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The structure-activity relationship study of the N-terminal domain in desert locust ion transport peptide… Zhao, Ying 2000

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The structure-activity relationship study of the N-terminal domain in desert locust ion transport peptide (ITP) b y YING Z H A O Bachelor of Medicine, Beijing Medical University, P.R.China, 1989 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF Z O O L O G Y We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 2000 © Y i n g Zhao, 2000 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 ~Z<o & L& f The University of British Columbia Vancouver, Canada Date ~£eA)• 2% . DE-6 (2/88) ii A B S T R A C T : The ileum and rectum of locust hindgut constitute the reabsorptive part of the excretory system. They are functionally analogous to a proximal and distal parts of convoluted tubule of the mammalian kidney tubule, respectively. Ion transport peptide (ITP) purified from locust nervous corpus cardiacum (CC) has been shown to stimulate salt and water reabsorption and inhibit acid secretion in the ileum of Schistocerca gregaria . The primary structure of ITP deduced from its cDNA suggests that it is a 72 amino acid peptide with C-terminal amidation and three disulfide bonds. Both synthetic and expressed ITP mimic the biological activity of ITP purified from S.gregaria CC. It has been demonstrated that ITP is a true member of the CHH (Crustacean Hyperglycemic Hormone) family. This study examines the structure-activity relationship of the N-terminal domain of ITP. Two questions are addressed: 1. Is the N-terminal domain of ITP consisting of the first six amino acids (SFFDIQ) important to bioactivity? 2. Which amino acids in the N-terminal domain of ITP are essential for ITP binding to the receptor and/or activating the receptor? Using site-directed mutagenesis and voltage-clamped locust ileum as bioassay, I found the ITP N-terminal domain (SFFDIQ) is important for its bioactivity. Among the six amino acid of ITP N-terminus, Phe2 and Phe3 are essential for ITP binding to the receptor, and Phe2 is also important to receptor activation. The other four amino acids SI, D4,15, and Q6 didn't contribute to the ITP bioactivity, even D4 is a highly conserved amino acid. Post-translational modification of conversion L- to D- amino acid probably occurs at Phe2 and Phe3 to yield two ITP isomorphs. Mutations on the ITP N-terminal domain didn't interfere with the dibasic cleavage site in spite of its immediate proximity to the dibasic cleavage site. Mutant F2A has the potential to be ITP antagonist. Ill T A B L E O F C O N T E N T S Page Abstract ii Table of contents iii List of Figures vi List of Tables viii List of Abbreviations ix Acknowledgments xii INTRODUCTION: Locusts as pests and potential strategy for their control 1 Anatomy and physiology of locust excretory system 2 Regulation of the excretory system by the neuroendocrine system 8 ITP structure and function 12 ITP homologues 14 Synthetic ITP structure and bioactivity 16 ITP-L structure, distribution and function 18 Expressed ITP structure and bioactivity 19 ITP C-terminal mutation analysis 20 Objective of this thesis 20 MATERIALS A N D METHODS: Subcloning 23 Oligonucleotides synthesis 23 Mutation strategy 23 PCR conditions 23 iv DNA electrophoresis 26 LMP (low melt point) agarose electrophoresis 26 Preparation insert and vectors 26 Ligation and transformation 28 Plasmid extraction and purification 29 Kcl cell expression system 29 Tris-tricine-SDS-PAGE and Western blotting system 30 Tris-tricine-SDS-PAGE 30 Tank transfer system 30 Antibody production 31 E C L Western blotting protocol 31 Measurement of expressed peptide concentration 33 Bioassay on locust ilea 33 Animals 33 Bathing saline 33 Short-circuit current (Isc) measurement 33 Dose-response curve 34 Competitive inhibition test 36 RESULTS: Mutation DNA fragments and mutation DNA sequencing 37 Western blotting analysis of mutation peptides expressed in K c l cells 37 Bioassay 42 Competitive inhibition assay 47 DISCUSSION: ITP N-terminus is essential for its bioactivity 53 Two Phes in the N-terminus of ITP are important for ITP binding to the receptor, and Phe2 also is important for receptor activation 53 Phe2 and Phe3 of ITP affect post-translation modification, and D-Phe2 or D-Phe3 results in a minor isomorph of ITP 55 Conserved amino acid Asp4 is not important for ITP bioactivity 57 The N-terminal domain of ITP does not interfere with the dibasic cleavage site 58 The structure of ITP antagonist should contain the first six amino acids of ITP 58 REFERENCES 60 vi L I S T O F F I G U R E S Page Figure 1. Overview of the locust excretory system 3 Figure 2. Specific solute and fluid movement in locust excretory system 5 Figure 3. The model of ion transport across locust ileum and its control with ITP 7 Figure 4. A model for the regulation of excretory system by diuretic and antidiuretic factors 9 Figure 5. A partial cDNA of ITP encoded a complete reading frame for preproITP and the amino acids sequence 15 Figure 6. Comparison of ITP with its homologues of C H H family 17 Figure 7. Site-directed mutation strategy 25 Figure 8. The structure of expression vector pZOP2F 27 Figure 9. S. gregaria ITP and ITP-L amino acid sequence map and their specific antibodies : 32 Figure 10a. A diagram of the chambers to detect CI" active transport 35 Figure 10b. The simplified model of short-circuit current 35 Figure 11. Mutation DNA fragments for point mutation F2A • 38 Figure 12a. Western blot with N- l antibody comparing SynlTP with KcITP 41 Figure 12b. Western blot with C- l antibody comparing DS-ITP with KcITP 41 Figure 12c. Western blot with C- l antibody comparing FD-ITP with KcITP 41 Figure 13 a. Western blot of SI A compared with KcITP 44 Figure 13b. Western blot of F2A and 15A compared with KcITP 44 Figure 13c. Western blot of F3A compared with KcITP 44 Figure 13 d. Western blot of D4A and Q6A compared with KcITP 44 Vll Figure 14a. Comparing the dose-response curve of SI A with KcITP 46 Figure 14b. Comparing the dose-response curve of D4A with KcITP 46 Figure 14c. Comparing the dose-response curve of 15 A with KcITP 46 Figure 14d. Comparing the dose-response curve of Q6A with KcITP 46 Figure 15. Maximum AIsc in ilea stimulated with KcITP and different mutated ITP peptides expressed in Kc l cells 48 Figure 16a. Competitive inhibition assay for DS-ITP and FD-ITP 49 Figure 16b. Competitive inhibition assay for F2A and F3A at low concentration ratio....49 Figure 17a. Competitive inhibition assay for F2A at high concentration ratio 50 Figure 17b. Competitive inhibition assay for F3A at high concentration ratio 50 Vlll L I S T O F T A B L E S Table 1 List of primer sequences and corresponding melting temperatures 24 Table 2 Different mutations in ITP N-terminus and their DNA sequencing results 39 Table 3 Concentration of mutation peptides expressed in K c l cells 45 Table 4 Summary of competitive inhibition test 52 ix LIST O F A B B R E V I A T I O N S + -positive charge 5-HT -5-hydroxytryptamine or serotonin [iL -microliter A -deoxyadenosine aa -amino acid Ab -antibody APS -ammonium persulfate ATP -adenosine 5' triphosphate BacITP -wild type LTP expressed in Sf9 cell bp -base pair C-1 -C-terminal antibody (ITP specific) C-2 -C-terminal antibody (ITP-L specific) C -deoxycytidine cDNA -complimentary DNA CHH -crustacean hyperglycaemic hormones CNS -central nervous system CRP -corticotropin releasing factor CTSH -chloride transport stimulating hormone Da -daltons DDT -l,l'-(2,2,2-trichloroethlidene) bis (4-chlorobenzene); -1,1,1 -trichloro-2,2-bis(p-clorophenyl)ethane D H -diuretic hormone DNA -deoxyribonucleic acid dNTP -deoxynucleotide triphosphates DP -diuretic peptide X DMSO -dimethyl sulfoxide DS-ITP -domain swap ITP DTT -dithiothreitol EDTA -ethylenediamineteraacetic acid EtBr -ethidium bromide EtOH -ethanol FD-ITP -mutation ITP replacing F3D4 with A A G -deoxyguanosine HPLC -high pressure (performance) liquid chromatography iel -immediate-early 2 promoter Isc -short circuited current ITP -ion transport peptide ITP-L -ion transport peptide-long Kcl cell -Drosophila cell line KcITP -wild type ITP expressed in Kcl cells KcITP-L -wild type ITP-L expressed in Kc l cells kD -kilodaltons K L H -keyhole limpet hemocyanin LMP -low melt point MCS -multiple cloning site mg -milligrams min. -minute(s) raM -millimolar N-l -N-terminal antibody (recognizes both ITP and ITP-L) NAPS -U.B.C. Nucleic Acid/Protein Service (unit) NCC -nervus corporis cardiaci Nps -neuroparsins xi ORF -open reading frame PA -polyadenylation PAGE -polyacrylamide gel electrophoresis PBS -phosphate buffered saline PCR -polymerase chain reaction PEG 8000 -polyethylene glycol PI-NSC -pars intercerebralis-neurosecretory cells PL-NSC -pars lateralis-neurosecretory cells pmoles -picomoles RACE -rapid amplification of complementary ends RNA -ribonucleic acid RT -reverse transcriptase RT-PCR -reverse transcriptase-polymerase chain reaction ScglTP -Schistocerca gregaria ion transport peptide SDS -sodium dodecyl sulfate Sf9 -Spodopterafrugiperda cell culture SOG -subesophageal ganglia SynlTP -synthetic ITP T -deoxythymidine TBE -tris-borate and E D T A buffer TBS-T -tris buffered saline with tween-20 T E M E D -N,N,N' ,N' -tetra-methyl-ethylenediamine Tm -melting temperature VGF -ventral ganglia hormone VIH -vitellogenesis-inhibiting factor Xll A C K N O W L E D G M E N T S I thank Dr. John Phillips for making the effort to allow me to pursue this study. I also gratefully thank him for his encouragement, support, guidance, understanding and generosity throughout. I must thank Dr. Hugh Brock for his excellent supervision and valuable discussion throughout this study. I also appreciate his consideration and spirit which motivates me. Thanks to him for the use of his equipment. I must say that Joan Martin is my mentor. She helps me to grow step by step in Canada. I am always motivated by her enthusiasm, expertise, open mind and kindness. I appreciate our friendship and the happiness to work with her. I thank Dr. Linda Matsuuchi for her advice, enthusiasm and suggestions on this manuscript and the use of her facilities. I also thank Dr. Chris Arries for his advice in this study. I would like to thank Yong-Jun Wang for the excellent training he gave me to start my research life. I also respect his intelligence, persistent and enthusiasm to science. I am glad to thank Andris Macins for his wise suggestions, friendship and many interesting conversation. I miss our pot luck lunch time. I must thank Dr. Jacob Hodgson for his knowledgeable discussion and his willingness to help me. I also thanks Holly Skeleton and Connie Hodson for their friendship and warmheart. Thanks for the good time we shared. I thank Dr. Terry Crawtford and Mike Chen for their kind support and understanding. I would like to thank Dr. Vanessa Auld and Dr. John Gosline for joining my defense committee and their kind understanding. Lastly, I would like to thank Xue-Feng Wang for his understanding, knowledgeable discussion, patience and his strong support. 1 I N T R O D U C T I O N Locusts as pests and potential strategy for their control In species diversity, insects outnumber all other forms of life combined. On the one hand, we depend on insects to pollinate many of our crops and orchards. On the other hand, insects are carriers for many diseases and compete with human for food. The desert locust (Schistocerca gregaria), the migratory locust and related orthopteans are major agricultural pests throughout the world. Locust plagues are a threat to crops and grazing lands in Africa, the Middle East and southwestern Asia and are occasionally a major contributor to famine (Bennet, 1993). Pests are a major problem in both developed and developing countries. Chemical insecticides are the most popular method of pest control, but they create three serious problems: 1) a great increase in the resistance of pests to the chemicals; 2) the death of many beneficial insects due to the chemicals' nonspecific activity; and 3) pollution of the environment. As a result, the pest-specific biological control strategies are now favored (Krall and Wilps, 1994). One such biological control strategy is the use of insect-specific hormones or their analogues as control agents. Neuropeptides hormones have the potential for use in locust control, because 1) neuropeptides can be highly selective in their action; 2) they are active at extremely low concentration (10"9 to 10"15 M); 3) they can be easily engineered by gene technology; and 4) the cDNA message for their synthesis can presumably be delivered directly to the insect pests through vector systems such as group specific viruses (Kelly, 1990). For the desert locust, maintaining a relatively constant internal environment, both osmotic and ionic, is critical to survival in an environment where the availability of water and ions can fluctuate widely and dehydration is a frequent threat. Insects are particularly susceptible to dehydration, because of their high surface area-to-volume ratio (Lehmberg et al., 1993). Locust antidiuretic peptide (ITP) is a neuropeptide found in corpus cardiacum (CC), and stimulates salt and water reabsorption in the excretory system. 2 Delivery of an ITP-receptor antagonist to pest populations using host-specific viruses could enhance the rate of kill by causing severe dehydration of locusts. Anatomy and Physiology of Locust excretory system The excretory system of insects consists of the Malpighian tubules and the hindgut (Fig. 1). The Malpighian tubules produce primary urine, usually rich in KC1, but low in Na + . The composition and volume of this primary urine are modified in both the ileal and rectal segments of hindgut by selective reabsorption of fluid and some secretion (e.g. FT and NH 4 + ) . In producing an isosmotic primary urine, Malpighian tubules have a role analogous to that of the glomerulus of the vertebrate nephron (Phillips, 1981). They arise from the midgut at its junction with the hindgut and generally lie free within the main body cavity bathed in hemolymph (Coast, 1994). The insect excretory process is unusual in that the primary urine is formed by active KC1 secretion rather by filtration (Phillips 1981)(Fig. 2). Fluid movement in Malpighian tubules is driven by active secretion of K + , and sometimes also Na + (reviewed by Nicolson 1993, Beyenbach 1995). Cation movement across the luminal membrane occurs via H + / K + or H + /Na + antiports, the driving force being a proton gradient established by a V-type proton ATPase. Cations enter the cell basolaterally through K + and Na + channels, and via a bumetanide-sensitive Na+/K+/2C1" cotransporter, the latter bringing CI" into cells. Chloride movement across the luminal membrane is favored by the lumen positive potential, consequently, KC1 is usually the predominant solute and the concentrations of most other solutes in the secretion are low relative to blood levels (Maddrell, 1978). Insect tubules can also actively secrete selected toxic molecules (organic anions and cations, plant alkaloids, S042", Mg 2 + ) which may be prevalent in the diet and would otherwise move slowly across the tubule wall by diffusion (Maddrell, 1971, and 1978). Fluid secretion is under endocrine control. The anterior (ileum) and posterior (rectum) segments of locust hindgut constitute the reabsorptive part of the locust excretory system (Phillips and Audsely, 1995) (Fig. 2). The entire enteron is lined by a single epithelial cell layer and is surrounded by a 3 M a l p i g h i a n t u b u l e s F i g . l l o c u s t a n d i t s e x c r e t o r y s y s t e m . T h e M a l p i g h i a n t u b u l e s a n d h i n d g u t c o n s t i t u t e l o c u s t e x c r e t o r y s y s t e m o u t l i n e d b y t h e s q u a r e . 4 Fig. 2 Diagram of the ion transport in Malpighian tubules and the hindgut. The primary urine is produced in Malpighian tubules by active transport KCL The reaborption of this isosmotic primary urine is driven by the electrogenic CI" pump located in the apical membrane of the ileum and rectum. The elaborate intracellular sinuses and channels in the rectum permit the rectum to extract an hypo-osmotic absorbate to create a strongly hyper-osmotic urine. 5 6 basement membrane and a variously developed muscle layer. The epithelial cells are connected by tight junctions and function as a barrier as well as a selective absorptive epithelium. The structural differences between the ileum and the rectum reflect their different physiological roles. The locust ileum is about a third as thick as the locust rectum (Irvine et al., 1988). Ileal reabsorption reduces volume of the urine without changing osmolarity, thus activities of the locust ileum are functionally analogous to those of proximal tubules of mammalian kidneys (review by Phillips, 1994). In the rectum, there are elaborate intercellular sinuses and channels that are absent in the ileum (Phillips, 1986). These elaborate intercellular sinuses permit the rectum to extract an hypo-osmotic absorbate to concentrate the lumen content to final osmotic concentrations several times that of the haemolymph; that is, this segment can create a strongly hyperosmotic urine. Moreover a high capacity proline pump (Meredith et al., 1988) in the apical membrane of the rectum (but not ileum) can drive additional water extraction. Proline recovered from the rectal lumen also provides the principal substrate for cellular respiration and ammoniagenesis leading to apical N H 4 + secretion in exchange for luminal Na + (Chamberlin and Phillips, 1983; Thomson et al., 1988). Locust ilea and recta, despite their structural differences, share many common epithelial transport mechanisms. Solute transport mechanisms in the posterior hindgut (rectum) of the desert locust have been studied in considerable detail leading to an epithelial model (reviewed by Philips et al. 1986, 1988, 1995). Fig.3 is a diagram of the ileal model. In both hindgut segments of the desert locust, the dominant transepithial active transport mechanism is an unusual electrogenic CI* pump located in the apical membrane (Phillips, 1996). Chloride exits the ileal cells passively via a basolateral conductance (putative channel). Potassium, the major cation absorbed, follows CI" passively by electrical coupling via cation channels with different properties in the apical and basolateral membrane, the former being opened by cAMP. The level of N a + in the primary urine is quite low (20 mM), and active reabsorption of this cation is therefore quantitatively less important. Na + enters ileal cells passively by several mechanisms (a 7 Fig. 3 A model of ion transport across the locust ileum and the control by ITP (Phillips, 1995). Ion transport peptide (ScglTP) acts via cAMP to stimulate CI", N a + , and K + entry at the apical membrane. Active secretion of H + is inhibited by ScglTP via an unidentified second messenger pathway. Thick arrows through circles, major ion pumps; thin arrow through circles, carrier-mediated co- or countertransport; arrows through gaps, ion channels. 8 conductance pathway; in exchange for N H 4 + and H + , and by cotransport with glycine) and is actively removed from cells basolaterally by a Na +/K +-ATPase pump. An electrogenic H + pump (probably an ATPase) in the apical membrane causes equal rates of acid secretion into the lumen in both hindgut segments and this is associated with passive exit of base equivalents (OH" and HC0 3") to the haemocoel side. Ammonia and H + secretion, and concomitant HC0 3 " absorption, contribute to acid-base regulation in the locust in the same way as do similar processes in the vertebrate kidney (reviewed by Phillips, 1994). In summary, ionic and osmotic regulation, as well as haemolymph pH regulation, in insects depends ultimately on selective, active and controlled reabsorption of solutes and water from primary urine in anterior (ileum) and posterior (rectum) hindgut segments. Regulation of the excretory system by the neuroendocrine system Early physiological observation on whole insects revealed very large changes in the solute composition of the final excreta in response to severe fluctuations in external conditions. For example, the desert locusts can survive several days without food or water. During such time, they excrete very few and very dry fecal pallets so as to conserve body water. In contrast, when feeding, locusts consume their own body weight daily of succulent plants and eliminate most of the ingested water and K + , thus suggesting there is neuronal or endocrine control of the excretory system (Phillips, 1998). •Structure of the neuroendocrine/neurosecretory system The principal neurosecretory tissues thought to control fluid balance in various insects are shown in Fig 4. Neuropeptides produced in median cells of the pars intercerebralis and subocillar regions of the brain are transported down axons for storage and release from the nervous corpus cardiacum (NCC). The neuroendocrine system controls the excretory system by release of both diuretic and antidiuretic factors. 9 PI-NSC PL-NSC Corpora Cardiaca Diuretic Hormone • CRF-DH • Kinnins ^J^w • Serotonin Anti-Diuretic Hormone CTSH NPS ITP Inhibitory Neuromodulator ITP-L Stimulation of gut stretch receptors could in turn activate the release of Diuretic Hormone from the CC. Osmosensitive cells in the Hemolymph could trigger the release of Anti-Diuretic Hormone from the CC. Figure 4. A simplified model of excretory control in the locust hindgut, highlighting diuretic and antidiuretic factors isolated to date. In the left upper corner, the locust brain and associated structures are represented. The regions marked PI-NSC and PL-NSC represent the pars intercerebralis- and the pars lateralis - neurosecretory cells respectively, (cited from Macins, 1997) 10 •Diuretic factors Diuretic factors isolated from insects fall into three categories: the corticotrophin-releasing factor related diuretic peptides (CRF-related DPs), insect kinin neuropeptides and the non-peptide 5-hydroxytryptamine (5-HT or serotonin; reviewed by Coast, 1996). Diuretic factors are widespread in tissues of central nervous system, and most diuretic hormones are neuropeptides (Coast, Kay and Wheeler, 1993). Diuretic factors act to increase Malpighian tubule secretion by stimulating ion transport (reviewed by Nicolson, 1993). CRF-related diuretic peptides Manduca-DH was first identified by Kataoka et al. (1989) from the tobacco hornworm. Manduca-DH shares 29-35% sequence identity with mammalian corticotrophin-releasing factor (CRF), and they are 40-47 amino acids long and have be identified in a number of species (Coast, 1998). The primary structure of insect CRF-related peptides is well conserved, especially in the N-terminal half of the molecules. The conserved region encompassing residues 6-12 of Maduca-DH is critical for receptor activation (Coast et al, 1994. Reagan, 1995a). The C-terminal amide group is also important for receptor binding. There is overwhelming evidence for cyclic AMP as a second-messenger in the action of CRF-related peptides (Coast, 1998). Receptors for Manduca-DH and Acheta-DP have been cloned (Reagan, 1994,1996). They belong to G-protein-coupled receptors, which have seven putative transmembrane domains and stimulate adenylate cyclase. Reagan (1996) reported that the amino acid sequence of the Acheta domesticus diuretic peptide receptor, deduced from its cDNA, consisted of 441 amino acids with seven putative membrane spanning regions. Insect kinin family of diuretic peptides 11 Kinins are small peptides (6-14 residues), and are characterized by the C-terminal sequence phe 1-Xaa 2-Xaa 3-Trp 4-Gly-NH 2, which is all that is needed for activity. This 'active core' probably adopts a p-turn when interacting with receptors, bringing together Phe'and Trp 4, which are critical for activity (Nachman et al., 1995, Coast, 1996). Kinin appear to act via a Ca2+-dependent mechanism (Coast, 1998). A G-protein-coupled kinin receptor has recently been cloned from the pond snail lymnaea stagnails (Coxetal., 1996). Synergism between diuretic hormones There is evidence that both Locusta migratoria and R. prolixus use two hormones (such as CRF-related peptide and kinin) which act synergistically to control diuresis (Coast, 1998). An important advantage of having two hormones act synergistically to control tubule secretion is that much lower amounts of both hormones will switch diuresis on, thereby reducing the cost of peptide synthesis. Serotonin Serotonin stimulates Malpighian tubule secretion in many insects, and acts via cAMP in R.prolixus (Montoreano et al., 1990), or by a cAMP-independent mechanism in locust and crickets (Morgan and Mordue, 1984, Coast, unpublished observation). •Antidiuretic factors There are three neuropeptides, which are reported to show anti-diuretic activity. Neuroparsins Neuroparsins are two proteins (NpA, NpB) isolated and sequenced from NCC of L. migratoria by Girardie et al. (1989). NpB is a homodimer of a 78 residue polypeptide. NpA is identical to NpB except for an additional heterogeneous N-terminal. NpB is thought to be formed from NpA by cleavage of the terminal amino acids. These peptides act via the PI-Ca 2 + second messenger system rather than the cAMP pathway (Fournier, 1991). Fouriner (1991) reviewed evidence that NpB increases Jv across rectal sacs of L. 12 migratoria, but it is not clear whether neuroparsins stimulate a long-term steady state Jv or some short-term and transient cell volume regulatory process (Phillips et al., 1995), because Jeffs (see Phillips et al. 1998) found no stimulation of rectal or ileal CI" transport (Isc) or Jv by neuroparsine in desert locusts (S. gregaria). CTSH By using CI" dependent Isc across a flat sheet preparation of Schistocerca gregaria recta as a bioassay, Spring et al. (1980a) have partially purified a neuropeptide stimulant from the CC, called CTSH (CI" transport stimulating hormone). However, biological activity is rapidly lost below pH 6, making separation by reverse-phase HPLC difficult. Using a size exclusion column, an active factor (CTSH) was eluted as a single peak with an apparent molecular weight of about 8000Da. CTSH appears to act on specific ion transport mechanisms via cAMP (Chamberlin and Phillips, 1988). Several observations suggest that CTSH is different from neuroparsins and ITP (see Phillips and Audsley, 1995). ITP ITP (ion transport peptide) is the subject of this thesis, and is discussed in the next section. ITP structure and function Schistocerca gregaria ion transport peptide (ScglTP) was isolated from locust N C C (Audsley et al., 1992). It acts as an antidiuretic hormone by stimulating CI", K + and fluid reabsorption in the ileum via cAMP and inhibits H + secretion by an unknown second messenger. It is proposed that ScglTP is a 72 amino acid neuropeptide matured from a prohormone by dibasic cleavage and C-terminal amidation (Meredith et al., 1996). • ITP isolation ScglTP was isolated from aqueous extracts of the corpus cardiacum by a four-step procedure, using reverse-phase high-performance liquid chromatography for 13 separation and voltage-clamped locust ilea as the bioassay. ITP was the first insect neuropeptide purified which was shown to act directly on the reabsorption of a specific ion in an insect excretory system, and the first neuropeptide isolated which influences an insect ileum (Audsley, 1992). Purified ITP at a dosage of 5pmol added to 2ml of bathing saline had the same range of actions as crude locust N C C extracts on the ileum: namely it caused a large increase in Isc, CI" transport (10-fold), N a + transport (2-fold), K + permeability (3-fold) and isosmotic fluid absorption (4-fold), and inhibited active acid secretion almost completely at high doses. Thus, a single neuropeptide (ITP) mimics all of the actions of crude nervous corpora cardiaca extracts on the ileum. ITP had no effect on ileal ammonia secretion. ITP has a reduced effect on rectal I s c and no effect on rectal J v or IK, suggesting that different factors (e.g. CTSH and/or neuroparsins) may regulate ion and fluid reabsorption in the rectum (Audsley, 1992). • cAMP is the second messenger of ITP ITP is thought to act via cAMP as its second messenger because all its effects (except inhibition of H + secretion) are mimicked by this cyclic nucleotide (Audsley and Phillips, 1990). In support of this view, forskolin (10-50(xmol/l) which stimulates adenylate cyclase, and the phosphodiesterase inhibitor, theophylline (5mmol/l), also stimulate ileal CI" dependent Isc (Audsley, 1990); moreover ITP increases intracellular levels of cAMP in the ileum (Audsley, unpublished observations). In summary, there is good evidence that an intracellular cAMP-mediated control system for NaCl and KC1 absorption is present in locust hindgut epithelia (Audsley and Phillips, 1990). Model for ITP action on the ileum A model was proposed by Audsley (1991) and modified by Phillips (1998) to summarize ITP control of ion transport across locust ileum (Fig.3). ITP binds to its receptor on the ileal haemolymph side, then acts via the second messenger cAMP to stimulate apical uptake of CI" (electrogenic pump), K + (ion channel) and Na + (ion channel). The ITP receptor was predicted to be a member of the G-protein family 14 because its second messenger was cAMP. ScglTP must bind to a different receptor or act to increase a second unidentified messenger system, which inhibits acid secretion. Electrophysiological studies by Richardson (reviewed by Phillips, 1998) support this model. • ITP amino acid sequence A partial amino acid sequence (33 amino acids) was determined for ScglTP purified from corpora cardiaca (Audsley, 1992). Meredith et al. (1996) used this partial amino acid sequence to clone a cDNA that exactly encoded the known partial amino acid sequences of ITP. The nucleotide sequence was extended by anchored PCR to the start and end of the ITP message using the 5' and 3' R A C E (rapid amplification of complementary ends) system. The resulting partial cDNA of 517 base pairs encoded a complete open reading frame for an ITP prepropeptide of 130 amino acid residues (Fig. 5). ITP is matured from its prepropeptide by cleaving the first 55 amino acids. The hydrophilicity plot (Kyte and Doolittle, 1982) shows that amino acids 25-40 are hydrophobic and may constitute the central or hydrophobic region of a signal peptide. The amino acid sequence immediately upstream from the putative amino-terminal cleavage site (position 54 and 55) is a dibasic cleavage site (Lys-Arg). The next 72 amino acids are believed to encode the native ITP. The prohormone has a second dibasic cleavage site at amino acid 129-130 (Lys-Lys), immediately C-terminus to the stop codon. These residues together with glycine (position 128) were predicted to provide the signal for amidation of the C-terminus of ITP (Meredith et al., 1996). ITP homologues On the basis of ITP partial sequence, Audsley et al. (1992b) first reported the considerable sequence similarity to a family of crustacean hormones, including hyperglycemic (e.g. CHH), moult-inhibiting (MIH) and vitellogenesis-inhibiting (VIH) hormones from several crustacean species. ITP is the first member of this protein family found outside crustaceans. Comparing the complete ITP amino acid sequences deduced 15 ACTCACCACCACCCCGTGGTCACGCTACTCGACGCCGCCACG 1 Met ATG His CAC His CAC Gin CAG Lys AAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Lys AAG Gin CAG Gin CAG Gly GGA Glu GAG 16 Ala GCT Pro CCG Cys TGC Arg CGA His CAT Leu CTC Gin CAG Trp TGG Arg CGG Lys TTA Ser TCA Gly GGG Val GTC Val GTC Leu CTC 31 Cys TGC Val GTC Leu CTC Val GTC Val GTA Ala GCT Ser AGC Leu CTC Val GTT Ser TCC Thr ACG Ala GCG Ala GCT Ser TCC Ser AGC 46 M Pro CCG Leu TTG Asp CAT Pro CCA His CAC His CAC leu CTT Ala GCC Lys \AA Arg AGG Ser TCC Phe TTC Phe TTC Asp GAC He ATC 61 Gin CAG Cys TGT Lys AAA Gly GGA Val GTT Tyr TAC Asp GAC Lys AAG Ser AGC He ATC Phe TTT Ala GCA Arg CGC Leu CTA Asp GAC 76 Arg CGC He ATC •a Cys TGC Glu GAA Asp GAT Cys TGC Tyr TAC Asn AAC Leu CTA Phe TTC Arg CGC Glu GAA Pro CCT Gin CAG Leu CTC 91 His CAC Ser TCT Leu CTG Cys TGC Arg AGA Ser TCT Asp GAC Cys TGT Phe TTC Lys AAG Ser AGC Pro CCA Tyr TAC Phe TTC Lys AAA 106 Gly GGT Cys TGT Leu CTT Gin CAG Ala GCA Leu CTA Leu CTT Leu CTG He ATT Asp GAT Glu GAA Glu GAA Glu GAA Lys AAA Phe TTT 121 no Asn AAC Gin CAA Met ATG Val GTG Glu GAA He ATA Leu CTG Gly GGG Lys AAG Lys AAG END TAG ACTGCACAAGAACTC CCTGCAGGCTGGGAGATAAATTACGGAAACATTCTAGTCTTTGAAAATATATGTTCGGAAGAGTT AGA Fig. 5 The partial cDNA of ITP encoded a comlete open reading frame for preproITP. The two dibasic cleavage sites are boxed. The numbers indicate the positions of amino acids in preproITP. 16 from its cDNA with the C H H protein family (Fig.6), it was found that these peptides are of similar length (72-78 residues), with the six cysteine residues conserved in all cases (Meredith et al., 1996). There are several other highly conserved sequences (residues 7-12, 16, 19-31, 39-43, 49 and 52-61). ITP and a majority of the C H H family exhibit terminal amidation, but greatest divergence is in the last 10 residues. Using Chou-Fasman and Robson-Garnier methods (as used by the protein analysis toolbox in Macvector software), the ITP C-terminus is predicted to be an alpha-helix. As a working hypothesis, it was assumed that there are three disulfide bridges in locust ITP at the same locations as have been determined for CHH (Kegel et al., 1989), namely residues 7-43,23-39 and 26-52 (Meredith et al , 1996). Several peptides with hyperglycemic activity have been found in individuals of different species indicating the presence of polymorphic forms of C H H (van Herp et al., 1998). The identification of two C H H preprohormones and four isoforms in the lobster (De Kleijin, 1995a) and the two isomorphs in the Mexican crayfish (Aguilar et al., 1995) points to a post-translational modification. The two isomorphs of CHH-I and CHH-II in Crayfish have identical sequences, and the difference between the two isomorphs consists in a post-translational modification of an L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus (Aguilar et al., 1995). Carcinus hyperglycemic hormone has high similarity (67%) to locust ITP, but had no effect on ileal Isc at 2.5x10"7M to 10"6M (Meredith et al., 1996). By contrast, purified ScglTP causes maximum response at 2.43x10"12M. The failure of Carcinus hyperglycaemic hormone and crab eyestalk extracts to stimulate locust ileal Isc indicates that the ITP receptor in the ileum is specific (Meredith et al., 1996). Synthetic ITP (synlTP) structure and bioactivity Based on the prediction of Meredith et al. (1996), synlTP was synthesized by King et al. (1998). SynlTP is a 72 amino acid peptide with amidation of residue 72 and has a molecular weight of 8558 Da with three disulfide bridges of which the location at 17 ITP AND Crustacean Hormone Alignment KRSFFDIQCKGVY. DKSIFARLDRICEDCYNLFREPQLHSLCRSDCFKSPYFKGCLQALLLIDEEEKFNQMVEILGKK KRSFFDIQCKGVY. DKSIFARLDRICTaX^TNLFREPQLHSLCRSDCFKSPYFKGCLQALLLIDEEEKFNQMVEILGKK KRSLFDPACTGIY. DRQLLRKLGRLCDDCTflNIOTREPKVATGCRSNCYHN^ KRSLFDPSCTGVF. DRQLLRPJjGRVCDrKlFNWREPWATECRSNCYNNPWRQCMAYVVPAHLHNEHREAVQMVGK SASFIDNTCRGVMGNRDIYKKVVRVC32DCTNIFRLPGLDGMCRNRCFYIffi KRQVFDQACKGIY. DRAIFKKLDRVCEDCYNLYRKPWATTCRQNCYANSWRQCLDDLLLIDVLDEYISGVQTVGK KRQVFDQACKGVY. DRmjFKKLDRVCETX^rMjYRKPFVATTCRENCYSNWVFRQCLDDLLLSDVIDEYVSNVQMVGK KRQVFDQACKGVY. DRNLFKKLDRVCEDCYNLYPJCPFVATTCRENCYSNWWRQCLDDLLLSDVIDEYVSNVQMVGK KRQVFDQACKGVY. DRNLFKKUSIRVCRr<rfNrLYRKPFIOTTC KRSLFDPSCTGVF. DRQLLRRLRRVCDDCFNVFREPWSTECRSNC^fNNEWRQCMEYLLPPHLHEEHRLAVQW K R R I F D T S C K G F Y . D R G L F A Q L D R V C E D C Y N L Y R K P H V A A E C R R D C Y T T E V F E SCLKDLMMHDFINEYKEMALMVS KREVFDQACKGIY. DRAI F K K L D R V C E D C Y N L Y R K P Y V A T T C R Q N C Y A N S V F R Q C L D D L L L I D V V D E Y I S G V Q T V G K KREVFDQACKGIY. DRAI F K K L D R V C E D C Y N L Y R K P Y V A T T C R Q N C Y A N S W R Q C L D D L L L IDWDEYISGVQTV KRDTFDHSCKGIY. D R E L F R K L D R V C E D C Y N V F R E P K V A T E C K S N C F V N K R F N V C V A D L R . RDV. SR FL KM A N SA L S Q V F D Q A C K G I Y . D R A I F K K L E L V C D D C Y N L Y R K P W A T T C R E N C Y A N S V F R Q C L D D L L L I N V V D E Y I S G V Q I V G K SFIDNTCRGVMGNRDIYKKWRVCI^TNIFRLPGLIXSMCRNRCFYNEWFLICLKAANREDEIEKFRW RYWEECPGVMGNRAVHGKOTRVC32DCYNWRDTDVIAGro AARVINDECPI^IGNRDLYKKVEWICEDCSNIFRKTGMASLC^ AARVINDIXTPNLIGNRDLYKKVEWICDDCM RVINDrXTPNLIGNRDLYKKVEWlCEDCSNIFRNTGMATLCRKNCFFNEDFLWC^ SAWFTN . C PGVMGNRDLYEKVAWCMDOVNIFRNNDVGVMCKKD^ ILR SAVffTOTECPGWGNRDLYEKVAWCNIX^IFR^ ITP S. gregaria ITP L. Migratoria SGP-III P.japonicus SGP-1 P. japonicus SGP-IV P. japonicus CHH 0. 1imosus CHH H.americanus CHH (A) H. americanus CHH (B) H. americanus CHH P.vannamei CHH Armadillidium CHH' P. c l a r k i i CHH P. bouvieri MIH-like P.vannamei MIH P. bovieri MIH P. japonicus MIH P. c l a r k i i MIH C. maenas MIH C. sapidus MIH C. pagarus VIH H. americanus GIH H. americanus Fig.6 ITP and its structural homologue CHH (crustacean hyperglycemic hormone) family alignment. The CHH family contains CHH, MIH (molt inhibiting hormone), and VIH/GIH (vitellogenin- or gonad-inhibiting hormone). ITP and CHH's have the similar lengths (72-78 amino acids) with six cystein residues (bold face) conserved in all members and the similarity is as high as 67%. Amino acids with bold face are highly conserved amino acids 18 cys 7-43 was established. This is the first member of this large family of arthropod neuropeptides to be synthesized. The biological activities of synlTP were consistently similar to those of ITP purified from locust N C C by Audsley et al. (1992b). Dose-response curves measuring active chloride transport (Disc) and time course of action in locust ilea are very similar for both synlTP and ScglTP, and the E C 5 0 differs by only two-fold (King et al., 1998). Antibodies made to amino acids 2-30 of ScglTP recognize synlTP, which co-migrates with a band from Schistocerca CC homogenates (Macins et al., 1999). These results show that synlTP and ScglTP are indistinguishable and that any further post-translational modification of ScglTP is not required for biological activity (King et al., 1998). ITP-L structure, distribution, and function A second related cDNA (ITP-L) was isolated from a brain cDNA library and is identical to ScglTP cDNA except for a 121-bp insert at position 95 to 135, suggesting alternative splicing of genomic DNA (Meredith et al, 1996). ITP-L has an open reading frame (134 residues) that is four residues longer than that of ITP. All six cysteines are conserved. ITP and ITP-L have the same N-terminal (amino acid 1-40), and most of the differences are among the last 20 residues. Using reverse transcription PCR, ITP-L mRNA was detected in many tissues (such as flight muscle, hindgut and Malpighian tubules) that have no stimulatory effect in the locust ileal Isc bioassay. In contrast, ITP mRNA is restricted to the brain and NCC, which do stimulate ileal Isc (Meredith et al., 1996). The function of ITP-L is unknown, but a working hypothesis was proposed that ITP-L might either act as an antagonist at the ITP receptor to shut off hindgut fluid reabsorption or act to reduce synthesis and release of ITP at the brain-NCC level (Phillips et al., 1998). ITP-L might be an antagonist of ITP because both peptides share an N -terminal sequence from amino acid 1-40. One in vitro experiment supports this idea. In a 19 competition assay, ITP-L expressed in K c l cells and used at high concentration (5nmol) was found to inhibit KcITP (84pmol) stimulatory activity by about 60%. The concentration ratio of KcITP-L to Kc-ITP is 60:1, suggesting KcITP-L has weak affinity for the ITP receptor (Wang et al., submitted). Expressed ITP structure and bioactivity Using the baculovirus/insect cell system, bacITP and bacITP-L were expressed •in a Sf9 cell line (Ring et al. 1998). BacITP is N-terminally extended by 11 amino acids compared to ScglTP. BacITP has biological activity when tested in vitro in the locust ileal bioassay but with a slower (12x) time course and reduced (270-fold) specific activity compared to synlTP. Ring et al. (1998) suggested that there were two possible explanations for these observations. First, the N-terminal extension may directly inhibit binding or result in a peptide that binds to ileal receptor with reduced affinity. Second, Sf9 cells may not produce the amidated C-terminal leucine as proposed by Meredith et al. (1996) and present in the synthetic peptide which is fully active in picomolar quantities (King etal, 1999). PreproITP cDNA and preproITP-L cDNA have been expressed in the Drosophila K c l cell line. The secreted peptides were named KcITP and KcITP-L respectively (Wang et al., submitted). Amino acid sequencing of the purified KcITP showed that KcITP has the identical N-terminus as ScglTP and comigrates with ScglTP on Western blots. In the bioassay, KcITP has stimulatory activity that is much higher than bacITP but lower than synlTP. The reduced activity of KcITP suggested that Kc cells do not carry out the amidation step, and that C-terminal leucine amide was necessary for full biological activity. In vitro amidation experiments of KcITP support this suggestion (Wang et al., submitted). KcITP-L has the same N-terminal nucleic acid and it is predicted to have the same N-terminal amino acid sequence as KcITP. KcITP-L has no stimulatory activity on ileal Isc, but it inhibits KcITP stimulation at a high concentration 20 ratio of 60:1. This supports the idea that the common N-terminus is involved in binding, but the different C-terminus of ITP-L prevents activation of the receptor. ITP C-terminal structure-function relationship The ITP C-terminus may be important for binding to the receptor and/or signal transduction. In order to understand ITP C-terminal functions, a series of amino acid deletions at ITP C-terminal were made using site-directed mutagenesis. Five deletions that removed K K , G K K , L G K K , ILGKK, and M V E I L G K K respectively from the C-terminus were expressed in Kcl cells (Wang et al., submitted). The dose-response curves of the mutants show that KcITP. K K has the same stimulating activity as KcITP, but KcITP "GKK has reduced stimulatory activity by two orders of magnitude. Further truncation (-LGKK, -ILGKK, and -MVEILGKK) abolished all biological activity. A competitive inhibition test shows that K c I T P . L G K K can reduce KcITP stimulation 40% at concentration ratio of 10:1. This suggests that K c I T P . L G K K can still bind to the receptor with reduced affinity (10 fold), and ITP C-terminal leucine is essential for signal transduction (Wang et al, submitted). Three point mutations of cysteine 23,26 and 43 (each replaced by alanine) were made. These point mutations abolish nearly all the stimulating activity, indicating that each of three disulfied bridges in ITP is required for the ITP bioactivity (King et al, 1999). Objective of this thesis The structure-activity relationship study of ITP will be useful to find a strong antagonist of ITP, which could be used for pest biological control. To further understand ITP structure-function relationship, my project focuses on the N-terminus of ITP. Comparing ITP and ITP-L primary structure, they have the same N-terminal amino acids 1-40, and competitive inhibition tests show KcITP-L can block KcITP stimulating activity in high concentration ratio, therefore, my hypothesis is that the ITP N-terminus contains a binding domain. Because the seventh amino acid of ITP is a conserved cysteine, that forms an essential disulfide bridge with the cysteine at the 43rd residue, my 21 study was limited to the amino acids 1-6 (SFFDIQ) at the N-terminus. The questions are: 1. Is the N-terminal domain, the first six amino acids (SFFDIQ), in ITP important to ITP bioactivity? 2. Which amino acids in the N-terminal domain of ITP are essential for ITP binding to the receptor and/or activating the receptor? The first 6 residues of this N-terminal domain may not be important for ITP activity, so an initial test of this prediction, I first carried out a domain swap mutation. •Domain swap ScglTP N-terminal domain (SFFDIQ) was changed to that of the P. japonicus CHH N-terminal domain (SLFDPA). This N-terminal domain swap changes 3 of the 6 amino acids in the N-terminal and directly tests whether this ITP N-terminal domain is important for ITP bioactivity. When the result showed this N-terminal domain was important for ITP stimulation of ileal Isc, then I made more specific mutations of the N -terminal domain. •conserved amino acids mutation The 3rd(F) and 4th(D) residues of ITP N-terminal are conserved amino acids compared with the C H H family, and were unchanged in the domain swap. Are these two conserved amino acids necessary for ITP function? To address this question, mutations of the conserved 3 r d and 4 th amino acids were made by replacing FD with A A . •Point mutations Stimulating activity was abolished by the above mutations, so each amino acid in the N-terminal domain (SFFDIQ) was changed individually to alanine. These mutants were tested for their bioactivity as stimulants or as inhibitors. 22 The results clearly show that ITP N-terminal domain (SFFDIQ) is important to ITP bioactivity. Among the six amino acid of ITP N-terminus, F2 and F3 are essential for ITP binding to the receptor, and F2 is also important to receptor activation. The other four amino acids SI, D4, 15 and Q6 don't contribute to the ITP bioactivity, even though D4 is a highly conserved amino acid. The post-translational modification of conversion L- to D- amino aicd probably occurred at F2 and F3 to yield two ITP isomorphs. Mutations in the ITP N-terminal domain do not interfere with the dibasic cleavage site in spite of its immediate proximity to the dibasic cleavage site. 23 M A T E R I A L S A N D M E T H O D S I Subcloning Oligonucleotide synthesis All gene-specific oligonucleotides were ordered from Gibcol BRL. Table 1 shows the sequences of primers used to produce different mutations. Some primers were designed with a restriction site (bold face, table 1) in the 5' flanking region to simplify cloning. Oligos used in PCR (polymerase chain reaction) were diluted in water to 100 ng/ul Mutation strategy Oligonucleotide-mediated mutagenesis was used to substitute nucleotides in the ITP N-terminal domain. The principle of the mutation method is showed in Fig.7. Two PCR procedures are required to complete the substitution. In the first step PCR, the template is the plasmid p2Zop2F containing preproITP cDNA, which consists of 405 bp (base pair). Two pairs of primers (Pa, P26 and P 2 5 , P2) were used to produce two D N A fragments, which are diagrammed in fig. 7, namely Pa-P 2 6 and P25-P2. Primers P 2 5 and P26 introduce the substituted nucleotides (solid square) into the two D N A fragments. Pa-P 2 6 fragment contains the substitute nucleotide and the upstream preproITP sequence. P25-P2 fragment contains nucleotide coding for the same substitution and down stream preproITP sequence. In the second step PCR, fragments Pa-P 2 6 and P25-P2 hybridize the length of the perfectly matched nucleotides (15-18 nucleotides) and form the sense and antisense primer to elongate the two DNA fragment to the full length of 405 bp. In addition, two primers, Pa and P 2 encoding the 5' and 3' ends of preproITP help to amplify the full length DNA which now contains the substituted nucleotides. This mutated DNA was subcloned into vector p2Zop2F for plasmid proliferation and transformation to insect cell line. PCR conditions 24 Table 1. List of primer sequences and corresponding melting temperatures (Tm) Peptide Primer Primer sequence (5'—>3') Tm KcITP A C G A C C T C G A G A C G A T G C A C C A C C A G A A G C A 60 P 2 C G G A A T T C T C T A G A C T A C T T C T T C C C C A G T 48 DS-ITP Pl9 T T C T T G T T C G A C C C C G C G T G T A A A G G A G T T T A C G A C 52 P20 C G C G G G G T C G A A C A A G G A C C T T T T G G C A A G 50 FD-ITP P23 T C C T T C G C C G C C A T C C A G T G T A A A G G A G T T 50 P 2 4 C T G G A T G G C G G C G A A G G A C C T T T T G G C A A G 54 F2A-ITP P25 A G G T C C G C C T T C G A C A T C C A G T G T 44 P26 G A T G T C G A A G G C G G A C C T T T T G G C A A G 46 I5A-ITP P27 T C G A C G C C C A G T G T A A A G G A G T T T A C 50 P28 T T T A C A C T G G G C G T C G A A G A A G G A C C T 46 Q6A-ITP P29 G A C A T C G C G T G T A A A G G A G T T T A C 50 P30 T C C T T T A C A C G C G A T G T C G A A G A A G G A 48 D4A-ITP P31 T T C T T C G C C A T C C A G T G T A A A G G A G T T 50 P32 A C A C T G G A T G G C G A A G A A G G A C C T T T T 46 S1A-ITP P33 A A A A G G G C C T T C T T C G A C A T C C A G 46 P34 G T C G A A G A A G G C C C T T T T G G C A A C G T G 46 F3A-ITP P35 T C C T T C G C C G A C A T C C A G T G T A A A 46 P36 C T G G A T G T C G G C G A A G G A C C T T T T G G C 46 25 M u t a t i o n s t r a t e g y Pa P25 5' ITP 3' ITP -—DLT "P2 P26 first step PCR f P25 P26 P2 second step PCR Pa P26 P25 P2 5' mutant 3' mutant Fig. 7 Mutation strategy showing the principle of site-directed mutagenesis. Point mutation F2A is used as an example. Two step P C R reactions were performed. In the first step P C R , two D N A fragments labeled as Pa -P26 and P25 -P2 are produced. The substituted nucleotides are introduced into the two fragments by primers P25 and P26. Each of the two fragments containing the substituted nucleotides is part of ITP cDNA. In the second step P C R , the whole length D N A of mutation F2A is produced by using the two D N A fragments (Pa-P26 and P25-P2) as templates and P a , P2 as primers. wild type ITP nucleotide ITP sequence elongated DNA substituted nucleotide • primer 26 The polymerase chain reaction was conducted in a total volume of 100 ul. This contained 1 ng DNA template, 50pmol of each primer, 0.2 mM of each dNTP, PCR buffer (20 mM Tris-HCI, 0.1 M KC1, 0.1 mM EDTA, pH 8) and 2.5 units of TaqDNA polymerase (Boehringer Mannheim). The reaction was carried out using a Perkin Elmer/Cetus DNA thermal cycler (MODEL 480) programmed to 30 cycles. Each cycle consisted of 94°C for 45 seconds (denature), 50-56°C for 45 seconds (anneal), and 72°C for 1 minute (extension). DNA electrophoresis PCR products were fractionated on 1% agarose gel containing 0.05 (ig/ml ethidium bromide, using a Biorad mini-sub or a wide mini sub DNA cell electrophoresis apparatus. The gel was made with agarose powder (Gibcol BRL) dissolved in T B E buffer (0.09 M Tris-borate and 0.002 M ethylene diaminetraacetic acid disodium salt solution (EDTA)). The DNA loading buffer was gel loading buffer III, containing 0.25% bromophenol blue, 0.25%) xylene cyanol FF and 30% glycerol in water. A 100-bp DNA ladder or XDNA Hindlll (Gibcol BRL) was used as the molecular weight standard. The power source for electrophoresis was a Pharmacia L K B GPS 200/400 power pack and was run at 60-100V. LMP (low melt point) agarose electrophoresis LMP agarose electrophoresis was used to purify the DNA fragments from both the first and the second PCR reaction. The gel was made from LMP agarose powder (Gibco BRL) in T B E buffer. After mnning this LMP agarose gel, the appropriate size D N A band was detected with U V light and the gel slice containing this D N A fragment was excised and digested with (3-agarase I (Biolabs) and the DNA fragment purified according to the manufacturer's instruction (Biolabs). Preparation insert and vectors Vector p2Zop2F (Fig. 8) was constructed to facilitate expression of heterologous proteins in insect cell lines (Hegedus et al, 1998 and Pfeifer et al, 1997). The Orgyia 27 CO CO DC E CO X CO < 0 5= "O O = £ CO 8 CO LU IE-2 Promoter on IE-2 Promoter Zeocin resistance SV 40 PA Fig. 8 Expression vector p2Zop2F for insect cell line protein expression system (modified from Hegedus D.D., 1998). P2Zop2F contains a zeocin resistance gene, iel promoter to direct the insert DNA and the zeocin resistance gene expression. The multiple cloning site (MCS) contains 13 unique restriction enzyme site, downstream of the iel promotor. The iel gene pA signal facilities the expression of genes lacking pA signals. 28 pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (iel) promoter mediates constitutive expression. P2Zop2F vector was derived from the basic cloning and shuttle vector p2ZeoKS, which uses an z'e2-synthetic bacterial EM-7 promoter to drive expression of the strepto-alloteichus hindustanus ble gene and confers resistance to Zeocin (Invitrogen,CA) in both insect cells and E. coli (Pfeifer et al, 1997). P2Zop2F has a multiple cloning site (MCS), containing 13 unique restriction enzyme sites, downstream of the iel promoter, and also has the iel PA (polyadenylation) signal sequences. In addition, this vector also contained translation stop codons in all three reading frames to allow expression of truncated genes (Hegedus et al, 1998) Both the vector (p2Zop2F) and PCR DNA fragments were digested with the EcoRI and Xhol enzymes (Boerhringer Mannheim). The reaction volume was 50 ul, containing 50 mM Tris-HCI, 100 mM NaCl, 10 mM MgCl 2 , 1 mM dithioerythritol (DTT), pH 7.5, 25 units EcoRI and 20 units Xhol. The digest reaction was at 37°C for 2 hours, and the digested insert and vector were purified by LMP agarose electrophoresis. Ligation and transformation The insert and vector digested by EcoRI and Xhol contain compatible cohesive termini. The ligation reaction had an insert-to-vector ratio of 3:1 in a reaction volume of 10 ul and used 1 unit of T4 DNA ligase (Gibcol BRL). The reaction mixture contained 50 mM Tris-HCI, pH 7.6, 10 mM MgCl 2 , 1 mM ATP, 1 mM D T T and 5% polyethylene glycol-8000. The ligation mixture was incubated at 16°C for 6 hours. Then 3 ul of this ligation reaction was used for transformation of 50 ul DH5-alpha competent cells (Gibcol BRL) using the manufacturer's suggested protocol. The transformed DH-5a cells were plated on LB culture plates containing 1.5% agar and 0.0025%) Zeocin as resistance selective marker. After culturing 12-16 hours at 37°C, each of 10 clones were picked and transferred to 3 ml LB culture medium containing 0.0025%) Zeocin. These were allowed 29 to grow in 3 ml LB medium for 12 hours at 37°C with vigorous shaking to replicate plasmid DNA. Plasmid extraction and purification Plasmids in cells of each clone were extracted by using the mini alkaline-lysis protocol (Ish-horowicz and Burke, 1981). Plasmids were screened using PCR and positives were cultured in 200 ml LB medium with 0.0025% Zeocin and large-scale plasmid extraction and purification was carried out using a Qiagen (manufacture) plasmid purification kit. An aliquot was digested to ensure that the appropriate sized fragment was in the cloning site of P2Zop2F. The insert sequence was confirmed by automated DNA sequencing using the ABI AmpliTaq (FSTaq) dye terminator cycle sequencing system and a 373 DNA automated sequencer (U.B.C. Nucleic Acid/Protein Service unit-NAPS). II. K c l cell expression system Plasmid (p2Zop2F) containing the mutated DNA fragments were transfected into Drosophila Kc l cells to obtain expressed and secreted peptides. K c l cells were grown in insect D22 medium without serum (Sigma) at 25°C. Purified plasmids and the transfection regent, Cellfectin (Gibcol BRL), were used according to the manufacturer's protocol for adherent cells. The transfection procedure was as follows: 3xl0 7 Kc l cells were allowed to grow to 80-90% confluence in a 75 ml T flask. Then DNA (20 ug) and cellfectin (160 |il) were incubated together 20 minutes at room temperature in 8 ml minimal Grace medium (Sigma). The cells were washed once with minimal Grace medium. The rninimal Grace medium containing the DNA and cellfectin was added into the T75 flask containing the washed cells, and incubation continued 8-10 hours at 25°C to allow DNA transfer into the cells. The minimal Grace medium was changed to D22 medium and the cells were cultured for another 48-60 hours. The cultured cells and supernatant were separated by centrifugation at 735 g for 3 min. and the supernant were concentrated 30 10-15 fold by polyethylene glycol (PEG 8000, Fisher) dialysis using dialysis tubing with a 3.5 kD cut off point. The cell pellet was resuspended in PBS and cells ruptured by sonication. III. Tris-tricine-SDS-PAGE and Western blotting system Tris-tricine-SDS-PAGE Proteins were separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Tricine-SDS-PAGE (Schagger and von Jagow, 1987 and Klafki et al, 1996) was developed for resolving small proteins, especially in the 5-20 kD range. The gel dimensions were 120x170x0.75 mm. The gel was cast in three layers: separating gel, spacer gel arid stacking gel. The separating gel was 16.5% polyacrylamide (16.5% total monomer concentration (%T)/2.4% crosslinking monomer concentration-bis-acrylamide (%C)) containing 1.0 M Tris (pH 8.45), 0.1% SDS, 0.06% ammonium persulfate (APS), 0.06% N,N,N',N'-tetra-methyl-ethylenediamine (TEMED) and 10.4% glycerol. The spacer gel, consisting of 16.5% polyacrylamide (16.5%T/1.0%C) containing 1.0 M tris (pH 8.45), 0.1% SDS, 0.06% APS, 0.06% T E M E D , was poured to 20 mm above the separating gel. Stacking gel was 3.96% polyacrylamide (3.96%T/0.24%C) containing 0.744 M Tris (pH 8.45), 0.074% SDS, 0.8% APS, 0.08% T E M E D and was poured to 10 mm below the bottom of the comb. Protein samples were boiled for 3 min. in SDS loading buffer (0.06 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol (v/v), 5% mercaptoethanol (v/v) with pyronin dye. The samples were laid under the cathode buffer. The molecular weight markers ranged from 3.5 KD to 37.6 K D (Bio-rad). The anode running buffer was 0.2 M Tris at pH 8.9 and the cathode buffer contained 0.1 M Tris, 0.1 M Tricine and 0.1% SDS (pH 8.25). Gels were run on a vertical gel apparatus (Tyler) at 60V for 16-20 hours. Tank transfer system 31 Proteins on SDS-polyacrylamide gels were transferred to 0.2 urn nitrocellulose membranes (Bio-rad) in mini trans-blot cell (tank blotting) apparatus (Bio-rad). The gel was cut to 8x10 cm size, and the nitrocellulose membrane was cut to the dimensions of the gel. Then the gel and membrane as well as Whatman papers and fiber pads were soaked 15 min. in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) and assembled as gel/membrane sandwich, which was put into the transfer tank (containing cold Towbin transfer buffer and an icebox). The gel was oriented on the cathode side of the membrane. The transfer system was run at 100V for 1 hour at 4°C using a Pharmacia L K B GPS 200/400 (Phamacia) power supply. Antibody production Polyclonal antibodies for ITP and ITP-L N-terminal (N-l, figure 9) and ITP as well as ITP-L specific peptide sequences (C-l , C-2, figure 9), were raised in New Zealand white rabbits (Ring et al., 1997). ECL western blotting protocol (Amersham) Nitrocellulose membranes were blocked 12-16 hours at 4°C in 5% milk powder in TBS-T (0.1% Tween 20 in Tris buffered saline, pH 7.5). The blot was then washed in TBS-T twice quickly, once for 15 minutes, and twice for 5 minutes with shaking at room temperature. The blot was then incubated with the primary antibody (ITP C-terminus specific antibody) at a 1/10000 dilution in 0.5%) milk powder in TBS-T for 2 hours with shaking at room temperature. The blot was then washed as above. The secondary antibody (anti-rabbit horseradish peroxidase linked-Amersham) was applied to the blot, also at a dilution of 1/10000 in 0.5%> milk powder in TBS-T, for one hour with shaking. Then the blot was washed as above, and twice more with lxTBS for 5 minutes, each at room temperature with shaking. The blot was quickly incubated in a 1:1 mixture of ECL reagents (Amersham) for 1 min. at room temperature. The excess liquid was removed and 32 1 I T P MHHQKQQQQQ KQQGEAPCRH L Q W R L S G W L C V L W A S L V S T A A S S P L D P H I T P - L MHHQKQQQQQ KQQGEAPCRH L Q W R L S G W L C V L W A S L V S T A A S S P L D P H 1 51 HLAKR'SFFDI H L A K R S F F D I ANTIBODY N-l QCKGVYDKSI QCKGVYDKSI F A R L D R I C E D F A R L D R I C E D C Y N L F R E P Q L C Y N L F R E P Q L H S L C R S D C F K H S L C R K D C F T l u i ANTIBODY C-l S P Y F K G C L Q A L L L I D E E E K F NQMVEILGKK S D Y F K G C I D V LLLQDDMDKI QSWIKQIHGA E P G V ANTIBODY C-2 Fig.9 S. gregaria ITP and ITP-L amino acid sequence map and their specific antibodies. Amino acids correlated with the boxes were used as antigens for the production of antibodies labeled N- l , C - l and C-2. The synthetic peptides C - l and C-2 have a C G G at their C-terminal ends for coupling to K L H (keyhole limpet hemocyanin). C - l is ITP specific and C-2 is ITP-L specific. The vertical arrow indicates a dibasic cleavage site. The amino acids in bold differ between ITP and ITP-L. 33 the blot was then wrapped in plastic-wrap and exposed (from 1 min. to 30 min. depending on signal strength) to Kodak X - O M A T RP XRP-1 film. Measurement of expressed peptide concentration The concentrations of expressed peptides were estimated by comparing Western blot band densities with band densities of known amounts of synlTP. Different volumes of supernatant containing expressed peptides and different amounts of synlTP were loaded on the same tris-tricine polyacrylamide gel, separated by SDS-PAGE, and proteins identified by Western blotting. Using the NIH imaging program, the density of each band was measured. By comparing the density of bands expressed peptides with that of the known concentrations of synlTP, the concentration of expressed peptides was estimated. IV Bioassay on locust ilea Animals The experimental ariimals were adult Schistocerca gregaria, 2-3 weeks past their final molt. They were reared at 28°C and 55% relative humidity under a 12:12 light: dark cycle, and fed a diet of lettuce and a mixture of dried grass, bran and milk powder. Ilea from females were used because of their larger size. Bathing saline The complex saline was based on the composition of locust haemolymph (Hanrahan and Phillips, 1983) and contained (mM): 100 NaCl, 5 K 2 S 0 4 , 10 MgS0 4 , 10 NaHC0 3 , 5.0 CaCl 2 , 10 glucose, 100 sucrose, 2.9 alanine, 1.3 asparagine, 1.0 arginine, 5 glutamine, 11.4 glycine, 1.4 histidine, 1.4 lysine, 13.1 proline, 6.5 serine, 1.0 tyrosine, 1.8 valine. The saline was bubbled with 95% 0 2 / 5% C 0 2 . This saline was used in all experiments. Short-circuit current (Isc) measurement To measure electrogenic ion transport, ilea were mounted as flat sheets between two modified Ussing chambers and voltage-clamped at zero mV, as described by 34 Hanrahan et al (1984) for locust rectum. Each chamber contained 2ml of saline which was stirred by vigorously bubbling with a mixture of 95% 0 2 / 5% C 0 2 at 22±2°C. More detail is given below. Ilea were removed from animals, cut longitudinally to produce a flat sheet and immediately (within 5 min. from start of dissection) secured over a 0.196 cm 2 opening by means of tungsten pins and an overlaying neoprene O-ring (Fig. 10). Edge damage was negligible with this technique (Hanrahan and Phillips, 1984a). To measure the transepithelial potential (Vt), 3 M KC1 agar bridges were placed near the tissue through ports on the side of the chambers and connected to a high input impedance differential amplifier (4253, Teledyne Philbrick, Dedham, Mass) which continuously monitored Vt. Short-circuit current (Isc), a direct continuous measure of electrogenic ion transport, was measured by maintaining Vt at 0 mV by a second amplifier (725, National Semi-conductor Corp. Santa Clara, Calif.) which passed current (Isc) between two Ag-AgCl electrodes at either end of the chamber. A third amplifier (308, Fairchild, Mountain View, Calif.) was used to measure Isc. Both Isc and Vt were monitored on a strip chart recorder (Soltec 1242, Slotec Corp., Sun Valley, Calif). Corrections were made for series resistance of the external saline and asymmetries between voltage-sensing electrodes (Hanrahan et al, 1984). Transepithelial potential under open-circuit conditions was monitored at intervals by stopping the voltage-clamp for 20-30 seconds. The increase in locust ileal Isc upon stimulation is a measure of electrogenic active CI" transport (Irvine et al, 1988). Dose-response curve The stimulatory action of expressed mutants on ileal Isc was tested at several dosages to obtain a dose-response curve. Concentrated supernatants of expressed peptides were added to the haemolymph side of the ileal preparations when these tissues had reached a steady-state Isc level 60-120 min. after dissection. The resulting increases in ileal Isc were followed until a maximum value was attained. Each dose was tested on 5-6 ileal preparations and results are reported as means ± 1 standard error of the mean (SE). 35 Fig.10 A diagram of the chamber for detecting CI" active transport and the principle of the circuit (A) Standard Ussing chamber assembly used for measurement of Vt and Isc. (1) flat-sheet ileum preparation, (2) plexiglass collar over which ileum is mounted, (3) neoprene O-ring for securing ileum attachment to collar, (4) neoprene chamber seal, (5) agarbridge port for measur ing vt, (6) gas inlet for saline aeration and mixing, (7) current sending electrodes, (8) rear chamber seal, (9) tungsten pins for attachment of ileum to collar (figure taken from Hanrahan et al, 1984) (B) A simpl i f ied mode l exp lan ing the measurmen t of short circuit current (Isc). Vt represents a voltmeter; Isc, microammeter. R is a var iable res is tance and E, battery. 36 Competitive inhibition test A competitive inhibition assay was used to detect binding of inactive mutant peptides to the ITP receptor. For example, inactive peptides (e.g. F2A or F3A) were added to the ileal haemolymph side, then after 1 hr, the expressed wild type ITP (KcITP) was added. If the inactive mutation peptide can bind to the ITP receptor, then the effect of the KcITP response should be reduced. In the control inhibition test, concentrated cell culture medium was added to the haemolymph side of ileal in the same volume as the inactive mutant peptides. Significant difference was determined by Student's t-test with P< 0.05 being considered significant. 37 RESULTS 1. Mutation DNA fragments and their DNA sequencing results Substitution at the N-terminus of ITP was achieved by a two step PCR procedure as discussed in Materials and Methods. Fig 11A shows two DNA bands produced from the first step PCR. Here, I used point mutation F2A as an example. Two DNA fragments both containing the substituted nucleotides are synthesized by PCR. The measured length of these fragments (240 and 200 bp) are close to the theoretical length (239 and 196 bp). Fig 11B shows 420 bp D N A band synthesized by second step PCR, the same length as cDNA of wild type ITP. D N A sequencing of this band confirmed it was mutant F2A cDNA. Table 2 shows the different mutations, together with their DNA sequence. Substitution are shown in bold. 2. Western blotting analysis of mutated peptides expressed in Kcl cells Fig. 12A compares Western blots of different amounts of synlTP and concentrated supernant from Kc l cells transformed with preproITP cDNA (named KcITP), probed with antibody N- l (1/10000). The KcITP contains two immunoreactive peptides: a major band that co-migrates with synlTP, and a minor band which migrates more slowly. The amino acid sequencing of the first 22 amino acids of the two bands of KcITP shows that they have exactly the same N-terminus (Pfeifer et al, 1999). The reason for the two bands will be discussed later. The major band concentration of KcITP was estimated by comparing the band density of KcITP with synlTP using NIH image program of densitometric analysis, and found to be 6pmol/(xl. Fig. 12B compares different amounts of KcITP with concentrated supernant of mutant DS-ITP expressed in K c l cells, probed with antibody C-l(l/10000). DS-ITP is a mutant changing ITP N-terminal domain (SFFDIQ) to a C H H N-terminal domain (SLFDPA). DS-ITP co-migrates with the major band of KcITP, so it was predicted that DS-ITP has the same size as the major band of KcITP. The reason for only one band of DS-ITP will be discussed later. Comparing ITP N-terminus with its homologue 38 -«420 Fig.11 Agrose gels seperating PCR products of point mutation F2A (A) 1% agrose gel seperate the two DNA fragment, Pa - P 2 6 and F2 5-P 2 (lane 2 and 3), resulting from the first step PCR. The 100bp DNA ladder is shown in lane 1. (B) The DNA fragment B 5 -P 2 6 (lane 2) is the second step PCR product for the mutation F2A. The 100bp DNA ladder is shown on lane 1. Estimated fragment sizes in base pairs are indicated by arrow heads. 39 T a b l e 2 . M u t a n t p e p t i d e s s e q u e n c e a n d D N A s e q u e n c i n g r e s u l t s Name N-terminal amino acid sequence DNA sequencing result KcITP SFFDIQ T C C T T C T T C G A C A T C C A G DS-ITP S L F D P A T C C T T G T T C G A C C C C G C G FD-ITP SFAAIQ T C C T T C G C C G C C A T C C A G S1A AFFDIQ G C C T T C T T C G A C A T C C A G F2A SAFDIQ T C C G C C T T C G A C A T C C A G F3A SFADIQ T C C T T C G C C G A C A T C C A G D4A SFFAIQ T C C T T C T T C G C C A T C C A G I5A SFFDAQ T C C T T C T T C G A C G C C C A G Q6A SFFDIA T C C T T C T T C G A C A T C G C G 40 Fig. 12 Western blotting analysis of supernatants containing ITP and mutant peptides expressed in K c l cells, probed with an antibody specific for the C-terminus of ITP (at 1:10,000 dilution). (A) Comparison of 10, 20, and 40 pmol synlTP (lanes 1-3) and 2, 4 and 8 ul KcITP (lanes 4-6). (B) A comparison of KcITP at 1, 2, 4, 6 ul (lanes 1-4) with DS-ITP at 1, 4, 6 ul (lanes 5-8). (C) Mutant FD-ITP at 1, 2, 4, 6 ul (lanes 5-8) compared with KcITP at 1, 2, 4, 6 ul (lanes 1-4). B 41 synlTP KD 36.9 27.6 15.3 8.7 3.5 KcITP DS-ITP KcITP FD-ITP 42 CHH, F3 and D4 are conserved amino acids, so a mutation at these two conserved amino acids was made, namely FD-ITP (F3D4—>A3A4). Fig. 12C shows the Western blotting of mutant FD-ITP compared with Kc-ITP and probe with antibody C - l (1/10000). This mutant FD-ITP was also expressed by Kc l cells, and the western-blotting shows the concentrated supernatant of FD-ITP has two immunoreactive bands which co-migrate with KcITP. The co-migration of DS-ITP and FD-ITP with KcITP suggests that they are the same size, and the changing of amino acids at the N-terminal of ITP does not interfere with the dibasic cleavage (KR at position 54,55), which result in wild type ITP N-terminus. Point mutations were made to each of the first six residues of ITP individually, namely SI A, F2A, F3A, D4A, 15 A, Q6A (each replaced with alanine), and these mutants were expressed at Kc l cells. Fig. 13 A-D show the Western blotting of each expressed product of six point mutations compared with KcITP and probed with antibody C- l (1/10000). Each point mutation co-migrates with KcITP and is predicted to have the same size as KcITP. This suggests that substitution in any of the first six amino acids at the ITP N-terminus had no effect on the dibasic cleavage site (KR at position 54, 55) in spite of their proximity to the dibasic sequence. Another interesting point is that mutants F2A, F3A have only one band which co-migrates with the major band of KcITP. The possible reason of F2A and F3A having only one major band will be discussed later. Table 3 shows concentrations of different mutation peptides after being concentrated 10-12 times by dialysis, estimated by densitometry as described above. 3. Bioassay Fig. 14 (A-D) compares the dose-response curves of Kc-ITP and mutants SI A, D4A, 15A and Q6A. KcITP or a mutant peptide were added to the hemolymph side of ilea, and Isc was recorded until the Isc reached a maximum. Each dose was tested on 5-6 ileal preparations, and the change of Isc (AIsc) was used to measure the peptide stimulatory activity. The slopes of these dose-response curves were not significantly 43 Fig. 13 Western blots of concentrated supernatants containing point mutant peptides expressed in K c l cells, probed with antibody to ITP C-terminus used at 1:10,000 dilution. (A) Point mutation peptide S1A ( 2, 4, 6, 8 ul; lanes 4-7) compared to KcITP (2, 4, 6 ul; lanes 1-3). (B) A comparison of KcITP (2, 4, 6 ul; lanes 1-3) with mutant F2A (2, 4, 6 ul; lanes 4-6) and mutant I5A (2, 4, 6 ul; lanes 7-9). (C) A comparison of mutant F3A (2, 4, 8 ul; lanes 4-6) to KcITP (4, 6, 8 ul; lanes 1-3). (D) A comparison of KcITP (2, 4, 6 ul; lanes 1-3) to mutant Q6A (2, 4, 6 ul; lanes 4-6) and mutant D4A (2, 4, 6 ul; lanes 7-9). 44 KcITP S1A 28.7-15.6— 8.2 — mm *m _ £SL ft&IHH 3.5 — B 37.6— 28.7— 15.6 — 8.2 -3.5 -KcITP F2A I5A KcITP Q6A D4A KcITP F3A 45 T a b l e 3. C o n c e n t r a t i o n o f m u t a n t p e p t i d e s e x p r e s s e d i n K c l c e l l c o n c e n t r a t e d 12 t i m e s b y d i a l y s i s N a m e C o n c e n t r a t i o n o f m a j o r b a n d ( p m o l / | l l ) SynlTP 5 KcITP 6 DS-ITP 11 FD-ITP 6.6 S1A 8.75 F2A 11 F3A 18 D4A 3 I5A 6 Q6A 2.2 46 Bioassay of mutant S1A, D4A, I5A and Q6A 12 10 E o ri-al S1A B -KcITP - S 1 A 0 1 2 3 concent ra t ion ( logpmol) 14 12 — 10 E o cr ^ 4 D4A • K c I T P • D 4 A 1 2 3 concent ra t ion ( logpmol) I5A 0 1 2 3 concent ra t ion ( logpmol) 12 10 E o & d> 2, o at < 4 Q6A - K c I T P - Q 6 A 0 1 2 3 concent ra t ion ( logpmol) Fig. 14 Dose-response curve showing the stimulatory activity of mutant peptides, compared to KcITP. A. Comparison of the specific biological activity of S1A to KcITP. B. Comparison of the dose response curve of mutant D4A with KcITP. C. Comparison of the mutant I5A increasing the Alsc with KcITP. D. Comparison of the dose-response curve of mutant Q6A with KcITP. There was no significant differece between the effect of mutant peptides with that of KcITP. 47 different, so I concluded the amino acid substitution replacing SI, D4, 15 and Q6 with alanine individually didn't affect KcITP activity. This suggests that the amino acids SI, D4, 15 and Q6 of ITP are not involved in ITP binding to its receptor or activating the receptor. Fig. 15 shows mutants DS-ITP, FD-ITP, F2A and F3A had no effect on ileal short-circuit current and no significant difference compared with control. Control is the effect of Kc l cell culture supernatant without any ITP on the ileal short circuit current. Control supernatant was added to the ileal hemolymph side at the same volume as for mutants. The results clearly show mutant DS-ITP, FD-ITP F2A and F3 A have lost all their stimulating activity. These results indicated that ITP N-terminal domain is required for ITP bioactivity. Substitutions at either F2 or F3 are common to all these mutant forms, thus identifying these two sites as essential. 4. Competitive inhibition test The loss of stimulatory activity in mutants F2A, F3A , DS-ITP and FD-ITP could result from loss of either binding to the receptor or loss of signal transduction ability. When performing competitive inhibition tests, inactive mutants were added to the hemolymph side of ileal one hour before adding KcITP. The short-circuit current (AIsc) for KcITP plus mutation peptide was compared with that of KcITP plus control supernatant (concentrated Kc l cell culture medium). Fig. 16A shows that DS-ITP at 1.26 nmol and FD-ITP at 0.99 nmol didn't reduce the stimulatory activity of KcITP at 0.08 nmol, and this suggests that neither DS-ITP nor FD-ITP binds to the receptor, or that the binding is very weak. Fig. 16B showed that mutant F2A at 0.8 nmol and F3A at 2.05 nmol didn't reduce the stimulatory activity of KcITP at 0.08nmol. The concentration ratio of F2A to KcITP was 10:1, and that of F3A to KcITP was 24:1. Fig. 17 shows the inhibitory effect of mutant peptides F2A and F3A tested at a higher concentration ratio (60:1). F2A did reduce KcITP stimulatory activity about 62%. This indicated that F2A can bind to the receptor but with low affinity. Although F2A can bind to the receptor, it had no stimulatory activity even at very high concentration 48 Biological activity of wild-type and mutated ITP 12 r mean+SE n=5-6 I KcITP 174 pmol Control DS-ITP FD-ITP 0 1862 pmol pmol 990 pmol T r 1 ! F2A F3A 794 2041 pmol pmol Fig.15 Maximum Alsc in ilea stimulated with wild-type ITP (KcITP) and mutated ITP peptides expressed in Kc1 cells. Control is the concentrated cell culture supernatant without any ITP peptide. * Significant difference (P<0.01) vs. control. DS-ITP, FD-ITP, F2A and F3A are not significantly different from the control. 49 DS-ITP and FD-ITP Inhibition Test 8 7 6 I4 # 3 v> < o 1 0 X Control 0.08 nmol KcITP DS-ITP 1.26 nmol I FD-ITP 0.99 nmol B F2A, F3A inhibition 0.08 nmol KcITP E o CT O o (A 1 control F2A F3A 0.8 nmol 2.05 nmol Fig.16 Competitive inhibition tests showing the effects of DS-ITP, FD-ITP (A) and F2A, F3A (B) on ileal short circuit current stimulation by KcITP (0.08 nmol). DS-ITP, FD-ITP, F2A and F3A were added to the hemolymph side of the ilea one hour before administration of KcITP. The concentration ratios are, DS-ITP to KcITP, 15:1; FD-ITP to KcITP, 12:1; F2A to KcITP, 10:1; and F3A to KcITP, 25:1. Control is the effect of KcITP (0.08nmol) in combination with cell culture supernatant without any ITP peptide on ileal short circuit current. No significant differences were found when comparing the effects of mutants with that of control. 50 F2A (3 nmol) inhibition test 10 E u & 1 u (A 4 H 3 2 0.05 nmol KcITP Control F2A 3 nmol B F3A (3 nmol) inhibition E u ri-al ^. o w 14 12 10 8 6 4 2 0 0.05 nmol KcITP I control F3A 3 nmol Fig.17 Inhibitory effect of F2A (A) and F3A (B) at higher amount (3nmol) on ileal short circuit current stimulation of KcITP (0.05nmol). The concentration ratio of F2A or F3A to KcITP is 60:1. Details is described in the legend of Fig.16. * Significant difference (p<0.05) vs. control. 51 (1.5uM); (Fig.15). This suggests that F2 is important for both receptor binding and receptor activation. In contrast, mutation of F3 of ITP affects ITP binding to the receptor. Fig. 17B shows that the AIsc of KcITP plus F3A is insignificantly different from the control. This indicates F3A can't reduce KcITP stimulatory activity, that is, no competition happens between F3A and KcITP even at a high concentration ratio (60:1), so F3 of ITP is essential for ITP binding to its receptor. Table 4 is a summary of results for different mutants bioactivity and the competitive inhibition tests for inactive mutants. Mutants of SI A, D4A, 15 A and Q6A keep the same stimulating activity as KcITP. There are no significant difference in the maximum AIsc and time course. Mutants of DS-ITP, FD-ITP, F2A and F3A show no stimulating activity. The competitive inhibition tests of F2A and F3A were performed at different concentration ratios. Only F2A at high concentration ratio of 60:1 can reduce the KcITP stimulatory activity 62%. 52 Summary of mutant peptide bioactivity and the competitive inhibition test Mutant amount of peptide in 2ml hemolymph side of ilea (nmol) stimulating activity ratio mutant: KcITP inhibition result KcITP 0.174 + S1A 0.174 + D4A 0.174 + I5A 0.174 + Q6A 0.174 + DS-ITP 1.26 — 15:1 — FD-ITP 0.99 — 11:1 — F2A 0.8 — 10:1 — F2A 3.0 — 60:1 + F3A 2.05 — 24:1 — F3A 3.0 — 60:1 — 53 DISCUSSION ITP N-terminus is essential for its bioactivity We have established that ITP is an antidiuretic peptide in Schistocerca gregaria and suggested ITP is important for locust homeostasis (Phillips, 1995). The ITP N-terminal domain (SFFDIQ) contains six amino acids preceding the first cysteine. To evaluate whether this SFFDIQ domain may be essential to bioactivity, I produced a domain swap mutation (DS-ITP) by site-directed mutagenesis, which replaces ITP N-terminal domain SFFDIQ with the N -terminal domain SLFDPA of C H H in Penaeus japonicus. Mutant DS-ITP showed no stimulating activity on ileal short circuit-current (Isc). This result indicated that ITP N -terminus (SFFDIQ) is essential for bioactivity. N-terminal structure-activity relationship has been studied by several groups for a variety of proteins. The N-terminal residues (1-8) of stromal cell-derived factor-1 (SDF-1) have been shown to be important for both receptor binding and functional activation (Crump et al , 1997). Reagan (1995) has demonstrated that the N-terminal region (residues 6-12) of Mas-DH is essential for receptor activation but not receptor binding. By using N-terminal truncated peptides, Wang et al. (1995) have shown that the decapeptide PDVDHVFLRFamide has separated binding and activation regions, and they found VFLRFamide is a strong antagonist of PDVDHVFLRFamide. My study is the first for any member of the large hormone family (CHH, MIH, VIH) to which ITP belongs. Two phenylalanines (F2, F3) in the SFFDIQ domain of ITP N-terminus are crucial for ITP binding to the receptor, and F2 is also important for receptor activation The domain swap mutation demonstrated that the N-terminal domain SFFDIQ is essential for its bioactivity. To evaluate which specific amino acids in the SFFDIQ domain are more crucial for ITP bioactivity, point mutations replacing each amino acid of the SFFDIQ domain with alanine were made by site-directed mutagenesis. Mutants SI A, D4A, 15 A, Q6A showed similar stimulatory activity as wild type KcITP. This indicates that the replacement of SI, D4,15, Q6 individually with alanine do not affect ITP bioactivity; therefore, the residues of 54 SI, D4,15, Q6 of ITP are not important for ITP bioactivity. In contrast, the mutant peptides F2A, F3A had no stimulatory activity, even at high concentration (1.5mM), so the hydrophobic side chains in both positions 2 and 3 are essential for ITP stimulatory activity. The reason for mutants F2A, F3 A and DS-ITP losing their stimulatory activity could result from loss of either binding or signal transduction ability. The competitive inhibition assay was used to test these inactive peptides for their ability to bind to the ileal receptor and thereby inhibit the stimulating activity of wild type KcITP. The inhibition tests show mutant F2A can reduce the KcITP stimulatory activity by 62% at a concentration ratio of 60:1; thus F2A can compete with KcITP for binding to the receptor at a very high concentration ratio of 60:1. In other words, F2A can bind to the receptor but its affinity is much lower than that of KcITP. Although F2A can bind to the receptor, this mutant didn't show any stimulating activity, so residue F2 is important for both receptor binding and activation. Mutant F3A didn't reduce KcITP stimulatory activity even at concentration ratio of 60:1, so mutant F3A can't bind to the receptor at this concentration ratio. This suggests F3 of ITP is essential for ITP binding to its receptor. In summary, the two phenylalanines in the SFFDIQ domain of ITP N-terminus are very important for ITP binding to the receptor. An interesting similarity is reported for the role of two phenylalanines in the integrin a6A subunit. De Melker et al. (1997) demonstrated that the two Phe residues in the conserved GFFKR motif are required for association of the cx6A subunit with (3) and for membrane expression, whereas the other three amino acids are irrelevant, even though KR are the charged amino acids. Further, they suggested the interaction between the subunit oc6A and P] is determined by hydrophobic bond between the non-polar residues in these subunits. Generally, hydrophobic and charged residues in the peptide chain play an important role in receptor binding (Keller et al., 1994). We propose that the two Phe residues in SFFDIQ domain of ITP interact with the receptor by hydrophobic bonds because of their hydrophobic phenol ring. Further mutation of these two residues by replacing Phe with Tyr will be useful to test this prediction. 55 Phe2 and Phe3 of ITP may affect post-translation modification, and D-Phe2 or D-Phe3 results in a minor isomorph of ITP Analyzing western blots of mutant F2A and F3A, another interesting finding is that F2A and F3A show only one immunoreactive band co-migrating with the major band of KcITP (Fig.l3B, 13C), whereas other point mutants such as SI A, D4A, 15 A, Q6A retained the two bands exhibited for KcITP, and had similar stimulatory activity as KcITP (Fig. 13). SynlTP, in contrast, yields only one band comigrating with the major band of KcITP (Fig. 12A), so the minor bands of KcITP and point mutants probably result from post-translation modification. This post-translation modification is most likely to have occurred at Phe2 and Phe3. There are several observations to support this prediction. First, amino acid sequencing of these two bands has shown they had exactly the same N-terminus (22 amino acid); (Wang et al, submitted). KcITP was expressed in Kc l cells transformed with preproITP cDNA. Thus there is no possibility of multiple form of mRNA in the Kc l expression system because no introns are present; therefore the difference between these two bands is probably due to post-translational modification. Second, three point mutations at cys23, cys26 and cys43, which disrupted the three disulfide bridge individually, showed the same two bands as KcITP (King et al., 1998), so the two immunoreactive bands were unlikely to be caused by alternate refolding of the peptides during electrophoresis. Third, truncation mutants of the ITP C-terminus always yielded two bands regardless of the number of amino acid truncated (Wang et al. submitted), suggesting that the post-translation modification occurs around the N-terminus of ITP. KcITP-L has the exactly the same N-terminus as KcITP (amino acid 1-40), and the greatest divergence is among the last 20 amino acids. Yet KcITP-L also showed two bands, which also support the view that modification occurs around the N-terminus of ITP. Finally, mutant F2A and F3A show only one band comigrating with the major band with KcITP. This indicates that either the post-translation modification occurred directly at Phe2 and Phe3 of ITP or that these two amino acids affect a distant post-translation modification. The replacement of Phe2 or Phe3 with Ala abolished this modification so that mutant F2A and F3A 56 had only one band. DS-ITP also had one band which co-migrated with the major band of KcITP. The reason was thought to be that the DS-ITP mutant replaced the Phe2 with Leu, so that the modification on Phe2 was abolished as discussed above. One possible post-translation modification at Phe2 or Phe3 of KcITP is the conversion of L-Phe to D-Phe. This prediction is strongly supported by comparing ITP with its CHH homologues. As described in the introduction, the two isomorphs of CHH-I and CHH-II in crayfish have identical sequences, and the minor isomorph is due to the D-Phe at the third position from the N-terminus (Aguilar et al., 1995). This kind of L-Phe to D-Phe post-translation modification probably also occurs in native ITP (ScglTP) because ScglTP shows two immunoreactive bands. There are a few examples of natural peptides containing a D-amino acid residues in animals. In the early 1980, Montecucchi et al. (1981) described the presence of a D-alanine in position 2 of the opioid peptide demorphin isolated from the skin of the frog Phyllomedusa sauvagei, and they demonstrated that the D-alanine is essential for the biological activity of dermorphin. The same phenomenon has been found in a number of related peptides (reviewed by Lazarus, 1993). A common feature of the D-amino acid residue containing peptides is that the D-amino acid is present at the second position of the end product (Kreil et al., 1994). Lobster CHHs differ, since the D-Phe occurred in position 3 (Soyez et al., 1994). What could be the mechanism of this most unusual posttranslational modification? Heck et al. (1994) reported that the venom of the funnel web spider contained an enzymatic activity that slowly converted L-serine at position 46 to the D-isomer in co-agatoxin, and they named the enzyme as a peptidyl aminoacyl L-D-isomerase. What remains completely puzzling is the specificity of this enzyme (Kreil, 1994). The presence of D-amino acids may serve several purposes. First, new three-dimensional structures can be formed that cannot be built from L-amino acids only. Second, the D-residue may modulate the biological activity of a peptide in a subtle way, thereby increasing the biological diversity encoded by a single gene. Last, the presence of D-amino acids may simply increase the biological half-life of peptides, as bonds adjacent to such residues are not 57 hydrolyzed by most exo- or endoproteases (Kreil, 1994). The functional difference between KcITP minor band and major band is unknown. Some evidence suggested the minor band didn't contribute to bioactivity (Pfeifer et al., 1999). Use of reverse-HPLC to separate and purify the two ITP bands will be essential to solve this problem. Residues of SI, D4, 15 and Q6 in the N-terminal domain are not important for ITP bioactivity, enen D4 is a highly conserved amino acid To identify structural features necessary for activity, many structure-activity relationship studies focus on the investigation of evolutionary conservation of sequence. The mutations at the conserved amino acids were mainly performed on the charged or aromatic residues. For example, in studying flavonol 3-sulfotransferase, Marsdais et al. (1997) found the conserved residues of Lys 134, Try 137, and Try 150 contributed to the structural stability of the enzyme, but Arg 140 is critical for substrate binding. By using site-directed mutagenesis, Roberge et al. (1999) had demonstrated the four conserved aromatic residues in family 10 xylanease were important for substrate binding and catalysis. On the other hand, the conserved amino acids are not always important for structure stability or bioactivity. The three charged amino acids (Glu243, Lys272 and Glu539) that are conserved in the Sacaharomyces cerevisiae uracil permease were studied by Pinson et al. (1999). They found only Lys272 of the three charged residues was important for the transport activity of the transporter. The dose-response curves show mutants SI A, D4A, 15 A and Q6A have the same stimulating activity as KcITP (Fig. 14). This suggests that the residues SI, D4, 15 and Q6 are not important for ITP bioactivity because the replacements of these four residues with Ala individually do not alter their stimulating activity. Comparing the amino acid sequence of ITP with CHH's, the amino acids Phe3 and Asp4 are the conserved amino acids (Fig.6). Mutation of these two conserved amino acids was made, named FD-ITP (F3D4 to AA). FD-ITP showed no stimulatory activity, and this was mainly due to by the replacement of F3 to A, because the mutant D4A still retained the same stimulatory activity as KcITP. In contrast, mutant F3A had no stimulatory activity, so the conserved amino acid D4 is not important for 58 ITP bioactivity. In addition, ITP is only a structural homologue to C H H family; for example, CHH does not stimulate the ITP bioassay. Functional homologues of ITP are not known, so whether D4 is conserved in ITP-like peptides is unknown. Mutations on the N-terminal domain of ITP do not interfere with the dibasic cleavage site ITP is a physiologically active peptide maturated from preproITP of 130 amino acid. The hydrophilicity plot (Kyte and Doolittle, 1982) of the predicted ITP prepropeptides sequences indicated that amino acid 25-40 are hydrophobic and may constitute the central or hydrophobic region of a signal peptide (Ring et al., 1997). ITP is produced by removing the first 55 amino acids from a prepropeptide by the dibasic cleavage at positions 54, 55. Factors that affect the proteolytic cleavage site are thought to be related with the conformation of the surrounding protein sequence. Analyzing the amino acid sequences situated around the putative proteolytic cleavage sites in twenty different biosynthetic precursors of peptide hormones, Rholam et al. (1986) hypothesize that (3-turns including the basic amino acid doublets, flanked by highly ordered secondary structures (either (3-sheet or a-helix) may constitute a minimal requirement for the recognition by the endoproteases involved in the processing of these precursors. Bek et al. (1990) reported that prohormonal cleavage sites are associated with a Q. loop. N-terminal residues of ITP (1-6) are predicted to form a [3-sheet (Ito et al., 1997). Mutations on ITP N-terminal domain do not interfere with the dibasic cleavage because all mutants whether DS-ITP or point mutation peptides, were secreted and had the same size with KcITP when compared by Western blotting, so any mutations at ITP N -terminal didn't alter the dibasic cleavage even the changing amino acid is close to the dibasic cleavage site. F2A has the potential to be ITP antagonist My study examines the structure-activity relationship of the N-terminal domain of ITP. The results show ITP N-terminus is necessary for ITP activity, especially the F2 and F3 are important for ITP binding to its receptor. This suggests that the first six amino acids are 59 the essential part of structure of ITP antagonist for its binding to the receptor. The mutational analysis on ITP C-terminus suggested that animated leucine at the end of ITP C-terminus is also essential for receptor activation (Wang et al., submitted). Further structure-activity relationship study on ITP central region will be useful to design the effective ITP antagonist. To date our studies here, ITP-L and mutant F2A are identified to be the potential antagonists that inhibit ITP stimulation in vitro and at high concentration. In summary, ITP N-terminal domain (SFFDIQ) is important to ITP bioactivity. Among the six amino acid of ITP N-terminus, Phe2 and Phe3 are essential for ITP binding to the receptor, and Phe2 is also important to receptor activation. The other four amino acids SI, D4,15, and Q6 didn't contribute to the ITP bioactivity, even D4 is a highly conserved amino acid. Post-translational modification of conversion L- to D- amino acid probably occurs at Phe2 and Phe3 to yield two ITP isomorphs. Mutation of the ITP N-terminal domain didn't interfere with the dibasic cleavage site in spite of its immediate proximity to the dibasic cleavage site. Mutant F2A has the potential to be ITP antagonist. 60 R E F E R E N C E Aguilar M.B., Soyez D., Falchetto R., Arnott D., Shabanowitz J., Hunt D.F., and Huberman A. (1995) Amino acid sequence of the minor isomorph of the crustacean hyperglycemic hormone (CHH-II) of the Mexican crayfish Procambarus bouvieri (ortmann): Presence of a D-amino acid. Peptides 16, 1375-1383. Audsley N. and Phillips J.E. (1990) Stimulants of ileal salt transport in neuroendocrine system of the desert locust. Gen. Comp. Endocrinol. 80, 127-137. Audsley N., Macintosh C , and Phillips J. E. (1992) Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport. J. Exp. Biol. 173, 261- 274. Audsley N., Macintosh C , and Phillips J.E. (1992) Actions of ion-transport peptide from locust corpus cardiacum on several hindgut processes. J. Exp. Biol. 173, 275-288. Audsley N. (1991) Purification of a neuropeptide from the corpus cardiacum of the desert locust which influences ileal transport. Ph. D Thesis, University of B. C. Bennett O. and Rowley J. (1993) Grasshoppers and Locusts: The plague of the Sahel, in Panos Dossier (Bennett O. ed), Panos Publications Ltd. London. Beyenbach K. W. (1995) Mechanism and Regulation of electrolyte transport in Malpighian tubules. J. Insect Physiol. 41, 197-207. Bungart D., Kegel G., Burdzik S., and Keller R. (1995) Structure-activity relationships of the crustacean myotropic neuropeptide orcokinin. Peptides 16, 199-204. Chamberlin M . E. and Phillips J. E. (1983) Oxidative metabolism in the locust rectum. J. Comp. Physiol. 151, 191-198. Chamberlin M . E. and Phillips J. E. (1988) Effects of stimulants of electrogenic ion transport on cyclic AMP and cGMP levels in locust rectum. J. Exp. Zool. 245, 9-16. Coast G. M . , Kay I., and Wheeler C. H. (1993) Diuretic peptides in the house cricket, Acheta domesticus (L): a possible dual control of Malpighian tubules. Mol. Comp. Endocrinol. 12, 38-66. Coast G. M . , Chung J.-S., Goldsworthy G. J., Patel M . , Hayes T. K., and Kay I. (1994) Corticotropin releasing factor related diuretic peptides in insects. Perspectives Comp. Endocrinol. 61-12. 61 Coast G. M . (1996) Neuropeptides implicated in the control of diuresis in insect peptides. Peptides 17, 327-336. Coast G.M. (1998) The regulation of primary urine production in insects. Recent adv. Arthropod Endocrinol. 189-209. Cox K. J. A., Tensen C. P., Van der Schors R., Van Heerikhuizen H., Vreugdenhil E. , Geraerts W. P. M . , and Burke J. F. (1996) Cloning characterization and expression of a G-protein coupled receptor from Lymnaea stagnalis and identification of a leucokinin-like peptide, PSFHSWGamide, as its endogenous ligand. J. Neurosci. 17, 1197-1205. Crump m.p., Gong J.H., Loetscher P., Rajarathnam K., Amara A., Arenzana-Seisdedos F., Virelizier J-L., Baggiolini M . , Sykes B.D., and Clark-Lewis I. (1997) Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J. 16, 6996-7007. De Kleijn D.P.V., De Leeuw E.P.H., Van Den Berg M.C. , Martens G.J.M., and Van Herp F. (1995) Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus. Biochem. Biophysic. acta 1260, 62-66. De Melker A.A. , Kramer D., Kuikman I., and Sonnenberg A. (1997) The two phenylalanines in the GFFKR motif of the integrin 6A subunit are essential for heterodimerization. Biochem. J328, 529-537. Fournier B. (1991) Neuroparsins stimulate inositol phosphate formation in locust rectal cells. Biochem. Physiol. 99B, 57-64. Francois V . H . (1998) Molecular, cytological and physiological aspects of the Crustacean hyperglycemic hormone family. Recent Adv. Arthropod Endocrinol. 53-70. Girardie J., Girardie A., Hute J. C , and Pernollet J.-c. (1989) Amino acid sequence of locust neuroparsins. FEBS left. 248, 4-8. Hanarahan J. and Phillips J. E. (1983) Mechanism and control of salt absorption in locust rectum. Am. J. Physiol. 244, 131-142. Hanarahan J. and Phillips J. E. (1984) KC1 transport across an insect epithelium: I. Tracer fluxes and the effects of ion substitution. J. Membr. Biol. 80, 15-26. Hanarahan J. and Phillips J. E. (1984) KC1 transport across an insect epithelium: II. Electrochemical potentials and electrophysiology. J. Membr. Biol. 80, 21-Al. 62 Harlow E. and Lane D.; Cuddihy J, editor. Antibodies: a Laboratory Manual. New York: Cold Spring Harbor;(1988) 726 Heck D., Siok C.J., Krapcho K.J., Kelbaugh P.R., Thadeio P.F., Welch M.J., Williams R.D., Ganong A.H. , Kelly M.E. , Lanzetti A.J.et al. (1994) Functional consequences of posttranslational isomerization of ser-46 in a calcium channel toxin. Science 266, 1065-1068. Hegedus D., Pfeifer T., Hendry J., Theilmann D., and Grigliatti T. (1998) A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines. Gene 207, 241-249. Irvine B., Audsley N., Lechleitner R., Meredith J., Thomson B., and Phillips J. E. (1988) Transport properties of locust ileum in vitro: effects of cyclic AMP. J. Exp. Biol. 137, 361-385. Ish-Horowicz D. and Burke J. F. (1981) Rapid and efficient cosmid cloning. Nucleic acids Res. 9, 2989 Ito M . , Matsuo Y., and Nishikawa K. (1997) Prediction of protein secondary structure using the 3D-ID comatibitliy algorithm. Comput. Appl. Biosci 13, 415-423. Joffe S.R. (1995) Desert locust management: A time for change. Washington, D. C. : world band Kataoka H., Toshi A., L i J. P., Carney R. L. , Schooley D. A., and Kramer S. G. (1989) Identification of an allatotropin from adult Manduca sexca. Science 243, 1481-1483. Kegel G., Reichwein B., Weese S., Gaus G., Peter-Katalinic J., and Keller R. (1989) Amino acid sequence of the crustacean hyperglycemic hormone (CHH) from the shore crab, Carcinus maenas. FEBSlett. 255,10-14. Kelly T.J., Masler E.P., and Menn J.J. (1990) Insect neuropeptides: New strategies for insect control. In Pesticides and alternatives (Casida J.E. ed), Elsevier Science Publishers, New York. King D.S., Meredith J., Wang Y.J., and Phillips J.E. (1998) Biological actions of synthetic locust ion transport peptide. Insect Biochem. Mol. Biol. 29, 11-18. Krall S. and Wilps H. (1994) New trends in locust control. In AnonymousEschborn, Federal republic of Germany: Deutsche Gesellschaft fur Technishe Zusammenarbeit, 63 Kreil G. (1994) Conversion of L- to D- amino acids: a posttranslational reaction. Science 266, 996-997. Kyte J. and Doolittle R.F. (1982) A simple method for displaying the hydropathic character of a protein. J Mol. Biol. 157,105-132. Lazarus L.H. and Attila M . (1993) The toad, ugly and venomous, wears yet a precious jewel in his skin (review). Prog. Neurobiol. 41, 473-507. Lehmberg E., Schooley D.A., Rerenz H.J., and Applebaum S.W. (1993) Characteristics of locusts migratoria diuretic hormone. Insect Biochem. Physiol. 133-140. Macins A., Meredith J., Zhao Y., Brock H.W., and Phillips J.E. (1999) Occurrence of ion transport peptide (ITP) and ion transport-like peptide (ITP-L) in orthopeteroids. Arch. Insect Biochem. Physiol. 40,107-118. Maddrell S.H.P. (1971) The mechanisms of insect excretory systems. Adv. Insect Physiol. 199-331. Maddrell S.H.P. (1978) Transport across insect excretory epithelia. Membr. Transport Biol. 239-272. Maddrell S.H.P. and Phillips J.E. (1978) Induction of sulfate transport and hormonal control of fluid secretion in Malpighian tubules of larvae of the mosquito Aedes taeniorhynchus. J. Exp. Biol. 72, 181-202. Marsolais F. and Varin L. (1997) Mutational analysis of domain II of flavonol 3-sulfortransferase. Eur. J. Biochem. 247, 1056-1062. Meredith J. and Phillips J.E. (1988) Sodium-independent proline transport in the locust rectum. J. Exp. Biol. 137, 341-360. Meredith J., Ring M . , Macins A., Marschall J., Cheng N. N., Theilmann D., Brock H.W., and Phillips J. E. (1996) Locust ion transport peptide (ITP): Primary structure, cDNA and expression in a baculovirus system. J. Exp. Biol. 199, 1053-1061. Montecucchi P.C., de Castiglione R., Piani s., Gozzini L. , and Erspamer V. (1981) Amino acid composition and sequence of dermorphin, a novel opiate-like peptides from the skin of phyllomedusa sauvagei. J. Pept. Protein Res. 17,275-283. Montoreano R., Triana F., Abate T., and Rangel-Aldao R. (1990) Cyclic AMP in the Malpighian tubule fluid and in the urine of Rhodnius prolixus. Gen. Comp. Endocrinol. 77, 136-142. 64 Morgan P. J. and Mordue W. (1984) 5-Hydroxytryptamine stimulates fluid secretion in locust Malpighian tubules independently of cAMP. Comp. Biochem. Physiol. 79c, 305-310. Nachman R. J., Coast G. M . , Roberts V. A., and Holman G. M . (1994) Incorporation of chemical/conformational components into mimetic analogs of insect neuropeptides. Insects :Chem. Physiol. Environm. Aspects 51-60. Nicolson S. W. (1993) the ionic basis of fluid secretion in insect Malpighian tubules: advances in the last ten years. J. Insect Physiol. 39, 451-458. Pfeifer T., Hegedus D., Grigliatti T., and Theilmann D. (1997) Baculovirus immediate-early promoter-mediated expression of the Zeocin resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines. Gene 188, 183-190. Phillips J.E. (1981) Comparative physiology of insect renal function. Am. J. Physiol. 241, 241-257. Phillips J.E., Hanarahan J., Chamberlin M . , and Thomson B. (1986) Mechanisms and control of reabsorption in insect hindgut. Adv. Insect Physiol. 19, 329-422. Phillips J.E., Thomson R.B., Peach J.L., Audsley N., and Stagg A.P. (1994) Mechanisms of acid-base transport and control in locust excretory system. Physiol. Zool. 67, 95-119. Phillips J.E. and Audsley N. (1995) Neuropeptide control of ion and fluid transport across locust hindgut. Am. Zool. 35, 503-514. Phillips J.E., Wiens c, Audsley N., Jeffs L. , Bilgen T., and Meredith J. (1996) Nature and control of chloride transport in insect absorptive epithelia. J. Exp. Zool. 275, 292-299. Phillips J.E., Meredith J., Audsley N., Ring M . , Macins A., Brock H.W., Theilmann D., and Littleford D. (1997) Locust ion transport peptide (ITP): function, structure, cDNA and expression. Recent adv. Arthropod Endocrinol. 210-226. Phillips J.E., Meredith J., Audsley N., Richardson N., Macins A., and Ring M . (1998) Locust ion transport peptide (ITP): A putative hormone controlling water and ionic balance in terrestrial insects. Am. Zool. 38,461-470. Phillips J.E., Audsley N., Lechleitner R., Thomson B., Meredith J., and Chamberlain M . (1988) Some major transport mechanisms of insect absorptive epithelia. Com. Biochem. Physiol. 90A, 643-650. 65 Pinson B., Chevallier J., and Urban-Grimal D. (1999) Only one of the charged amino acids located in membrane-spanning regions is important for the function of the Saccharomy Cerevisiae Uracil permease. J. Biochem. 339, 37-42. Reagan J.D. (1994) Expression cloning of an insect diuretic hormone receptor member of the calcitonin/secretion receptor family. J. Biol. Chem. 269, 9-12. Reagan J.D. (1995) Functional expression of a diuretic hormone receptor in baculovirus-infected insect cells: evidence suggesting that the N-terminal region of diuretic hormone is associated with receptor activation. Biochem. Mol. Biol. 25, Reagan J.D. (1995) Functional expression of a diuretic hormone receptor in baculovirus-infected insect cells: evidence suggesting that the N-terminal region of diuretic hormone is associated with receptor activation. Insect Biochem. Mol. Biol. 25, 535-539. Reagan J.D. (1996) Molecular cloning and functional expression of a diuretic hormone receptor from the cricket, Acheta domesticus. Insect Biochem. Mol. Biol. 26, 1-6. Ring M . , Meredith J., Wiens c, Macins A., Brock H.W., and Phillips J. E. (1998) Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-L) in a baculovirus/insect cell system. Insect Biochem. Mol. Biol. 28, 51-58. Roberge M . , Shareck F., Morosoli R., Kluepfel D., and Dupont C. (1999) Characterization of active-site aromatic residues in xylanase. Protein Engineering 12, 251-257. Schagger H. and Von Jagow G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-379. Soyez D., Van Herp F., Rossier J., Le caer J-D., Tensen C P . , and Lafont R. (1994) Evidence for a conformational polymorphism of invertebrate neurohormones. J. Biol. Chem. 269, 18295-18298. Spring J H. and Phillips J.E. (1980) Studies on locust rectum. I. Stimulants of electrogenic ion transport. J. Exp. Biol. 86, 211-223. Thomson R.B., Thomson J.M., and Phillips J.E. (1988) NH4 + transport in an acid secreting insect epithelium. Am. J. Phsiol. 254, 348-356. Wang Z., Orchard I., Lange A.B., and Chen X. (1995) Binding and activation regions of the decapeptide PDVDHVFLRFamide (SchistoFLRFamide). Neuropeptide 28,261-266. 66 Williams D., Phillips J.E., Peince W., and Meredith J. (1978) The source of short-circuit current across locust rectum. J. Exp. Biol. 11,107-122. Ziltener H.J., Clark-Lewis I., Hood L.E. , Kent S.B., and Schrader J.W. (1987) Antipeptide antibodies of predetermined specificity recognize and neutralize the bioactivity of the pan-specific hemopoietin interleukin 3. J. Immunol. 138, 1099-1104. 

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