylated protein predicted to be 28 kDa). The size of the IgG portion did not vary between isoforms of CD45 suggesting that the observed differences in apparent molecular weight between fusion proteins containing different isoforms of CD45 were due solely to differential post-translational modifications of CD45. In addition, the size of the IgG portion was the same in fusion proteins expressed by Cos 7 cells and X63-Ag8.653 cells, suggesting that the IgG region was similarly processed by these two cell lines prior to secretion. As it has been determined that there is 5 kDa of N-linked glycosylation (out of a total of 16-21 kDa for CD45R0 and 60-75 kDa for CD45RABC in X63-Ag8.653 cells and Cos 7 cells respectively), the majority of apparent carbohydrate must be O-linked to serine or threonine. Digestion of CD45R0:MuIgG expressed in both Cos 7 cells and 79 X63-Ag8.653 cells with O-glycosidase revealed a decrease in apparent molecular weight of approximately 10-12 kDa. The predicted molecular weight of CD45R0:MuIgG is 34 kDa, so the addition of 5 kDa of N-linked carbohydrate and 11 kDa of O-linked carbohydrate by X63-Ag8.653 cells results in the apparent molecular weight observed - 50 kDa- by SDS-PAGE analysis and western blotting. On the other hand, digestion of CD45RABC:MuIgG expressed by both Cos 7 and X63-Ag8.653 cells did not appear to be complete. A decrease in apparent molecular weight of 30 kDa for CD45RABC:MuIgG in X63-Ag8.653 cells and 14 kDa for CD45RABC:MuIgG in Cos 7 cells was observed upon O-glycosidase digestion. These numbers do not account for the total amount of O-linked glycosylation expected. However, it is possible that O-glycosidase digestion of CD45RABC:MuIgG was not complete due to the presence of terminal sialic acid resides, which act to prevent cleavage by O-glycosidase. It has been determined that fusion proteins expressed by different cell lines have slightly differing apparent molecular weights and antibody reactivity. In addition, it has been suggested that fusion proteins expressed by different cell lines may have differential carbohydrate modifications. Therefore, in order to determine the identity of specific carbohydrate residues, each fusion protein was tested for reactivity with various sugar residue-specific lectins (Figure 18). The results are summarized in Table VII. In general, it was determined that fusion proteins expressed by different cell lines but expressing the same isoform of CD45 had different lectin binding properties. In some cases, lectin reactivity was different between CD45RABC:MuIgG and CD45R0:MuIgG expressed by the same cell line. Lack of binding to Vicia villosa (VVA) lectin and Concanavalin A (Con A) revealed the absence of a-linked N-acetygalactosamine, mannose, and glucose on all fusion proteins. The lack of mannose can be explained by the fact that high mannose structures are commonly associated with N-linked sugar chains, which were shown by PNGase F digestion to contribute to just 6% of total post-translational modifications of CD45RABC:MuIgG. In addition, two glucose residues 80 are often removed from precursor N-linked chains to allow for association with calnexin, a protein found in the endoplasmic reticulum (ER) that functions to prevent incompletely folded or misfolded glycoproteins from further processing [59]. Calnexin binds to monoglucosylated, incorrectly folded glycoproteins in the ER, thus preventing further transport to the cell surface. Removal of the final glucose residue from correctly folded glycoproteins is required for release of calnexin and subsequent downstream processing. Ricinus communis 120 (RCA) is a group II galactose-specific phytohemagglutinin that commonly binds to carbohydrate chains containing terminal N-linked galactose linked (3-1,4 to N-acetyglucosamine [60]. Peanut agglutinin (PNA) is a group I lectin that commonly binds carbohydrate chains containing O-linked galactose linked (3-1,3 to N-acetylgalactosamine. RCA reactivity of CD45:MuIgG fusion proteins expressed by Cos 7 cells was higher than P N A reactivity, suggesting that the majority of N-linked chains end in galactose whereas the majority of O-linked chains have an additional sugar present that acts to mask galactose-specific P N A reactivity. O-linked carbohydrate chains commonly have N -acetylneuraminic acid or sialic acid as the ultimate residue. Lectins that have been previously shown to recognize a-2,6 and a-2,3 N-linked sialic acid, SNA and M A A , [50, 51] did not react significantly with fusion protein expressed by Cos 7 cells, confirming previous conclusions from RCA and P N A data that suggest that the majority of N-linked chains on CD45:MuIgG fusion proteins expressed by Cos 7 cells end in a galactose residue. Unfortunately, a lectin that binds with high affinity to O-linked sialic acid could not be found in order to further confirm the outlined conclusions. Sialic acids often act as to mask antigenic sites. For example, the surfaces of trophoblast cells are rich in sialic acids which are thought to serve as an immunobarrier between mother and embryo [14]. Partial loss of this barrier has been proposed as one of the causes of autoimmune disease. In a similar manner, 81 the presence of terminal sialic acid residues on CD45:MuIgG fusion proteins acts to mask reactivity of galactose-specific peanut agglutinin (PNA). Therefore, removal of sialic acid by digestion with neuraminidase (sialidase) wil l expose galactose residues that wil l then react with PNA. Digestion of CD45R0:MuIgG expressed in Cos 7 or X63-Ag8.653 cells with neuraminidase did not result in an increase in subsequent P N A reactivity, suggesting that very little sialic acid was added to CD45R0 during processing and secretion (Figure 19). On the other hand, digestion of CD45RABC:MuIgG expressed by Cos 7 cells with neuraminidase resulted in a significant increase in subsequent P N A reactivity while digestion of CD45RABC:MuIgG expressed by X63-Ag8.653 cells also resulted in an increase in PNA reactivity, but to a lesser extent than that observed with Cos 7 expressed fusion proteins. This data suggests that although sialic acid appears to be added to CD45RABC:MuIgG, a much larger amount is added by Cos 7 cells than X63-Ag8.653 cells. This data correlates with the previous observation that fusion proteins expressed by Cos 7 cells have a slightly higher apparent molecular weight. The increased addition of sialic acid can account for the observed increase in apparent molecular weight of fusion proteins expressed by Cos 7 cells. The next logical question to consider was whether or not the increased addition of sialic acid to fusion proteins expressed by Cos 7 cells accounts for the observed reactivity of B220 isoform-specific antibody RA3 6B2. Digestion of CD45RABC:MuIgG with Vibrio cholerae neuraminidase did not affect RA3 6B2 reactivity of fusion proteins expressed by either Cos 7 or X63-Ag8.653 cells (Figure 19). Hence, the binding of this antibody does not appear to depend on the presence of sialic acid. Vibrio cholerae neuraminidase is known to cleave cc-2,6, ot-2,3, and a-2,8 sialic acid residues so this enzyme should remove most residues from CD45. However, it is possible that some sites may be more accessible than others and thus it would be better in future experiments to metabolically label sialic acids and show that they are all removed by this treatment. However, it is clear from lectin analysis 82 that fusion proteins expressed by different cell lines have differential carbohydrate addition. It is likely that RA3 6B2 reactivity depends on the formation of an epitope containing sugars other than sialic acid. In agreement with RA3 6B2 reactivity, CD45RABC:MuIgG expressed by Cos 7 cells reacted with wheat germ agglutinin (WGA) whereas the equivalent fusion protein expressed by X63-Ag8.653 cells did not bind to WGA. Although the binding properties of W G A are complex, it is generally thought to bind to N-acetylglucosamine, poly-N-acetyl-lactosamine, and sialic acid residues[61]. Interestingly, carbohydrate structures on endogenous CD45 are thought to be rich in poly-N-acetyl-lactosamine [62] and N-acetylglucosamine. Whether the reactivity of RA3 6B2 correlates with the presence of N-acetylglucosamine and/or poly-N-acetyl-lactosamine on fusion proteins remains to be fully elucidated. Fusion proteins expressed by both Cos 7 and X63-Ag8.653 cells were observed to form dimers in non-reducing SDS-PAGE conditions. In the absence of dithiothreitol (DTT), a reducing agent known to break disulfide bonds between cysteine residues, CD45:MuIgG fusion proteins migrated to an apparent molecular weight of approximately two fold of that observed in the presence of DTT - 50-55 kDa in reducing conditions versus 120 kDa in non-reducing conditions for CD45R0:MuIgG whereas CD45RABC:MuIgG migrated to 110-125 kDa in reducing conditions versus approximately 240 kDa in non-reducing conditions. In X63-Ag8.653 cells, CD45R0:MuIgG was also expressed in a form with an apparent molecular weight of approximately 190 kDa, which is consistent with the formation of a multimer. The expression and characterization of isoform-specific CD45:MuIgG fusion proteins revealed the following observations. Firstly, the levels of CD45:MuIgG fusion protein expression was in the same range between Cos 7 and X63-Ag8.653 cells. The fusion protein containing the smallest isoform of CD45, CD45R0, was expressed approximately 2 fold more than the fusion protein containing the largest isoform of CD45, CD45RABC, which contains alternatively spliced exons A, B, and 83 C. The apparent molecular weight of CD45:MuIgG expressed by Cos 7 cells was 5-10 kDa higher than the equivalent fusion protein secreted by X63-Ag8.653 or T28 cells. A l l isoform-specific fusion proteins reacted with anti-CD45 exon-specific antibodies as predicted with the exception of RA3 6B2, which was observed to react with both transiently expressed CD45RABC:MuIgG and CD45RA:MuIgG. In addition, RA3 6B2 reacted with stable fusion proteins secreted by Cos 7 cells whereas this antibody did not react with fusion proteins secreted by X63-Ag8.653 or T28 cells. Isoform-specific fusion proteins are extensively O-glycosylated on serine and threonine residues. PNGase F digestion revealed that although minimal N-linked glycosylation was present, N-linked glycans that were present were most likely located on the murine IgG portion of the fusion protein, determined to be 34 kDa by thrombin cleavage. Lectin analysis of fusion proteins expressed by Cos 7 cells revealed that the majority of N-linked carbohydrate chains terminated in a galactose residue, whereas the majority of O-linked chains had been further modified by the addition of sialic acids. Removal of sialic acid by digestion with neuraminidase resulted in a significant increase in subsequent P N A reactivity of CD45RABC:MuIgG from Cos 7 cells whereas only a slight increase in P N A reactivity was observed for the equivalent fusion protein expressed by X63-Ag8.653 cells, suggesting that a larger amount of sialic acid was added to fusion proteins expressed by Cos 7 cells. The increased addition of sialic acid may account for the slightly higher apparent molecular weight but does not account for the differences observed in B220-specific RA3 6B2 reactivity. The work presented in this thesis has established the feasibility of generating isoform-specific CD45-immunoglobulin fusion proteins that can now be used in the search for an isoform-specific ligand for CD45. A CD45-immunoglobulin fusion construct has been developed which will allow for secretion of sufficient amounts of fusion protein for purification and subsequent use in ligand identification. It has been established that CD45-immunoglobulin fusion proteins can be easily purified in one step by precipitation of fusion proteins with protein-A-sepharose from cells 84 grown in serum free hybridoma media. In addition, it has been determined that the same fusion protein can be differentially glycosylated by expression in different cell lines. Moreover, the presence of carbohydrate residues on the protein backbone was shown to affect antibody reactivity, and therefore, could affect subsequent ligand interactions. Given the observations presented, it is now possible to produce large amounts of purified fusion protein from cells and use this fusion protein as a diagnostic tool in immunoadherence assays to identify isoform-specific ligand interactions of CD45. In particular, it would be interesting to determine if CD45-immunoglobulin fusion proteins expressed by different cell lines bind to the B cell specific molecule, CD22. 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