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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  ZHAO  Bachelor of Medicine, Beijing Medical University, P.R.China, 1989 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF T H E REQUIREMENTS FOR  T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES D E P A R T M E N T OF Z O O L O G Y  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A February ©Ying  2000  Zhao, 2000  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  University  of  British  Columbia, I agree  available for  copying  of  this  department publication  or of  reference and study.  thesis by  this  for  his thesis  scholarly  or  her  for  Department  of  ~Z<o & L&  f  The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  ~£eA)•  2%  .  I further  purposes  the  requirements that the  for  Library  an  It  gain shall not  be is  granted  by  understood be  allowed  advanced  shall make  agree that permission for  may  representatives.  financial  permission.  of  the that  without  it  extensive  head  of  my  copying  or  my  written  ii ABSTRACT:  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 C H H (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  TABLE OF CONTENTS Page Abstract  ii  Table of contents  iii  List of Figures  vi  List of Tables  viii  List of Abbreviations Acknowledgments  ix 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  D N A electrophoresis  26  L M P (low melt point) agarose electrophoresis  26  Preparation insert and vectors  26  Ligation and transformation  28  Plasmid extraction and purification  29  K c l 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 D N A fragments and mutation D N A 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 Two Phes in the N-terminus of ITP are important for ITP binding to the  53  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  REFERENCES  58  60  vi  LIST O F FIGURES 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 D N A fragments for point mutation F2A  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  •  38  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 K c 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  LIST OF TABLES Table 1  List of primer sequences and corresponding melting temperatures  Table 2  Different mutations in ITP N-terminus and their D N A sequencing results  24  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 ABBREVIATIONS +  -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 D N A  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  DH  -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  K c l cell  -Drosophila  KcITP  -wild type ITP expressed in K c l cells  KcITP-L  -wild type ITP-L expressed in K c l cells  kD  -kilodaltons  KLH  -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  cell line  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  TEMED  -N,N,N' ,N' -tetra-methyl-ethylenediamine  Tm  -melting temperature  VGF  -ventral ganglia hormone  VIH  -vitellogenesis-inhibiting factor  Xll  ACKNOWLEDGMENTS 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 knowledgeable discussion, patience and his strong support.  understanding,  1  INTRODUCTION 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" to 10" M); 3) they can be easily engineered 9  15  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 N a . 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 N H ) . +  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 N a (reviewed by Nicolson 1993, Beyenbach 1995). +  across the luminal membrane occurs via H / K +  +  or H / N a +  +  Cation movement  antiports, the driving force  being a proton gradient established by a V-type proton ATPase. Cations enter the cell basolaterally through K and N a 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, S0 ", M g ) which may be 2  2+  4  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  Malpighian  Fig.l  l o c u s t a n d its e x c r e t o r y s y s t e m .  tubules  T h e M a l p i g h i a n tubules and  constitute 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 the  square.  hindgut  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 K C L  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 hyperosmotic 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 N a (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. N a 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 ") to the haemocoel side. 3  Ammonia and H  +  secretion, and concomitant H C 0 " absorption, contribute to acid-base regulation in the 3  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 p H 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. water.  For example, the desert locusts can survive several days without food or  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  Stimulation of gut stretch receptors could in turn activate the release of Diuretic Hormone from the CC.  Diuretic Hormone • CRF-DH • Kinnins  ^J^  w  • Serotonin  Anti-Diuretic Hormone CTSH NPS  Osmosensitive cells in the Hemolymph could trigger the release of Anti-Diuretic Hormone from the CC.  ITP  Inhibitory Neuromodulator ITP-L  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 CRFrelated 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 A M P as a second-messenger in the action of CRF-related peptides (Coast, 1998). Receptors for Manduca-DH and AchetaDP 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 -Xaa -Xaa -Trp -Gly-NH , which is all that is needed for activity. 1  2  3  4  2  This 'active core' probably adopts a p-turn when interacting with receptors, bringing together Phe'and Trp , which are critical for activity (Nachman et al., 1995, Coast, 1996). 4  Kinin appear to act via a Ca -dependent mechanism (Coast, 1998). 2+  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). A n 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 N C C 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 C C , 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.  C T S H 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 fourstep 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 and no effect on rectal J sc  v  or I , suggesting that different factors (e.g. C T S H and/or neuroparsins) may regulate ion K  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 channel).  +  (ion channel) and N a  +  (ion  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 His His Gin Lys ATG CAC CAC CAG AAG  Gin Gin Gin Gin Gin Lys Gin Gin Gly Glu CAG CAG CAG CAG CAG AAG CAG CAG GGA GAG  16 Ala Pro Cys Arg His Leu Gin Trp GCT CCG TGC CGA CAT CTC CAG TGG 31 Val Ala Cys Val Leu Val GTC CTC GTC GTA GCT TGC  Arg Lys Ser Gly CGG TTA TCA GGG  Ser Leu Val AGC CTC GTT  Val Val Leu GTC GTC CTC  Ser Thr Ala Ala TCC ACG GCG GCT  Ser Ser TCC AGC  46 Pro Leu Asp Pro His His leu Ala CCG TTG CAT CCA CAC CAC CTT GCC  M Lys \AA  Arg Ser AGG TCC  Phe Phe TTC TTC  Asp He GAC ATC  61 Gin Cys Lys CAG TGT AAA  Ser He Phe AGC ATC TTT  Ala Arg GCA CGC  Leu Asp CTA GAC  Gly Val Tyr Asp Lys GGA GTT TAC GAC AAG  76 Glu Asp Arg He Cys Cys CGC ATC TGC GAA GAT TGC  Tyr TAC  Asn Leu AAC CTA  Phe Arg Glu TTC CGC GAA  •a  91 Ser Leu Cys His Arg Ser Asp Cys CAC TCT CTG TGC AGA TCT GAC TGT  Phe TTC  106 Gly Cys GGT TGT  He Asp Glu Glu ATT GAT GAA GAA  Leu Gin Ala Leu Leu CTT CAG GCA CTA CTT  Leu CTG  Pro Gin Leu CCT CAG CTC  Lys Ser Pro Tyr AAG AGC CCA TAC  121 no Asn Gin Met Val Glu He Leu Gly Lys Lys AAC CAA ATG GTG GAA ATA CTG GGG AAG AAG  END TAG  Phe Lys TTC AAA  Glu Lys GAA AAA  Phe TTT  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 712, 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 C H H (Kegel et al., 1989), namely residues 7-43,2339 and 26-52 (Meredith et a l , 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" M to 10" M (Meredith et al., 1996). By contrast, 7  6  purified ScglTP causes maximum response at 2.43x10" M. 12  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  ITP  S. g r e g a r i a  KRSFFDIQCKGVY. DKSIFARLDRICTaX^TNLFREPQLHSLCRSDCFKSPYFKGCLQALLLIDEEEKFNQMVEILGKK  ITP  L. M i g r a t o r i a  KRSLFDPACTGIY. DRQLLRKLGRLCDDCTflNIOTREPKVATGCRSNCYHN^  SGP-III  KRSLFDPSCTGVF. DRQLLRPJjGRVCDrKlFNWREPWATECRSNCYNNPWRQCMAYVVPAHLHNEHREAVQMVGK  SGP-1  P.japonicus P. japonicus  SASFIDNTCRGVMGNRDIYKKVVRVC32DCTNIFRLPGLDGMCRNRCFYIffi  SGP-IV  KRQVFDQACKGIY. DRAIFKKLDRVCEDCYNLYRKPWATTCRQNCYANSWRQCLDDLLLIDVLDEYISGVQTVGK  CHH  0. 1imosus  KRQVFDQACKGVY. DRmjFKKLDRVCETX^rMjYRKPFVATTCRENCYSNWVFRQCLDDLLLSDVIDEYVSNVQMVGK  CHH  H.americanus  KRQVFDQACKGVY. DRNLFKKLDRVCEDCYNLYPJCPFVATTCRENCYSNWWRQCLDDLLLSDVIDEYVSNVQMVGK  CHH (A) H. americanus  KRQVFDQACKGVY. DRNLFKKUSIRVCRr<rfNrLYRKPFIOTTC  CHH (B) H. americanus  P. japonicus  KRSLFDPSCTGVF. DRQLLRRLRRVCDDCFNVFREPWSTECRSNC^fNNEWRQCMEYLLPPHLHEEHRLAVQW  CHH  P.vannamei  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  CHH  Armadillidium  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  CHH'  P. c l a r k i i  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  CHH  P. b o u v i e r i  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. S R F L K M A N S A L S  MIH-like  P.vannamei  QVFDQACKGIY. DRAIFKKLELVCDDCYNLYRKPWATTCRENCYANSVFRQCLDDLLLINVVDEYISGVQIVGK  MIH  P. b o v i e r i  SFIDNTCRGVMGNRDIYKKWRVCI^TNIFRLPGLIXSMCRNRCFYNEWFLICLKAANREDEIEKFRW  MIH  P. japonicus  RYWEECPGVMGNRAVHGKOTRVC32DCYNWRDTDVIAGro  MIH P. c l a r k i i  AARVINDECPI^IGNRDLYKKVEWICEDCSNIFRKTGMASLC^  MIH  C. maenas  AARVINDIXTPNLIGNRDLYKKVEWICDDCM  MIH  C. sapidus  RVINDrXTPNLIGNRDLYKKVEWlCEDCSNIFRNTGMATLCRKNCFFNEDFLWC^ SAWFTN  . C PGVMGNRDLYEKVAWCMDOVNIFRNNDVGVMCKKD^  SAVffTOTECPGWGNRDLYEKVAWCNIX^IFR^  MIH ILR  VIH  C. pagarus 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  Amino acids with bold face are highly conserved  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 show that synlTP  C C homogenates (Macins et al., 1999).  These results  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 c D N A library and is identical to ScglTP cDNA except for a 121-bp insert at position 95 to 135, suggesting alternative splicing of genomic D N A (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 N C C , 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 Drosophila K c l cell line.  in the  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 K c l cells (Wang et al., submitted). The dose-response curves of the mutants show that K c I T P .  KK  has the same stimulating activity as KcITP, but  KcITP "GKK  has reduced stimulatory activity by two orders of magnitude.  Further  truncation (-LGKK, -ILGKK, and - M V E I L G K K ) abolished all biological activity. competitive inhibition test shows that K c I T P .  LGKK  A  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 C H H 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 3 (F) and 4 (D) residues of ITP N-terminal are conserved amino acids rd  th  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 and 4 amino acids were made by replacing F D with A A . rd  th  •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. were tested for their bioactivity as stimulants or as inhibitors.  These mutants  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  MATERIALS AND METHODS 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, P 6 and P , P ) were used to produce two D N A 2  25  2  fragments, which are diagrammed in fig. 7, namely Pa-P and P 5-P . Primers P 26  2  2  2 5  and P 6 2  introduce the substituted nucleotides (solid square) into the two D N A fragments. Pa-P  26  fragment contains the substitute nucleotide and the upstream preproITP sequence. P 5-P 2  fragment contains nucleotide coding for the same substitution preproITP sequence. In the second step PCR, fragments Pa-P  26  2  and down stream  and P 5-P hybridize the 2  2  length of the perfectly matched nucleotides (15-18 nucleotides) and form the sense and antisense primer to elongate the two D N A fragment to the full length of 405 bp. In addition, two primers, Pa and P encoding the 5' and 3' ends of preproITP help to 2  amplify the full length D N A which now contains the substituted nucleotides. mutated D N A was subcloned into vector p2Zop2F transformation to insect cell line. PCR conditions  This  for plasmid proliferation and  24  Table 1. List of primer sequences and corresponding melting temperatures (Tm) Peptide  KcITP  DS-ITP  FD-ITP  F2A-ITP  I5A-ITP  Q6A-ITP  D4A-ITP  S1A-ITP  F3A-ITP  Primer  Primer sequence (5'—>3')  Tm  A  CGACCTCGAGACGATGCACCACCAGAAGCA  60  CGGAATTCTCTAGACTACTTCTTCCCCAGT  48  Pl9  TTCTTGTTCGACCCCGCGTGTAAAGGAGTTTACGAC  52  P20  CGCGGGGTCGAACAAGGACCTTTTGGCAAG  50  P23  TCCTTCGCCGCCATCCAGTGTAAAGGAGTT  50  P  CTGGATGGCGGCGAAGGACCTTTTGGCAAG  54  P25  AGGTCCGCCTTCGACATCCAGTGT  44  P26  GATGTCGAAGGCGGACCTTTTGGCAAG  46  P27  TCGACGCCCAGTGTAAAGGAGTTTAC  50  P28  TTTACACTGGGCGTCGAAGAAGGACCT  46  P29  GACATCGCGTGTAAAGGAGTTTAC  50  P30  TCCTTTACACGCGATGTCGAAGAAGGA  48  P31  TTCTTCGCCATCCAGTGTAAAGGAGTT  50  P32  ACACTGGATGGCGAAGAAGGACCTTTT  46  P33  AAAAGGGCCTTCTTCGACATCCAG  46  P34  GTCGAAGAAGGCCCTTTTGGCAACGTG  46  P35  TCCTTCGCCGACATCCAGTGTAAA  46  P36  CTGGATGTCGGCGAAGGACCTTTTGGC  46  P  2  24  25  Mutation  strategy  P25  Pa  3' ITP 5' ITP  -—DLT  "P2  P26  first step PCR  f  P25  P26  P2  second step PCR Pa  P26  P25  P2  3' mutant 5' mutant  Fig. 7 Mutation strategy showing the principle of site-directed mutagenesis. Point mutation F 2 A is used a s an example. T w o step P C R reactions were performed. In the first step P C R , two D N A fragments labeled a s P a - P 2 6 and P 2 5 - P 2 are produced. T h e substituted nucleotides are introduced into the two fragments by primers P 2 5 and P 2 6 . E a c h of the two fragments containing the substituted nucleotides is part of ITP c D N A . In the s e c o n d step P C R , the whole length D N A of mutation F 2 A is produced by using the two D N A fragments ( P a - P 2 6 and P25-P2) a s templates and P a , P 2 a s primers. wild type ITP nucleotide substituted nucleotide  ITP s e q u e n c e •  primer  elongated D N A  26  The polymerase chain reaction was conducted in a total volume of 100 ul. This contained 1 ng D N A template, 50pmol of each primer, 0.2 m M of each dNTP, P C R buffer (20 m M Tris-HCI, 0.1 M KC1, 0.1 m M E D T A , pH 8) and 2.5 units of TaqDNA polymerase (Boehringer Mannheim).  The reaction was carried out using a Perkin  Elmer/Cetus D N A 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 D N A 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 D N A 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 D N A ladder or X D N A 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 L M P agarose electrophoresis was used to purify the D N A fragments from both the first and the second PCR reaction. The gel was made from L M P agarose powder (Gibco BRL) in T B E buffer. After mnning this L M P 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 D N Afragmentpurified 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  IE-2  Promoter  DC  E  5= CO CO "0 O O X <  = £  CO 8 CO LU  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 D N A 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 E M - 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 D N A fragments were digested with the EcoRI and Xhol enzymes (Boerhringer Mannheim). The reaction volume was 50 ul, containing 50 m M Tris-HCI, 100 m M NaCl, 10 m M MgCl , 1 m M dithioerythritol 2  (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 L M P 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 D N A ligase (Gibcol BRL). The reaction mixture contained 50 mM Tris-HCI, pH 7.6, 10 m M MgCl , 1 m M A T P , 1 m M D T T and 5% polyethylene 2  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 D H - 5 a cells were plated on L B 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 L B culture medium containing 0.0025%) Zeocin. These were allowed  29  to grow in 3 ml L B medium for 12 hours at 37°C with vigorous shaking to replicate plasmid D N A . 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 L B medium with 0.0025% Zeocin and large-scale plasmid extraction and purification was carried out using a Qiagen (manufacture) plasmid purification kit. A n aliquot was digested to ensure that the appropriate sized fragment was in the cloning site of P2Zop2F.  The insert sequence was confirmed by automated  D N A sequencing using the ABI AmpliTaq (FSTaq) dye terminator cycle sequencing system and a 373 D N A automated sequencer (U.B.C. Nucleic Acid/Protein Service unitNAPS). II. K c l cell expression system Plasmid (p2Zop2F) containing the mutated D N A fragments were transfected into Drosophila K c 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 K c l cells 7  were allowed to grow to 80-90% confluence in a 75 ml T flask. Then D N A (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 D N A and cellfectin was added into the T75 flask containing the washed cells, and incubation continued 8-10 hours at 25°C to allow D N A 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-bisacrylamide (%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 K D 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). were run on a vertical gel apparatus (Tyler) at 60V for 16-20 hours. Tank transfer system  Gels  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 m M 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 E C L reagents (Amersham) for 1 min. at room temperature. The excess liquid was removed and  32  1 I  T  P  ITP-L  MHHQKQQQQQ  KQQGEAPCRH  LQWRLSGWL  CVLWASLVS  TAASSPLDPH  MHHQKQQQQQ  KQQGEAPCRH  LQWRLSGWL  CVLWASLVS  TAASSPLDPH  1  51  ANTIBODY N-l  HLAKR'SFFDI  QCKGVYDKSI  HLAKRSFFDI  QCKGVYDKSI  l  u  i  FARLDRICED FARLDRICED  CYNLFREPQL  HSLCRSDCFK  CYNLFREPQL  HSLCRKDCFT  ANTIBODY C - l  SPYFKGCLQA  LLLIDEEEKF  NQMVEILGKK  SDYFKGCIDV  LLLQDDMDKI  QSWIKQIHGA  EPGV  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 S 0 , 10 M g S 0 , 10 2  4  4  N a H C 0 , 5.0 CaCl , 10 glucose, 100 sucrose, 2.9 alanine, 1.3 asparagine, 1.0 arginine, 5 3  2  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 . This saline was used in all 2  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 at 22±2°C. 2  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 opening by 2  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 56 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 m e a s u r i n g 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 simplified m o d e l e x p l a n i n g the m e a s u r m e n t of short circuit current (Isc). V t r e p r e s e n t s a voltmeter; Isc, m i c r o a m m e t e r . R is a v a r i a b l e r e s i s t a n c e a n d 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 D N A bands produced from the first step PCR. Here, I used point mutation F2A as an example. Two D N A 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 D N A 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 K c 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 and F2 -P (lane 2 and 3), resulting from the first step PCR. The 100bp DNA ladder is shown in lane 1. (B) The DNA fragment B -P 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. 26  5  2  5  2  39  Table 2. M u t a n t peptides sequence and D N A sequencing results  Name  N-terminal amino acid sequence DNA sequencing result  KcITP  SFFDIQ  TCCTTCTTCGACATCCAG  DS-ITP  SLFDPA  TCCTTGTTCGACCCCGCG  FD-ITP  SFAAIQ  TCCTTCGCCGCCATCCAG  S1A  AFFDIQ  GCCTTCTTCGACATCCAG  F2A  SAFDIQ  TCCGCCTTCGACATCCAG  F3A  SFADIQ  TCCTTCGCCGACATCCAG  D4A  SFFAIQ  TCCTTCTTCGCCATCCAG  I5A  SFFDAQ  TCCTTCTTCGACGCCCAG  Q6A  SFFDIA  TCCTTCTTCGACATCGCG  40  Fig.  12  Western blotting analysis  of  mutant peptides expressed in K c l cells,  supernatants  containing  probed with an antibody  ITP and specific  for the C-terminus of I T P (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 58). (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).  41  synlTP  KcITP  B  KD 36.9  27.6 15.3  8.7 3.5  KcITP  DS-ITP  FD-ITP  42 C H H , F3 and D4 are conserved amino acids, so a mutation at these two conserved amino acids was made, namely FD-ITP (F D —>A A ). 3  4  3  4  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 K c l cells, and the westernblotting 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 K c 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 I T P 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  B KcITP  S1A  F2A  I5A  KcITP  Q6A  D4A  37.6— 28.7—  28.715.6— 8.2  KcITP  15.6 — — mm *m _ £SL ft&IHH  8.2 -  3.5 — 3.5 -  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 cell concentrated  Name  12  times by  in K c l  dialysis  Concentration o f major band  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  (pmol/|ll)  46  Bioassay of mutant S1A, D4A, I5A and Q6A B  S1A  12  D4A  14 12  10 —  10  -KcITP E o rial  -S1A  E o cr  •KcITP •D4A  ^  0  1 2 concentration  4  1  3 (logpmol)  2  concentration  I5A  3  (logpmol)  Q6A  12  10  -KcITP  E o  -Q6A  &  d> 2, o at  4  <  0  1  2  concentration  3 (logpmol)  0  1  2  concentration  3 (logpmol)  Fig. 14 Dose-response curve showing the stimulatory activity of mutant peptides, compared to KcITP. A. Comparison of the specific biological activity of S 1 A 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 Q 6 A 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 K c 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 K c 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 mean+SE n=5-6 r  I  T KcITP  Control  DS-ITP  FD-ITP  174 pmol  0 pmol  1862 pmol  990 pmol  F2A  794 pmol  r ! 1  F3A  2041 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 0.08 nmol KcITP  8 7 6  I  I  X  4  #  3  v> < 1o 0 Control  B  DS-ITP 1.26 nmol  FD-ITP 0.99 nmol  F2A, F3A inhibition 0.08 nmol KcITP  1  E o CT  O  o  (A  control  F2A  F3A  0.8 nmol  2.05 nmol  Fig.16 Competitive inhibition tests showing the effects of DS-ITP, FD-ITP (A) and F 2 A , F 3 A (B) on ileal short circuit current stimulation by KcITP (0.08 nmol). DS-ITP, FD-ITP, F2A and F 3 A 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; F 2 A to KcITP, 10:1; and F 3 A 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 0.05 nmol KcITP 10  E u  &  1  4H  3(A  2  u  Control  F2A 3 nmol  B  F3A (3 nmol) inhibition  14  0.05 nmol KcITP  12 10  E u  8  rial  ^.  6  o w  4  I  2 0  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 F 2 A or F 3 A 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  mutant peptide bioactivity and the competitive inhibition test  amount of peptide in 2ml hemolymph side of ilea (nmol)  stimulating activity  KcITP  0.174  +  S1A  0.174  +  D4A  0.174  +  I5A  0.174  +  Q6A  0.174  +  ratio mutant: KcITP  inhibition result  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 (DSITP) by site-directed mutagenesis, which replaces ITP N-terminal domain SFFDIQ with the N terminal domain SLFDPA of C H H in Penaeus japonicus. stimulating activity on ileal short circuit-current (Isc).  Mutant DS-ITP showed no  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 MasD H 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 G F F K R 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 K R 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 K c l cells transformed with preproITP cDNA.  Thus  there is no possibility of multiple form of mRNA in the K c l expression system because no introns are present; therefore the difference between these two bands is probably due to posttranslational 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 C H H 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 posttranslation 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 A A ) .  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, C H H 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. 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