Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies of a sperm antigen recognized by two monoclonal antibodies McChesney, Patricia 1994

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1994-0515.pdf [ 3.1MB ]
Metadata
JSON: 831-1.0087540.json
JSON-LD: 831-1.0087540-ld.json
RDF/XML (Pretty): 831-1.0087540-rdf.xml
RDF/JSON: 831-1.0087540-rdf.json
Turtle: 831-1.0087540-turtle.txt
N-Triples: 831-1.0087540-rdf-ntriples.txt
Original Record: 831-1.0087540-source.json
Full Text
831-1.0087540-fulltext.txt
Citation
831-1.0087540.ris

Full Text

STUDIES OF A SPERM ANTIGEN RECOGNIZED BY TWO MONOCLONALANTIBODIESByPatricia McChesneyB.A. The University of Colorado, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEINTHE FACULTY OF GRADUATE STUDIESTHE DEPARTMENT OF OBSTETRICS AND GYNAECOLOGYREPRODUCTIVE AND DEVELOPMENTAL SCIENCES PROGRAMMEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1994® Patricia McChesney, 1994Signature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. it is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of ) ( 4The University of British ColumbiaVancouver, CanadaDate X4- 9/DE.6 (2188)Signature(s) removed to protect privacyAbstractThe monoclonal antibodies HSA-5 and HSA-6 were generated by others in Dr. Lee’s labagainst ionophore-treated acrosome reacted human sperm in 1989. They were submitted tothe second WHO workshop for interlaboratory evaluations of their potential to beimmunocontraceptive vaccine candidates, and were concluded to be Thigh priority candidateswin 1989.The two antibodies recognize identical bands on a western blot of crude human spermextract, which indicate that they recognize the same protein (HSAg-516). Indirectimmunofluorescent assays performed in our lab have shown that the HSAg-5!6 is located inthe equatorial region of methanol-fixed human sperm, as well as on the head and tail ofmouse sperm. The antigen was immunolocalized on human sperm by Homyk et al (1993)who showed that HSAg-5/6 was located on both the inner and outer acrosomal membranes.The corresponding antigen from mouse sperm which binds to HSA-5 and HSA-6 was foundto have characteristics identical to those of the human protein as analyzed by western blotassay.The purpose of my project was to study the molecular nature of human HSAg-5/6, andisolate cDNA clones of the corresponding antigen from mouse. This characterizationincluded studying the purified human antigen, and molecular cloning of the homologousprotein from a mouse testis cDNA library.From work performed by Dr. T. Yoshiki, it was established that purified HSAg-5!6 is aprotein with a molecular mass of approximately 6OkD. My studies established that theUisolated cDNA clone expresses the C-terminal region of the protein and accounts forapproximately 13% of the total molecular weight, based upon the approximated molecularweight of the cloned fusion protein compared to the molecular weight of the native HSAg5/6. However, antigenic sites for both monoclonal antibodies lie in this tail end, whichsuggests that the C-terminus of HSAg-5/6 is exposed to the environment such that an immuneresponse occurs.The studies of HSAg-516 described in this thesis not only supplement our currentknowledge regarding sperm-specific proteins, but also aid in development of animmunocontraceptive vaccine.inTable of ContentsAbstract iiTable of Contents ivList of Abbreviations viiList of Figures ixAcknowledgment xIntroduction. 1Natural and Induced Immunoinfertility 1Anti-fertility Vaccines and Advantages 2Criteria for Candidates of Immunocontraceptive Vaccines 2The Anti-Sperm vaccines 4Potential Sperm Antigens for Antifertility Vaccine Development 6HSAg-5/6 9Objectives 17Materials and Methods 18Chemicals 18Iminunofluorescent Assays of Mouse Sperm and Mouse Testis 18Immunoaffinity Chromatography 19Peptide Sequencing of Purified Antigen 20Immunoscreening of Mouse Testis eDNA Library 20Fusion Protein Analysis 22Western Blot 23ivDNA Sequence Analysis.23Results 24Purification of HSAg-516 from Human Semen Extract 24Peptide Sequencing 27Immunoscreening of a Mouse Testis eDNA Library 27Characterization of Isolated cDNA clones 31Fusion Protein Analysis 31DNA Sequencing 34Deduced Amino Acid Sequence 37Sequence Homology To Known Proteins 37Deduced Molecular Size of Fusion Protein 37Predicted Antigenic Regions of Peptide Insert 41Discussion 44Studies of the Monoclonal Antibodies - HSA-5 and HSA-6 44Localization of HSAg-5/6 on Human and Mouse Sperm 44Purification and Studies of HSAg-5/6 46Western Blot analysis of HSAg-516 48Cloning and Molecular Biology of HSAg-5/6 48Immunoscreening 49eDNA Sequence Analysis 49The Deduced Amino Acid Sequence and Fusion Protein Analysis 51Structural Analysis of HSAg-5/6 52Future Studies for HSAg-516 52Conclusions 54Bibliography 56Appendix A 65VList of AbbreviationsHSA-5 Human Sperm monoclonal antibody, group A- 5HSA-6 Human Sperm monoclonal antibody, group A -6HSAg-5/6 Cognate antigen recognized by HSA-5 and HSA-61gM Immunoglobulin, M classIgA Immunoglobulin, A classIgG Immunoglobulin, G classSDS-PAGE Sodium dodecylsulfate-Polyacrylamide Gel ElectrophoresisDNA Deoxynucleic acidRNA Ribonucleic acidcDNA Complementary Deoxynucleic acidPBS Phosphate-buffered salineFITC Fluorescein isothiocyanateHCI Hydrochloric acidLB Luria BenaniE. Coli Escherichia CoilTBS Tris Buffered salineTBS-T Tris buffered saline with 0.5% Tween-20SDS Sodium dodecylsulfateEDTA Ethylenediaminetetraacetic aciddH2O Deionized watervurpm Rotations per minuteHBS-9505 Monoclonal antibody, class M, against Hepatitis BdATP deoxy Adenosine triphosphatedCTP deoxy Cytosine triphosphatedTTP deoxy Thymidine triphosphatedGTP deoxy Guanidine triphosphate.gt11 Species of phage used as a vector for cloningTaq polymerase DNA polymerase isolated from Thermus Aquaticusbp Base pairskb kilobases, or 1000 base pairsUV light Ultraviolet lightPCR Polymerase Chain ReactionTM,Perkin-Elmer CetusBLAST Basic Alignment Search ToolkD kilodaltonEcoRl Restriction Enzyme, also site of cDNA insertion in )gt11 phageUTR Untranslated Region following the termination codon in mRNAASA Anti-sperm antibodyWHO The World Health OrganizationIPTG isopropyl 3-D-thiogal actopyranosidePVDF Polyvinylidene difluorideviiiList of FiguresFig.1 Immunofluorescent Assay of HSA-5 binding to the tail of mouse sperm. [11]2 Indirect immunofluorescent assay of HSA-6 monoclonal antibody binding to frozentesticular sections of adult mouse. [14]3 An SDS-PAGE gel (12% acrylamide) of HSAg-516 purified on an immunoaffinitychromatography column. [25]4 Nitrocellulose membranes displaying plaques of E.Coli lysate containing 8-galactosidasefusion protein which have positively bound to polyclonal HSA-516 antibody and tomonoclonal HSA-5/HSA-6 antibody cocktail. [29]5 Western blot analysis of 6-galactosidase fusion protein. [32]6 Nucleotide sequence of Clone #5 and deduced amino acid sequence. [35]7 Diagram illustrating the 6-galactosidase protein, and the predicted size of the resultingfusion protein. [39]8 Hydrophilicity plot of the deduced amino acid sequence of Clone #5. [42]lxAcknowledgmentWhile several people deserve credit for helping me accomplish this goal, there are two specialpeople who have earned extra gratitude. First, I would like to thank my lab partner and dearfriend Letticia. Without her, I would have not been able to continue my work. To mybeloved husband Darrell, who has supported me through the best and worst of times, I ameternally indebted.Additionally, I would like to thank Dr. T. Yoshiki for allowing me to display his data priorto journal publication (Fig. 1 and Fig. 2).Thank you.xIntroductionNatural and Induced ImmunoinfertilityIt is well known that some sperm anitgens are auto- or isoimmunogenic inhumans.It has been known for almost one hundred years that spermatozoa can cause animmune response when introduced into areas of the body which are not part of thereproductive tract (Landsteiner, 1899; Metchnikoff, 1899). Approxomately 70% ofvasectomized men form anti-sperm antibodies, and 30% of infertility is associatedwith anti-sperm antibodies in either the male or female partner (Naz, 1990).Furthermore, it has been shown that treatment of immunoinfertility withimmunosuppressive agents results in lower antibody titres and often leads to successfulconception (Hendry et al, 1979; Shulinan, 1986).Sperm antigens which lead to antibody formation are expressed after meiosis,when the sperm are protected from systemic circulation by the blood-testis barrier. Asa result, these antigens are foreign to the immune system (Witkin, 1988). The spermare also exposing ‘loreign” antigens to the female reproductive tract. However, itwas observed that ‘autoantibodies to spermatozoa in males and isoantibodies infemales are inhibited both by the physical isolation of spermatozoa from the systemicimmune system and by active immunosuppression mechanisms* (Witkin, 1988)Immune reactions are inhibited in men by T-suppressor cells in semen and in womenby activation of T-suppressor cells following coitus, as well as physical barriers inboth genders (Witkin, 1988).IHowever, introduction of sperm or sperm antigens into the systemic circulationleads to an immune response.Anti-fertility Vaccines and AdvantagesExperiments using spermatozoa as a vaccine to prevent fertility were conductedin the 1920’s and 1930’s. In 1932, Baskin published a report of 20 women who hadreceived three intramuscular injections of fresh semen, seven days apart. In all casesbut one the injections appeared harmless, and sperm cytotoxicity in serum wasmaintained for up to twelve months. There were no pregnancies reported under theseconditions. However, the follow-up period was too short to assess subsequent fertilityor health (Baskin, 1932).Immunocontraception refers to utilizing the immune system to inhibitreproduction, and has a number of advantages over traditional methodsof preventingpregnancy. Vaccines are not pharmacologically active and are specific tothe immunesystem. Use and administration of vaccines can be accomplished by nurses orparamedical personnel with relative ease (Jones, 1982).Criteria for Candidates of Immnnocontraceptive VaccinesDevelopment of an immunocontraceptive vaccine requires considerationof avariety of factors. The most important is identifying an antigen whichwill not inducean immunopathological condition in the vaccine recipient. Therefore, theantigen mustbe specific to the tissues in the reproductive system. It has been suggested that thereare a number of proteins found on sperm surface that are shared with somatic cell2plasma membranes (Naz et al, 1990). For example, a varietyof laboratory inducedASA’s have been shown to cross react with brain(Freund et al, 1955), kidney(Chaffee and Schachner, 1978) and erythrocytes (Kerek, 1974). There is also a riskthat non-specific antibodies could react with soluble proteins normally found in bodyfluids such as milk, saliva and serum. It has beengenerally agreed that the utility ofan antigen as a contraceptive vaccine is contingent upon its tissue specificity, itsinvolvement in fertility and upon a sufficient immune response within the genital tractssuch that fertility is intercepted (Naz et al, 1990).The antigen chosen for immunocontraception should not normally be found inthe recipient (Jones WR, 1982; Griffin D, 1990), or the antigen should be presenttransiently (as in the case of sperm presence in the female reproductive tract).Alternatively, a consistently present antigen may be shown to be in such lowconcentrations that a “low-grade immune response” would be insignificant to thevaccine recipient.The capability of an antibody to intercept fertilityrequires that the antigen beaccessible to the antibody and that the local immune response in the genital tract issufficient to prevent conception. This requirement was demonstrated when a vaccineagainst acrosin was tested (De loannes et al, 1990). Acrosinis a sperm proteinessential to fertilization. The resulting antiserawas shown to prevent in vitrofertilization in mice (Dc loannes et al, 1990). However, when the antibody was usedas a passive vaccine, it was ineffective when systemic immunization was induced infertile female mice (Dc loannes et at, 1990). Despite a high serum antibody titre, theimmune response in the reproductive tract was insufficient to prevent conception invivo (Naz et at, 1990).3The final requirement of an contraceptive vaccine is that it must be produced inmass quantities. The antigen used for the vaccine must be produced under artificialconditions in order to accomodate thehigh demand. For example, it may beexpressed as a fusion protein in a bacteria culture or it may be manufacturedin apeptide synthesizing apparatus. Natural sources of antigen such as human tissues orfluids are impractical for large-scale vaccine production.The World Health Organization’s (WHO) criteria for an ixnniunocontraceptivevaccine suggested that a good vaccinecandidate will (Griffin, 1990):1. provide effective, safe fertility regulation when antigen is eliminated2. be specific to its intended target3. insure that the immune reaction ispresent in a controlled site anddoes not lead to immunopathology4. include an antigen which is presenttransiently or at low levelscompared to the immune response it elicits5. will not elicit undesired responseseg. IgE production or an allergic reaction6. can be chemically characterized andproducedThe Anti-Sperm vaccinesOne of the areas of active research in antifertility vaccines is antisperni agents.The World Health Organization (WHO) has recognized five steps involved in vaccinedevelopment (Griffin, 1990):1. identification of events in reproduction which are accessible toimmune intervention4For example, sperm-zona pellucida bindingand entry of sperm into thefemale reproductive tract are events that are exposed to theimmunesystem and are accessible to intervention.2. identification of molecules which are necessary for reproduction whoseelimination will result in safe, effective antifertility3. development of vaccines utilizing those molecules4. pre-clinical testing of the vaccine for safety and efficacy on lab animals5. clinical testing of the vaccine for safetyand efficacy in humansOne approach to identify sperm proteinswhich are involved in the fertilizationprocess has been antifertility studies or in vitro fertilization experiments. The firststep of the process can be accomplished by immunizing mice with human sperm.Ifthe sperm are fresh then the antigens exposed to the murine immune system are thosefound on the surface of the sperm. The antigens and resulting antibodies haveprovided valuable information about the epididymal maturation of a spermatozoa(Myles et al, 1990), but more research is requiredto prove their efficacy (Mitchison,1990). On the other hand, if the sperm have undergone the acrosome reaction priorto immunizing the mice, then the antigens exposed to the immune system may includethose components which are essential forfertilization or sperm-egg plasma membraneassociations.The procedure of immunizing mice withtarget materials is the primary step ofmonoclonal antibody production. The resulting immune response provides valuableantibody producing fl-cells which can beharvested from the munne spleen. Thesecells are fused with immortal mouse myeloma cells according to established5procedures (Davis et al, 1982).The new hybridoma cellline is cultured and testedfor antibody production (Lee etal, 1984; Lee et al, 1984; Lee et al, 1984). Onceantibody production has been established, then research into the nature of the targetantigen commences.The second step of the vaccine development process (Griffm, 1990) includesstudying those antigens which illicit an immune response, and verification that theantibody production will prevent fertility. After experimenting with a variety ofantibodies and cognate antigens, Lee and Wong (1986) concluded that inonoclonalantibodies which bind tothe acrosomal region of asperm most effectively preventfertilization in vitro and in vivo.Potential Sperm Antigens for Antifertility VaccineDevelopmentThe development of anti-sperm vaccine candidateshas thus far progressed tothe second step of WHO’s program (Griffin, ]entifying antigens which illicitan immune response. Several candidates have Lcflider4 and s;:i.Since 1983, Naz has beenstudying ant ióund on the surface of intactspermatozoa. Antisera against Fertility Antigen-i (FA-l)has been found to reducehuman sperm fusion withzona-free hamster oocytes(Naz et al, 1984). It also inhibitsfertility in female rabbitsimmunized with FA-1 (Naz et al, 1987).Goldberg (1990) reported extensively on lactate dehydrogenase-C4(LDH-C4).This enzyme is a sperm-specific (Wheat et al, 1983; Lianget al, 1986) isomer oflactate dehydrogenase and is found mainly on e tail of sperm (Goldberg, 1963;Blanco et al, 1963). This enzyme is not produced in females, and is isolated from the6male immune system by the blood-testis barrier. When used as a vaccine, LDH-C4provokes an immune response in bothgenders. However, active immunization againstthis protein in female mice, rabbits andbaboons resulted in only partial fertilitysuppression (Goldberg, 1975; Wheat et al, 1983).Two antigens found in guinea pig sperm, called PH-20 and PH-30 have beenstudied by Primakoff and Myles (Myles et al1984, Primakoff et al 1985). PH-20 is a64kD glycoprotein located on the postacrosome of guinea pig sperm. Nucleicacid/peptide sequence analysis revealedthat it is a cell adhesion related protein(Blobelet al 1992). Active immunization against PH-20 resulted in complete, reversiblesterilization of either male or femaleanimals (Primakoff et al 1988). PH-30 consistsof two subunits, and is essential for sperm-oocyte fusion (Blobel et al 1992).Immunization against PH-30 resulted inpartial fertility suppression.Leyton et al (1992) have studied a 95kD mousesperm protein called P-95.This protein has been identified as a putative receptor with tyrosine kinase activity. Itbinds ZP-3 on mouse zonae pellucidae, and may participate in regulation ofgameteinteraction. P-95 is considered an excellent target for inimunoregulation offertility.However, a homologous human antigenhas not been fully studied.Rabbit Sperm Autoantigen (RSA) is a zona-pellucida binding protein isolatedfrom rabbits by O’Rand (O’Rand et al, 1988).This lectin-like protein binds to thezona pellucida with high affinity (O’Rand et at, 1988) and has a molecular weight of65-7OkD. Western blot analyses usinganti-RSA antibodies have shown thatsimilarproteins are present in human and pig sperm (O’Rand et al, 1988). Interestingly,O’Rand has found a ten amino acid peptide sequence, called P1OG, withinthe RSAfamily which represents an autoantigenic epitope that specifically binds torabbit7autoantisera (O’Rand and Widgren, 1989). They have shown by ELISA thatantibodies to RSA and P1OG bind to human sperm lysate in a dose dependent manner(O’Rand et al, 1990). More studies are required to isolate the human RSA analogprotein to determine its zona binding capabilities and structural similarity toRSA andthe P1OG peptide (O’Rand et al, 1990).Herr et al have been studying a spermspecific protein called SP-10, recognizedby the monoclonal antibody MHS-10(Herr et al, 1990 a-d; Homyk et al, 1990; Writeet al, 1990). This antigen is located in the intra-acrosomal region of human sperm.Western blot analysis demonstrated SP-10 to be a group of proteins ranging froml8kD to 34kD (Herr et al, 1990a). WHO-sponsored interlaboratory studies of theMHS-10 monoclonal antibody showedthat it inhibited interactions betweensperm andzona-free hamster oocytes (Anderson et al, 1987).MSA-63 is an antigen found in the acrosome of mouse sperm and binds tothemonoclonal antibody HS-63 (Liu et al, 1989).HS-63 was shown to inhibit in vitrofertilization of mouse (Lee et al, 1990). HS-63 was also shown to inhibit bindingofhuman sperm to zona-free hamster oocytes. The purified MSA-63 is comprised ofthree major soluble glycoproteins with molecular weights of 5OkD, 43kD and 42k1)(Liu et al, 1989; Lee et al, 1990). MSA-63 has also been cloned from a mouse testiscDNA library. The full length eDNA is 1067 bases in length and includesa poly-Atail region and an open reading framewith 261 amino acids. The predictedproteinsize of MSA-63 is 27.9kD (Liii et al, 1992).A spernilplacenta cross reacting antigen called STX-10 has been studied(Leeet al, 1993). This 75kD group.of glycoproteins is found on the acrosome ofmethanol-fixed human sperm and is recognized by the monoclonal antibodyHSA-108(Lee et al, 1993). Human spermpenetration of zona-free hamster oocytes wasreduced by nearly 75% in the presence of HSA-1O when compared to an unrelatedantibody used as a negativecontrol (Lee et al, 1993). HSA-1O was labeled as awhighpriority” vaccine candidateby the second WHO workshop (Anderson, 1992).HSAg-516The monoclonal antibodiesHSA-5 and HSA-6 were developed in 1989. Theywere two of seven antibodies generated against ionophore-treated acrosome reactedhuman sperm. Both antibodies are 1gM class and have been propagated in BALB/cmice as ascites fluid.Following purification by 40% ammonuum sulfate precipitation and passagethrough a Sephacryl S-300gel filtration column, theantibodies were conjugated toAffigel-lO agarose according to the protocol provided by BioRad Lab. Thisimmunoaffinity column wasused to purify the antigenfrom human semen extract.Rabbit antisera against theaffinity-isolated cognate antigen were raised according tothe standard protocol (Liii et al,1989). The monoclonal antibodyfrom ascites fluidand rabbit antisera were utilized to study the cognate antigen.The HSA-5 and HSA-6 antibodies were submitted tothe second WHOWorkshop for interlaboratory evaluations of their potential to be immunocontraceptivevaccine candidates. Of the69 submissions, only five were considered “high prioritycandidates,” including HSA-5 and HSA-6 (Anderson, 1990).The interlaboratoryevaluations determined thatthe antibodies do not reactwith adult somatic tissue of9human and mouse. It wasalso determined that HSA-5 and HSA-6 react with thesperm of human, mouse and chimpanzee. The antibodies were designated S71 andS72 by WHO Workshop (Anderson, 1992).Furthermore, the monoclonalantibodies were shown to recognize identicalbands on a western blot of crude human sperm extract,which indicated that theyrecognize the same protein (Anderson, 1992; Yoshiki et al, unpublished). However,sandwich enzyme imniunoasssays (SEIA) determined that eachmonoclonal antibodymay recognize different antigenic regions (Yoshiki et al, unpublished).Indirect immunofluorescent assays, performed by Dr. T. Yoshiki, of humanand mouse sperm have shown that the antigen is locatedin the equatorial region of theacrosomal of methanol-fixedhuman sperm (data not shown).Similar assaysdemonstrated binding of themonoclonal antibodies to anantigen localized to the headand tail of mouse sperm. Fig. 1 shows HSA-5 binding to the tail of mouse sperm.10Fig. 1 Immunofluorescent Assay of HSA-5 binding to thetail of mouse sperm.A: Sperm as seen under light microscopyB: Sperm as seen underUltraviolet light400X Magnification, photographic enlargementData courtesy of Dr. T. Yoshiki.11zTIIHSAg-5/6 protein has been localized in the acrosome of thehuman sperm andprimarily on the tail of mouse sperm. The antigen was immunolocalized on humansperm by Homyk et al (1993) using light and transmission electron microscopy. Theirresearch showed that HSAg-516 was located on both the inner and outer acrosomalmembranes.Frozen sections of mouse testis were examined by indirect immunofluorescentassay for antigen binding to HSA-5 and HSA-6. Immunohistological studies of frozenmouse testis sections were performed using the publishedprotocol of Lee and Wong(1986) by Dr. T. Yoshiki (Fig. 2). These studies demonstrated that HSAg-516 isexpressed post-meiotically in the mouse testis.13Fig. 2 Indirect immunofluorescent assay of HSA-6 monoclonal antibodybinding to frozen testicular sections of adult mouse.Note the staining of the sperm tail is apparent in the center of the photo.400X Magnification, phographic enlargement includedData courtesy of Dr. T. Yoshiki.14Ma.t%)Western blot assays were utilized to determine the molecular weight of theantigen recongized by the HSA-5 andHSA-6 monoclonal antibodies (Dr. T. Yoshiki,unpublished observations).Western blot assay revealed that a 60kilodalton (lcD) protein band wasimmunoreactive with monoclonal antibodies HSA-5 and HSA-6 (not shown). Parallelwestern blot analysis of crude spermextract confirmed an immunoreactive band at6OkD. A similar western blot was probed with rabbit antisera raised against thepurified cognate antigen. The antisera recognized multiple bands in crudehumansperm extract ranging from 11.5kD tolO6kD. Both monoclonal and polyclonalantibodies recognized the 60k1) band.The corresponding antigen from mouse sperm which binds to HSA-5 and HSA6 was also found to have characteristics identical to those of the human protein asanalyzed by Western blot assay.Following assessment of the molecular size and location of HSAg-5/6 onhuman and mouse sperm, it became important to study the protein on the molecularlevel. Such studies reveal the molecular nature, sequence, and structure of aprotein.In collaboration with our lab, Dr Mona Homyk, in the laboratory of Dr. J.C.Herr at the University of Virginia have cloned HSAg-516 from a human testis cDNAlibrary (Homyk et al, unpublished observations). They have obtained a partial lengthsequence of the cDNA for this antigen. However, their cDNA fragmentis only 600base pairs (bp) in length and constitutes the C-terminal region of the protein. Forourpart of the collaboration, we set outto perform a similar study using a mouse testiseDNA library.16ObjectivesThe outcome of this project is to characterize the cognate antigen recognizedby the monoclonal antibodies HSA—5 and HSA-6.Characterization of this proteinwill not only supplement current knowledge of sperm physiology, but it will also aidin developing an immunocontraceptive vaccine.The purpose of my portion of the project was to study the molecular nature ofhuman HSAg-5/6, and isolate cDNA clones of thecorresponding antigen gene frommouse. This characterization included examining the antigen as isolated from humansperm extract, and molecular cloning the homologous protein from a mouse testiscDNA library.17Materials and MethodsChemicalsThe monoclonal antibodies HSA-5 and HSA-6 (in both ascites fluid andpurified solution), as well as the polyclonal antisera were generously donated by Dr.C.Y.G. Lee. Triton X-100 was purchased from Sigma (St. Louis, MO). Allanalytic-grade reagents for gel electrophoresis, nitrocellulose and PVDF membranesfor protein blot were obtained from BlO-RAD (Richmond,CA). Embedding mediumfor testicular sections was Tissue TeK1I, O.C.T. Compound No. 4583, from Lab-Tek.Bacterial culture media supplies were fromDifco (Burlington, ON, Canada).Disposable petri dishes were purchased from Fisher Scientific (Vancouver, BC).Molecular biological supplies, fluorescein-isothiocyanate (FITC)-labeied goat anti-mouse/rabbit IgG+IgA+lgM, and horseradish peroxidase labeled goat anti-mouse/rabbit IgO+IgA+IgM were obtained from Gibco/BRL (Burlington, ON,Canada). The )bgtll phage mouse testis cDNA library was a gift from Dr. C. Lau ofthe University of California, San Fransisco. Immunoscreening kits were fromClontech (Palo Alto, CA). Sequencing primers for Agtl 1 were purchased from NewEngland Biolabs (New Hampsire).Immunofluorescent Assays of Mouse Sperm and Mouse TestisFresh semen samples were obtained from the epididymus’ of sacrificed mice.Sperm were washed three times in Ham’sF-b, and allowed to swim up for 30minutes at 37°C, and were diliuted to 1Osperm/mi. Drops of 51d each were appliedto 8-well slides and dried on a hot plate at37°C for approximately five minutes.18Once dry, the sperm were fixed to the slides by immersion in methanol for tenminutes. Blocking solution consisting of 0.5% Bovine Serum Albumin and 0.1 %Thimersol in Phosphate buffered saline (PBS-BSA-TMS) was used to wash the slidesthree times prior to incubation in the solution for ten minutes. The sperm wereprobed with mouse monoclonal HSA-5 orHSA-6 ascites fluid in diluted in blockingbuffer for one hour at 37°C. Following probing the slides were washed three times inthe blocking solution. The primary antibody was detected by Goat Anti-mouse IgG +IgA + 1gM conjugated to FITC for one hour at 37°C.After three final washes, theslides were examined under white and fluorescent light for antibody binding.Similar assays were performed with mouse testicular sections. For theseexperiments, testes were removed from male mice 21 days after birth and frozenimmediately at liquid nitrogen temperatures(approx. - 196°C). They were then placedin an embedding medium at -60°C. Sections of 10am thickness were cut with acryotome and placed on slides. After air drying, the slides were rinsed with PBSBSA-TMS and used for indirect immunofluorescence assay as described above.This work was performed by Dr. T. Yoskiki and is displayed in Fig. 1 and Fig.2.Immunoaffinity ChromatographyAn immunoaffinity column was prepared topurify HSAg-5/6 by Dr. C.Y.G.Lee. Monoclonal antibodies HSA-5 and HSA-6 were conjugated to Affigel-lOagarose according to the protocol provided by BioRad Lab.In my experiments, the column was washed with 20m1 of 0. 1M glycine-HCI(pH =2.2) and equilibrated with 40m1 PBS. Approximately 500nil of crude human19semen extract from frozen stocks, containing an average of 37mg/mi protein, waspassed through the column. After rinsing the column thoroughly with PBS, the boundproteins were eluted with 0.1Macetic acid, or glycine-HCI (pH=2.2).Afterneutralizing the pH with 1M Tris—Ci (pH=8.O) the fractions were quantitatedspectrophotometrically at absorbance of 280nm (O.D.). Samples containing thehighest concentration of proteinwere pooled and further concentrated employingAMICON Centriprep concentrators. The protein samples wereexamined by SDSPAGE using published methods(Laemmli, 1971).Peptide Sequencing of PurifiedAntigenPurified HSAg—5/6 was obtained and run on an SDS-PAGE (12 % acrylamide)gel. The protein bands were electrophoretically transferred to aPVDF membrane in aBlO-RAD transblot apparatus at60 V for three hours. Following transfer, themembrane was rinsed with deionized water and stained with Cooniassie Fast Blue(.025 %) for five minutes. The membrane was then de-stained overnight in a solutioncontaining 50 % methanol and 50% deionized water. The visible bands at l9kD andI 1kD were cut out and allowedto dry at room temperature before being sent to theBiochemistry Department at theUniversity of Victoria for peptide sequencing by theEdman degradation method.Immunoscreening of Mouse Testis cDNA LibraryA single colony of E. Coil strain Y1090 was isolated and grown overnight inLB broth containing 2% maltoseat 37°C with constant agitation.Two hundredmicrolitres of the liquid culture was inoculated with approximately 7.5 x 1020recombinant Agtl 1 phage. The mixture was incubated at 37°C for 20 minutes. LBsoft top agar was added to a volume of7 ml and poured onto LB agar platescontaining 50g/ml Ampicillin. The culture was then allowed to grow at 42°C for2.5 - 3.5 hours. This procedure was repeated for 10-12 150mm LB agar plates.Following the initial incubation, 150mm nitrocellulose filters, previously saturatedwith 10mM IPTG, were overlayed ontothe plates. The plates were then incubated inan incubator at 37°C for an additional 3.5hours to allow growth of phage, expressionof fusion protein, and lysis of the E. Coil. The filters were then removed fromtheplates and incubated in a blocking solution consisting of Tris-buffered salinecontaining 0.5% Tween-20 and 5% powdered skim milk for 1 hour at roomtemperature. Antibody probing of the filters began when both monoclonal antibodiesHSA-5 and HSA-6 (ascites fluid) were added to the blocking solution to aconcentration of 1 l/ml. The filters were incubated in the antibody solutionovernight at room temperature with constant agitation. The next morning, thefilterswere washed in Tris-buffered saline containing 0.5% Tween-20 three times for 5minutes each and incubated in the blocking solution containing goat anti-mouseIgG+IgM+IgA conjugated with horseradish peroxidase (1 iUml) for 3-4 hours.After three further washes with TBS-T, and one with TBS the filters were incubated ina peroxidase substrate solution containing 3,3’-diaminobenzidine for 10 - 20 minutesto develop color.This procedure was repeated with modifications for immunoscreening with therabbit polyclonal antibody HSA-5/6. After the initial filters were removed, a secondIPTG saturated filter was overlayed ontothe same plate. The incubation times andconditions for the filters remained as above. However, antibody probing involved21rabbit antiserum diluted in blocking solution (1 1d/ml) followed by incubation withgoat anti-rabbit IgG+IgM+lgA conjugated with horseradish peroxidase (1 ui/mi).Positive plaques were identified and isolatedfor a second screening by thesame method. Only those plaques which were positive for both antibody probes werechosen for subsequent screenings. AU clones were screened at least four times oruntil plaques were 100% positive.Following immunoscreening, positive clones were confirmed by an additionalscreening using antibodies which had beenpre-absorbed in E. Colt lysate according tothe published method (Sainbrook et al, 1989).Fusion Protein AnalysisRecombinant fusion protein consisting of 6-galactosidase with an insertedpeptide was expressed in the process of the iminunoscreening protocol. Followinggrowth, inoculation by positive Agtl 1 phage clones, and induction of 13-galactosidaseexpression by IPTG, fusion protein was eluted from the LB top agarose for westernblot analysis. Briefly, the top agarose was scraped into a Sml culture tube andincubated in buffer containing sucrose, SDS, Tns, sodium-EDTA, and dHO. Afterovernight incubation at room temperature,the mixture was homogenized andsonicated. The samples were then centrifuged at 10,000 rpm to remove the agaroseand cellular debris. The protein laden supernatant (lysate) was removed and evaluatedby spectrophotometry (O.D.) and western blot analysis (Towbin, 1979).22Western BlotLysate samples containing recombinant fusion protein were obtained and runon an SDS-PAGE gel (10% acrylarnide). The samples were electrophoreticallytransferred to a nitrocellulose membrane in a BlO-RAD Trans-Blot apparatus at 60Vfor 3 hours. Following transfer, the membrane was incubated in a blocking solutionof 5% powdered skim milk and 0.1 % Tween-20 in Tris-buffered saline TBS-T) for 30minutes at room temperature. The membrane was probed with a cocktail of HSA-5and HSA-6 (ascites fluid) diluted to a concentration of 2 tlJml in 5% milk-TBSTovernight at room temperature with constant agitation. The mouse 1gM monoclonalantibody HBS-9505 was used in a parallel experiment as a negative control. Afterwashing the membrane three times in TBST, the antibodies were detected byincubation in Goat Anti-mouse lgG + igA + 1gM conjugated with horseradishperoxidase, 1 jLlJml in blocking buffer for 1.5 hours at room temperature withconstant agitation. Color development was achieved by incubating the membrane in ahorseradish peroxidase substrate solution containing 3,3’-diaminobenzidine.DNA Sequence AnalysisThe cDNA sequence and corresponding predicted peptide sequence of clone #5were sent to the Basic local alignment search tool (BLAST) for a search of publicsequence databases. Databases offered both nucleotide and peptide sequences forcomparison.23ResultsPurification of HSAg-516 from Human Semen ExtractHSAg-5/6 was purified from crude human semen extract by immunoaffinitychromatography. Protein fractions were eluted with 0. 1M glycine HC1 (pH=2.2) or a0. IM acetic acid and were examined by SDS-PAGE (Laemelli, 197 1)(Fig. 3). Threemajor protein bands of molecular weights 11k]), 19k]), and 6OkD were observed,respectively. The results of this analysis are presented in Fig. 3.24Fig. 3 An SDS-PAGE (Laemmli, 1971) gel (12% acrylamide) of HSAg-5/6 purifiedon an immunoaffinity chromatography column. The gel was stained with asilver stain kit purchased from BioRad. Note the bands appearing at 6OkD,l9kD and l2kD.An immunoaffinity column (lOmi) was prepared to purify HSAg-516 by Dr.C.Y.G. Lee, using the monoclonal antibodies HSA-5 and HSA-6.For the experiment shown, I washed the column twice with 20m1 of 0.1 Mglycine-HCI (pH =2.2) and equilibrated it with 40m1 PBS. Approximately500m1 of crude human semen extract, containing an average of 37mg/miprotein, was passed through the column. After rinsing the column thoroughlywith 300 ml of PBS, the bound proteins were eluted with glycine-HCI(pH=2.2). Upon neutralizing the pH with 1M Tris-CI (pH=8.0) the fractionswere quantitated spectrophotometrically at absorbance of 280nm (O.D.2)Samples containing the highest concentration of protein were pooled andlyophilized. The fractions were resuspended in 10mM NaOH to aconcentration of 0.4 g/jl. Approximately 8g of protein was run in eachlane of the gel.Samples of purified HSA-5 and HSA-6 1gM immunoglobulin was donated byDr. C.Y.G. Lee, and run in the gel to compare the purity of antigen andantibody. Approximately 1O0g of 1gM was run in each lane.Lane 1 HSAg-5/6 Fractions #4 and #5Lane 2 HSAg-5/6 Fractions #6 and #7Lane 3 HSA-5 purified 1gM ImmunoglobulinLane 4 HSA-6 purified 1gM IminunoglobulinLane 5 Standard Molecular Weight Marker,Top to Bottom: lO6kD8OkD49.5kD32 .5kD27.5kD18.5kD25•11()I1tt1’tmPeptide SequencingThe lower molecular weight protein bands of purified HSAg-5/6, observed bySDS-PAGE were originally thought to be the sperm antigen which specifically bindsto HSA-5 and HSA-6 monoclonal antibodies. As a result, these bands weretransferred to a PVDF membrane and were subjected to peptide sequencingprocedures. Despite several attempts, the Edman degradation sequencing revealedonly seven amino acids on the N-terminal region of the 18k]) protein.KQEGRDDraeflfleenenefleaflflflnnneaennen.Lys Gin Glu Gly Mg Asp AspThe short sequence was analyzed using a local database in the BiomedicalResearch Center. It was determined that the proteins are degradation products of aprecursor to Semenogelin 1 and Semenogelin 2 (Lilja et al, 1987; Lilja et al, 1989).Western Blot analysis of the 60k]) protein was carried out by Dr. T. Yoshikiin Dr. Lee’s lab. His data confirmed the true size of the HSAg-5/6 protein.Immunoscreening of a Mouse Testis cDNA LibraryThe cDNA segment which codes for expression of the antigen recognized byHSA-5 and HSA-6 has been cloned from a mouse testis eDNA library. The cDNAlibrary was constructed in a ).gtll bacteriophage expression vector. Varying27quantities of bacteriophage were inoculated into E. Coli strain Y1090, and expressionof the fi-galactosidase fusion protein was induced. The expressed fusion protein wasblotted onto two identical nitrocellulose membranes. The membranes were subjectedto an immunoassay using an HSA-5/6 monoclonal antibody cocktail (HSA-5/HSA-6)or rabbit antisera as primary probes for detecting the antigenic region of the 8-galactosidase fusion protein. Sixteen independently derived clones have been isolatedby this method.Of the sixteen clones, eight were selected at random for further analysis oftheir cDNA inserts and characterization of the expressed fusion protein. Theauthenticity of these clones was verified by immunoassay using antibody probes whichhad been pre-adsorbed with E.Coli lysate according to published methods (Sambrooket al, 1989). Seven of the eight clones demonstrated 100 % binding to bothmonoclonal and polyclonal antibody probes under these conditions. This procedureprovided conclusive evidence that the clones express fusion protein with antigenicepitopes not otherwise found in E.Coli. FIg. 4 shows clone #5 as an example.28Fig. 4 Nitrocellulose membranes displaying plaques of E.Coli lysate containing 8-galactosidase fusion protein which have positively bound to polyclonal HSA5/6 antibody and to monoclonal HSA-5/HSA-6 antibody cocktail. Detection ofantibody binding was achieved by incubation in goat anti-rabbit or goat anti-mouse lgG+IgA+lgM conjugated to horseradish peroxidase. Colordevelopment resulted from peroxidase substrate binding in the presence of 3,3’-diaininobenzidine.29Fig. 4-1• -... I. •:7’cmHSA-5 pHSA-5/6mHSA-630Characterization of Isolated eDNA clonesFusion Protein AnalysisThe fusion protein generated during immunoscreening was examined byWestern blot analysis to determine the molecular size. E.Coli were induced to expressfusion protein with IPTG, and were lysed. The lysate containing fusion protein wasrun on an SDS gel and transferred to PVDF membrane which was probed with acocktail of monoclonal HSA-5 and HSA-6 antibodies. The antibodies demonstratedspecific binding to the fusion protein, a protein with molecular weight ofapproximately 1 ISkD - l2OkD (Fig. 5).31Fig. 5 Western blot analysis of fi-galactosidase fusion protein.Lane A: E.Coli lysate containing clone #5 fusion protein probed with HSA5/HSA-6 antibody cocktailLane B: Negative control blot probed with HBS-9505 monoclonal antibodyagainst hepatitis B virus.The estimated molecular weight of the fusion protein is I l5kD - l2OkD.32I1.DNA SequencingThe cDNA insert segment for Clone #5 was amplified by PCR usingsequencing primers designed for use with 2gt1 1 vector. The resulting 900bp cDNAfragment was then submitted to the DNA sequencing facility operated by the CanadianGenetic Diseases Network. “Dye-Deoxy” cycle sequencing method provided anucleotide sequence 860 base pairs in length (Fig. 6).34Fig. 6 Nucleotide sequence of Clone #5 and deduced amino acid sequence.Note the RNA degradation site and poly-A tail signal sequence as denoted by** and ***, respectively.35Fig. 6HSA2-5/6 Cloned Sequence #5Deduced Amino Acid SequenceGGAATTCCGQMTTCCGGAATTCCTmTGTACAATAMG1TIAATCACAATTATAAAAATGTGGJGITGGGTTICACTATTAAC OSGlu Phe Arg Asn Set Gly lie Pro Pbe Cys Thr lie Lys Phe Asn His Asn Tyr Lys Asn Val Val Leu Gly Phe Thr lie Asn 28AlT ATG TAC MT CCA GCC CAA CM GM GGG CCT GCC CCC CAC CAT CCT GGC CGA CTG AGC CAG AGC CT GAG ACA cCA GGG 166Xe Met Tyr Mn Pro Ala Gi Gin Glu Gly Pro Ala Pro His H Pro Gly Pro Leu Set Gbi Ser Pro Glu Thr Pio Gly 55TGGGAGGGTGAC CCA CAG GGT CTG GCA GGA ACA CAT GAG GCA GGG TIC CAG GCA CAG ACT TCC CAG GAC TAG 238Trp Glu Gly Asp Pro Gin Gly Leu Ala Gly Thr Asp Glu Ala Gly Phe GIn Ala Gin Set Set Gin Asp TEA 79GGGCCAAAGGCcCCCCCAGGGGGAGMCAT GGCCACTGCC CCAGAGACCC CTAAACTGGG GCGGGGTGCA CATAGGGCAG318AAGGGGMGT CcCCATIAA CATGAAGCM AGGGCTATGC AGGCcCCAAG MTAGCATCTA TACAGGCTGGTACCCCAGGfl 398TCCACAGGAA ACAGCTGCTG GCAGCTACAA MCA1TTACA GC1TCTICTI CGCAAAGAAA M.MM1TAA GAGGGTGITT478G3q4TGGGG CATAGGGGAGGCAGCACATG CCTCAMGGA CTGAGGCTCC CTTGTTGGCCTCAGTGCTCC CTATCCCACG558!GAAGGTGGGGGCAGTGGG cCCTGCTGAGC GCAATAAGGCTGGGGTGCTGG GAGC1TAGGG CACCAAGGGG GAATAAATAG 636GGGTGAGGGCAccGGTCCTGcCcCAcCTCA GTIGAGGGAG GCAGGCAA C1TICAGTGC CGCACAGACA cGGAATCTIGTTGTCTTCT 728AGTGGGAACT TGTAGTCGTAGGTGATCTCC ICATCCACGCCGATGGGCTG CTTCGAGTAGATGACAATCT TITICTGAGA808CTCGGAA1TC CAGCTGAGCG CCGQTCGCTA CCATTACCAG TIGGTCTGGT IC 860RNA Degradation SitePolyA Tail Signal Sequence36Deduced Amino Acid Sequence.Each of the clones was screened with monoclonal antibodies and polyclonalantisera, thus an expressed fusion protein was instrumental in locating the desiredclones. Translation of the fusion protein from mRNA included codons of the clonedinsert directly following the translated codons of the 8-galactosidase in precise order.Logically, the amino acid sequence of the cloned insert was deduced by knowing theamino acid sequence of 8-galactosidase and the cDNA sequence of the insert.Clone #5 is 76 amino acids in length. It represents the C-terminal end of theHSAg-5/6 protein and includes a termination codon, an RNA degradation site and apoly-A tail signal sequence (see Fig. 6).Sequence Homology To Known Proteins.The cloned cDNA insert sequence and resulting amino acid sequence weresubjected to comparison with known sequences listed in public databases. Computeranalysis of both sequences revealed that HSAg-5/6 has no significant homology to anyother known protein listed in public databases.The results of the computer database search for the amino acid sequence of thecloned insert is shown in Appendix A.Deduced Molecular Size ofFusion Protein.When ?.gt1 1 phage containing the cDNA insert for HSAg-516 is inoculated intoE.Coli and induced by IPTG, a fusion protein is expressed. This protein was detected37by both monoclonal antibodies and polyclonal antisera in the immunoscreeningprocedure.The fusion protein consists of fi-galactosidase with an inserted amino acidsegment, in this case a fragment of the HSAg-516 protein. In its native state, flgalactosidase is a large protein with a molecular size of 1 l2kD. The insert site hasbeen specifically engineered within the DNA to be the six base sequence recognizedby the EcoRl restriction enzyme, located 1.7kb from the termination codon.The deduced peptide sequence of this insert (clone #5) is 76 amino acids inlength, and includes a termination codon, an RNA degradation site and a poly-A tailsignal sequence (Fig. 5).However, because the HSAg-5/6 insert segment includes a termination codon,approximately 1 .7kD of the C-terminal region of fi-galactosidase was not expressed.Rather, the HSAg-5/6 fragment insert occupied the C-terminal region of the fusionprotein (as well as representing the C-terminal region of the HSAg-5/6 protein).Based upon these facts, the total fusion protein has an estimated molecular size of1 l9kD.This hypothesis is represented in graphical form in Fig. 7.38Fig. 7 Diagram ifiustrating the fi-galactosidase protein, the EcoRl insertion site foreDNA, the deduced amino acid sequence of clone #5, and the predicted size ofthe resulting fusion protein. The N-terminus is on the right side of thediagram, the C-terminus is on the left. The fusion protein was estimated to be1 l9kD based upon the deduced amino acid sequence and the publishedinformation on 8-galactosidase.39Fig.71.7 kD--ll2kDb- ga I actos I dase\cDNA Insertion SiteCloned Sequence #576 Amino Acidsnc I udes term I nat i oncc donFusion ProteinEstimated MW ll9kD40Predicted Antigenic Regions of Peptide Insert.Additionally, the cloned amino acid sequence was analyzed for predictedhydrophilic segments. As certain amino acid residues are hydrophilic and others arehydrophobic, segments of the total amino acid sequence can be evaluated for theirionic properties. Those regions which are hydrophiic would probably be exposed tosolution, and would constitute possible antigenic regions where antibodies such asHSA-5 and HSA-6 are most likely to bind. The plot in Fig. 8 shows probableantigenic regions within hydrophilic areas of clone #5 insert site.41Fig. 8 Hydrophilicity plot of the deduced amino acid sequence of Clone #5. Areasshown above 000 are bydrophilic and possibly antigenic for HSA-5 and HSA6. The antigenic region is estimated to be within the final 35 amino acids.(Graph courtesy of BioYang Un, consultant from Dr. Michael Hayden’slaboratory)42Fig. 8Translation of PCR5 Protein Toolbox Plot Page 1Monday, April 18, 1994 4:12 PMHydrophilicity Window Size = 75.004.00: 2.001.00E —1.00E1 —2.00—3.00—4.00—5.00Scale = Kyte-DoolittleIbN__10 20 30 40 50 60 7043DiscussionStudies of the Monoclonal Antibodies - HSA-5 and HSA-6The monoclonal antibodies HSA-5 and HSA-6 were generated againstionophore-treated acrosome reacted human sperm in 1989. Both antibodies are 1gMclass and were produced in BALB/c mice as ascites fluid.The HSA-5 and HSA-6 antibodies were submitted to the second WHOworkshop for interlaboratory evaluations of their potential to be immunocontraceptivevaccine candidates and were among five considered Thigh priority candidates,”(Anderson, 1990). The evaluations determined that the antibodies donot cross-reactwith somatic tissue.Furthermore, they were shown to recognize identical bands on a Western blotof crude human sperm extract, which indicated that theyrecognize the same protein(HSAg-516). It was suggested that these two antibodies were identified from cell lineswhich originated from the same hybridoma. However, sandwich enzymeimmunoasssays (SEIA) later determined that the antibodies recognize differentantigenic epitopes of HSAg-5/6 (Yoshiki et al, unpublished).Purification of the antibodies led to conjugation with Affigel-lO and theformation of an immunoafflmty column. This unmunoaffinity column was used topurify the antigen from human semen extract, wherebyrabbit antisera were raisedaccording to the standard protocol (Liu et al, 1989). Both monoclonal antibodyascites fluid and rabbit antisera were utilized to study thecognate sperm antigen.Localization of HSAg-5/6 on Human and Mouse Sperm44Indirect immunofluorescent assays of human sperm were utilized extensively todetermine the location of expression of HSAg-5/6. Assays using methanol-fixedhuman sperm, as well as live human and mouse sperm were performed. It wasclearly established that the antigen recognized by the HSA-5 and HSA-6 monoclonalantibodies is located in the equatorial region of acrosome-reacted sperm (data notshown). Live sperm assays were observed to show little binding to fresh spermpreparations, while the binding increased significantly (>38%) following a 24-hourincubation. Positive staining increased further subsequent to incubation of the spermwith calcium lonophore A23187 to induce the acrosome reaction (data not shown).Indirect immunofluorescent assays with epididymal mouse sperm were alsoperformed. In this case, the monoclonal antibodies HSA-5 and HSA-6 demonstratedbinding to antigens localized on the principal piece of the tail and the head of thesperm, rather than the acrosomal region as seen in human sperm (Fig. 1). A fewsperms showed positive staining on both the head and tail regions.The iinmunofluorescent assays demonstrated that HSA-5 and HSA-6 bind tothe acrosomal region of human sperm and the head and tail of mouse sperm. To date,we have little explanation for these observation. However, O’Rand et al (1984) foundthat their monoclonal antibodies bound to the head region of rabbit sperm and themid-piece of the human sperm. It has been hypothesized that HSAg-5/6 in the mouseis expressed in late spermiogenesis as an intracellular protein which is shed among thecytoplasmic droplets and later attaches to the sperm tail. Goldberg (1963) noted thatLDH-X expression in sperm follows a similar pattern of migration.This hypothesis was supported by indirect immunofluorescent assays on frozensections of mouse tissues and developing mouse testes. Using procedures developed45by Lee and Wong (1986), Dr. T. Yoshiki (unpublished) observed that the antigenrecognized by HSA-5 and HSA-6 is expressed only in testicular and epididymal spermof the adult mouse, but not in the immature testes, nor in somatic tissues.Furthermore, positive staining of the sperm occurred on the head and tail regions inthe testis, but primarily on the tail region of epididymal sperm (unpublished results).Transmission electron microscopy was performed by Homyk et al (1992) toprecisely localize the HSAg-5/6 protein on human sperm. The epitopes for theseantibodies were adversely affected by fixative chemicals. Therefore, the sperm werepermeablized and incubated in HSA-5 and HSA-6 monoclonal antibodies prior toembedding. In separate experiments, HSA-5 and HSA-6 were bound to human spermand detected with a secondary antibody conjugated to gold particles. Aftercounterstaining with a silver enhancer to monitor the bound gold particles, the spermwere observed under and electron microscope. The results showed that the HSAg-5/6protein is located on both the inner and outer acrosomal membranes.Purification and Studies of HSAg-516HSA-5 and HSA-6 were conjugated with Affigel-lO to form an immunoafflnitycolumn according to the protocol provided by BioRad Lab and loaded into a modifiedsyringe. This inununoaffinity column was used to purify the HSAg-5/6 from humansemen extract. The resulting protein fractions were examined by SDS-PAGE (Fig. 3).Three major protein bands of molecular weights 1 lkD, l9kD, and 6OkD wereobserved.46The lower molecular weight protein bands of purified HSAg-5/6, observed bySDS-PAGE were originally thought to be the sperm antigen which specifically bindsto HSA-5 and HSA-6 monoclonal antibodies. Consequently, these bands weretransferred to a PVDF membrane and were subjected to Edman degradationsequencing at a service laboratory in the University of Victoria. Unfortunately, onlyseven amino acids on the N-terminal region of the protein were elucidated (K Q E GRDD).The short sequence was analyzed and it was determined that the proteins aredegradation products of a precursor to Semenogelin 1 and Semenogelin 2. Theseproteins are secreted by the seminal vesicle and are structural contributors to theformation of a gelatinous matrix within human semen (Lilja et al 1989). Prostatespecific antigen (PSA) is an abundant prostatic protease also found in human semen.This protease has been shown to progressively fragment Semenogelin proteins duringand after liquefaction of semen, following ejaculation (Lilja et al, 1987; Lee et al,1989). Fragments of semenogelin proteins including antigenic epitopes have beenlocalized to parts of the spermatozoa associated with locomotion. It is thought thatthese fragments may participate in activation of progressive sperm movement as thegelatinous matrix is disassembled by PSA following ejaculation.We believe that seminogelin protein fragments in the preparation of the crudesemen extract were co-purified along with HSAg-516 in the inununoafflnity isolationprocess. It is possible that seminogelin proteins bind to the Fc regions of antibodiesin a manner similar to those described by Kamada (Kamada et al, 1991). Theyhypothesized that Fc binding proteins which coat spermatozoa, provide protection forthe sperm from immuno-attack by the host. However, the l6kD-2OkD proteins47described by Kamada bound only to IgG class antibodies, not to 1gM class antibodiessuch as HSA-5 and HSA-6.Western Blot analysis of HSAg-5/6Western blot analysis was performed by Dr. T. YoshIki with purified HSAg5/6 and compared to similar Western blot analyses of crude sperm extract from humanand mouse. The analyses revealed that only the 6OkD band of purified HSAg-5/6 isimmunoreactive with the HSA-5 and HSA-6 monoclonal antibodies (unpublisheddata).The analyses which involved crude human and mouse sperm extractsdemonstrated a range of immunoreactive proteins from 4OkD to 6OkD (unpublisheddata). When compared to the purified antigen, with a molecular weight of 6OkD, hehypothesized that the 6OkD band on the western blot of crude extract(s) is HSAg-5/6,while the other protein bands are degradation products. The presence of proteases insemen (Lee et a!, 1989) suggests the possibility that HSAg-516 is digested intofragments prior to enzyme neutralization during the extract preparation. Dr. C.Y.G.Lee further speculated that the acidic conditions under which HSAg-5/6 is affinitypurified eliminate the degradation products from the purified fractions; only the intactform of HSAg-516 binds to the immunoaffinity column.Cloning and Molecular Biology of HSAg-516Having verified the size and location of HSAg-5/6 on human and mousesperm, it became important to study the protein on the molecular level. Theseprocedures reveal the molecular nature, sequence, and structure of a protein.48In collaboration with our lab, Homyk and Herr (unpublished) have clonedHSAg-5/6 from a human testis eDNA library. They have obtained a 600bp sequenceof the eDNA, which represents the C-terminal region of the protein. As a result oftheir limited success, we set out to perform a similar cloning study in a mouse testiscDNA library. It was thought that once the cDNA sequence of the mouse gene iselucidated, then the DNA fragment could be used to isolate a full length human cDNAclone.ImmunoscreeningA cDNA library cloned into Agtl 1 phage vectors (provided by Dr. C. Lau ofthe University of California, San Francisco) was subjected to screening for the HSAg5/6 epitopes. The library contained approximately 1 x 106 independent eDNA inserts.Following adsorption of the phage in E.Coli, expression of a fusion protein consistingof 8-galactosidase with a peptide insert was induced. The cells were then lysed andthe proteins were blotted to nitrocellulose where they were accessible to binding bythe HSA-5 and HSA-6 monoclonal antibodies, or polyclonal rabbit antisera.Using these antibodies to detect expressed fusion protein, sixteen independentclones were isolated in the immunoscreening procedure. Eight of those clones wereselected at random and subjected to further examination to determine the nature of thecDNA inserts.cDNA Sequence AnalysisSequencing of the eDNA insert demonstrated that the clones isolated inimmunoscreening represented the C-terminus of the HSAg-5/6 protein plus part of the49untranslated region (UTR) of messenger RNA (mRNA). The DNA sequence was 860base pairs in length. Included in the sequence was a termination codon T A G,which was 235 bases from the origin (Fig 3).Among the bases of the 3’ UTR was a segment which may represent an RNAdegradation site - A T T T A (Fig. 3) (Liu et al,1992). Shaw and Kamen (1986)found that insertion of nucleotide AT sequences into the 3’ UTR of a humanlymphokine gene caused the otherwise stable mRNA to become highly unstable invivo. The sequence TTATTTAT was identified (Caput et al, 1986) as being aconserved region in the 3’ UTR of mouse and human Tumor Necrosis Factor geneswhich may play a role in regulating expression of mRNA. The five base pair segmentin the HSAg-5/6 eDNA could play a regulatory role in the expression of HSAg-516,and the stability of the mRNA, in the mouse testis during spermatogenesis.Also included in the 3’ UTR of the HSAg-516 eDNA insert was a signal forcleavage of the raw RNA transcript and addition of a poly-A tail. The sequence A AT A A A (Fig. 3) signals Poly-A Polymerase to add 100-200 adenosine residues to the3’ end of the RNA chain. This is one of the first modifications which an RNAtranscript undergoes in the nucleus of a cell (Alberts et al, 1989).The cDNA sequence in its entirety and the deduced amino acid sequence weresubjected to comparison of known sequences stored in the public databases accessedby the Basic Local Alignment Search Tool (BLAST) at the National Institutes ofHealth, Maryland (Altschul et al, 1990). There were no significant homologies foundto any known DNA or peptide sequence found in the public databases. The results ofthe BLAST search for the deduced peptide sequence are shown in Appendix A.50The Deduced Amino Acid Sequence and Fusion Protein AnalysisThe amino acid sequence of the cloned insert for HSAg-516 was deduced basedupon the fact that it was expressed as a peptide insert within the 8-galactosidaseprotein in the Agtl 1 phage. Translation of the fusion protein from mRNA includedcodons of the cloned insert directly following the translated codons of the 8-galactosidase in precise order. Therefore, the amino acid sequence of the clonedinsert was deduced by examining the flanking DNA sequences of 8-galactosidase(Glover, 1985) and the cDNA sequence of the insert.The clone #5 is 76 amino acids in length. It represents the C-terminal end ofthe HSAg-516 protein and includes a termination codon, an RNA degradation site anda poly-A tail signal sequence.Human and mouse native HSAg-516 was determined by Western blot to have amolecular weight of 6OkD. Such a protein would contain roughly six hundred aminoacids, and would be coded by an mRNA of approximately three kilobases in length.Using this reasoning, we estimate that the cDNA clone of HSAg-516, which wasisolated by immunoscreening represents 76 out of 600 amino acids, or 13% of thetotal protein.As stated, the isolated clone is seventy six amino acids in length and includes atermination codon. It has a molecular weight of approximately 7.6kD. When thisquantity is added to the known molecular weight of 8-galactosidase (Glover, 1985),taking into account the termination codon in the insert and accordingly eliminating thetail segment of the carrier protein, the molecular weight of the fusion protein isestimated at 1 l9kD (Fig. 7). This figure corresponds well to the molecular weight ofthe fusion protein demonstrated by Western blot in Fig. 5 at 1 l5kD - l2OkD.51Structural Analysis of HSAg-5/6The deduced amino acid sequence of the cDNA insert was subjected to ahydrophilicity analysis. This process determined regions of the peptide which werelikely exposed to antibody reaction, versus those regions which were hydrophobic andprobably hidden from solution.The hydrophilicity plot in Fig. 8, demonstrates that there are several areas ofthe C-terminal region of HSAg-5/6 which could be exposed to immune reaction invivo. These areas elicited an immune response in a vaccinated host mouse, wherebythe monoclonal antibodies HSA-5 and HSA-6 were originally raised. Thehydrophilicity analysis suggests that the C-terminus of the HSAg-5/6 protein isexposed to the exterior environment in mouse sperm and similarly uncoveredsubsequent to the acrosome reaction in human sperm. This hypothesis is supported bythe findings of Homyk (unpublished) who cloned the C-terminal region of humanHSAg-5/6 from a human testis cDNA library by immunoscreening.The clones which were isolated by immunoscreening were shown to beidentical to each other (Fig. 10 and Fig. 12). The use of antibodies, particularlymonoclonal antibodies, to screen fusion protein expression in the cloning processexplains why the clones are identical. In order for the antibodies to hybridize to thefusion protein, the antigenic epitopes of HSAg-5/6 were required to be intact. Thisrequirement greatly restricted the screening process to a limited number of cDNAinserts. This forced the clone selection to identical, or nearly-identical features.Future Studies for HSAg-51652The antigen recognized by the monoclonal antibodies HSA-5 and HSA-6requires further study to determine its true potential as an antifertility vaccinecandidate.The exact epitopes which bind the HSA-5 and HSA-6 monoclonal antibodiesneed to be elucidated. The hydrophilicity plot in Fig. 8 demonstrates which sequencesof the peptide are likely epitopes. Short peptides based upon epitope sequences can besynthesized in an automated peptide synthesizing apparatus. These peptides could betested for their antigenicity to the monoclonal antibodies by ELISA. Once the exactepitope is known, it alone could be a candidate for immunocontraceptive vaccine.The advantage of using a short peptide antigen as a vaccine (rather than an entireprotein) lies in a lesser chance of other protein epitopes being common to host tissues.Northern Blot assay of the rnRNA from mouse testis must be achieved. Usingthe 860bp eDNA insert from HSAg-5/6 as a probe to visualize the native mRNA frommouse testis will establish the size of the full length cDNA. This assay could confirmor disconfirm the current belief that the native HSAg-5/6 protein has a molecularweight of 6OkD.The full length cDNA needs to be isolated from the mouse testis cDNAlibrary. Again, using the 860bp eDNA as a probe, the Agtl 1 phage library could bescreened for complementary cDNA inserts. If the screening does not rely on an intactprotein epitope, then the likelihood of encountering a larger segment of the cDNAinsert increases substantially.Once the full length cDNA insert is isolated, then the expressed fusion proteincould be isolated. An immunoaftinity chromatography procedure involving eitheranti-A-galactosidase antibodies or the HSA-5 and HSA-6 monoclonal antibodies could53be utilized to isolate the fusion protein from a crude E.Coli/phage lysate. Isolatingthe entire fusion protein lends the advantage of raising antisera to further confirm thevalidity of the eDNA insert. Once the fusion protein is isolated, it can be used tovaccinate an animal host, which will result in a protein specific antisera. The antiserawill be subjected to ELISA, indirect immunofluorescent assays of human and/ormouse sperm and Western blot assays to confirm that it recognizes the same protein asthe original HSA-5 and HSA-6 monoclonal antibodies.The isolated fusion protein can also be subjected to partial peptide sequencingto confirm its homology to the original HSAg-5/6 protein.ConclusionsUnderstanding the nature of molecules which have been chosen for antifertilityvaccine candidates is essential. Subsequently, the outcome of this project is tocharacterize the cognate antigen recognized by the monoclonal antibodies HSA-5 andHSA-6, which are considered “high priority candidates” for immunocontraceptivevaccine.From the results of my work on the molecular nature of HSAg-516, thefollowing conclusions can be drawn:1. Analysis of the isolated cDNA clones and expressed fusion protein suggest thatthe antigenic epitopes recognized by the monoclonal antibodies HSA-5 andHSA-6 are localized in the C-terminal region of the HSAg-516 protein.542. Sequence analysis of the cDNA insert (including the 3’ UTR), as well as thededuced peptide sequence demonstrated that the HSAg-5/6 protein has nosignificant homology to any other known protein or gene (Appendix A).55BibliographyAlberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1989):”MolecularBiology of the Cell, Second Edition”, New York, NY, Garland Publishing,Inc.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local aligmnentsearch tool. J Mol Bio, 215:403-410Anderson DJ (1990) World Health Organization sperm antigen workshops and spermantigen nomenclature project. In: Gamete Interaction, Prospects forImmunocontraception (Alexander NJ, Griffin D, Spieler JM, Waites GMH,eds), New York, Wiley Liss, A John Wiley and Sons, Inc. Publications; 103-106Anderson DJ (1992) WHO sperm antigen workshop, Task Force on Vaccines forFertility RegulationBaskin MJ (1932) Temporary sterilisation by the injection of human spermatozoa.Caput D, Beutler B, Hartog K, mayer R, Brown-Shimer S, Cerami A (1986)Identification if a common nucleotide sequence in the 3’ untranslated region ofmRNA molecules specifying inflammatory mediators. Proc Nati Acad SciUSA, Mar; 83(6):1670-4Chaffee JK, Schachner M (1978) NS-6 (Nervous System antigen-6): a new cellsurface antigen of brain, kidney and spermatozoa. Dev Bio., 63: 173-18456Cross NL, Morales P (1978) Two simple methods for detecting acrosome-reactedhuman sperm. Gamete Res, 115:213-226Davis TM, Pennington JE, Kubler AM, Conscince JF (1982) A simple, single-steptechnique for selecting and cloning hybridomas for the production ofmonoclonal antibodies. J Immun Methods, 50:161-168De loannes AE, Becker MI, Perez C, Capote C, Barros C (1990) Role of Acrosin andAntibodies to Acrosin in Gamete Interactions. In: Gamete Interaction,Prospects for Immunocontraception (Alexander NJ, Griffin D, Spicier JM,Waites GMH, eds), New York, Wiley Liss, A John Wiley and Sons, Inc.Publications; 185-195Dohr G, Biaschitz A, Hirsch T, Kohlstadt S, Uchanska-Ziegler B, Ziegler A (1990)Immunoelectron microscopy of human spermatoma. Andrologia, 22(suppl 1):92-100Dondero F, Lenzi A, Gandini L, Lombardo F (1993) Immunological infertility inhumans. Exp din Immunogenetics, 10(2): 65-72Fann CH, Lee CYG (1992) Monoclonal antibodies affecting sperm-zona bindingand/or zona-induced acrosome reaction. J Repro Immun, 21:175-187Fichorova R, Anderson DJ (1991) Use of sperm viability and acrosomal status assaysin combination with immunofluorescence technique to ascertain surfaceexpression of sperm antigens. J Repro Immun 20: 1-13Freund 3, Thompson GE, Lipton MM (1955) Aspermatogenesis, anaphylaxis, andsutaneous sensitization induced in the guinea pig by homologous testicularextract. 3 Exp Mcd, 101:591-60357Glover DM (1985): “DNA cloning Volume 1 a practical approach”, Oxford, England:IRL Press LimitedGoldberg E (1963) Lactic and malic dehydrogenases in human spermatozoa. Science,139:602-603Goldberg E (1975) Lactate dehydrogenase-X (crystalline) from mouse testes. In:Methods in Enzymology Vol XLI: Carbohydrate Metabolism, Part B (WoodWA, ed), New York, Academic Press; 3 18-322Griffin D (1990) Strategy of Vaccine Development. In: Gamete Interaction, Prospectsfor Immunocontraception (Alexander NJ, Griffin D, Spieler JM, Waites GMH,eds), New York, Wiley Liss, A John Wiley and Sons, Inc. Publications; 501-522Hendry WF, Stedronska J, Hughes L, Cameron KM, Pugh RCB (1979) Steroidtreatment of male subfertility caused by antisperm antibodies. Lancet, ii: 498-501Herr JC, Write RM, John E, Klotz K, Homyk M, Foster J, Flickinger CJ (1990a)Monoclonal antibody MHS-10 and its cognate antigen SP-1O. In: GameteInteraction, Prospects for Iminunocontraception (Alexander NJ, Griffin D,Spieler JM, Waites GMH, eds), New York, Wiley Liss, A John Wiley andSons, Inc. Publications; 13-36Herr JC, Flickinger CJ, Homyk M, Klotz K, John E (1990b) Biochemical andmorphological characterization of the intra-acrosomal antigen SP-10 fromhuman sperm. Bio Reprod, 42:181-193Herr JC, Write RM, Flickinger CJ (1990c) Biochemical, ultrastructural and geneticcharacterization of human acrosomal antigen SP-10. In: Proceedings of the 4thInternational Congress of Reproductive Immunology (Mettler L, BillingtonWD, eds.), Amsterdam, Elsevier, 5 1-5958Herr JC, Write RM, Foster J, Flickinger CT (1990d) Identification of humanacrosomal antigen SP-10 in primates and pigs. Bio Reprod, 42: 377-382Homyk M, Anderson DJ, Wolff H, Herr JC (1990) Defferential diagnosis ofimmature germ cells in semen utilizing monoclonal antibody MHS-10 to theintra-acrosomal antigen SP-10. Fertil Steril, 53: 323-3290Homyk M, Herr JC (1992) Light and electron microscopic immunolocalization ofsperm proteins identified by monoclonal antibodies from the World HealthOrganization TaskForce on Sperm Antigens. J Reprod Immun 22:237-256Kerek G (1974) Distribution of the blood group antigens A and B on humanspermatozoa. Tnt 3 Fertil, 19: 181-191Jones WR (1982): “Immunological Fertility Regulation”, Victoria, Australia:Blackwell Scientific PublicationsJones WR (1990): Lessons From an Antihuman Chorionic Gonadotropin ContraceptiveVaccine Trial. Wiley-Liss, Inc., Gamete Interaction: Prospects forImmunocontraception, pp595-605Kamada M, Liang Z, Koide SS (1991) IDENTIFICATION OF IgG AND Fe-BINDING PROTEINS IN HUMAN SEMINAL PLASMA AND SPERM.Archives of Andrology, 27:1-7Landsteiner K (1899) Zur Kenntnis der spezifisch auf bluthorperchen wirkenden Sera.Zb Bakt, 25: 546-54959Laemmli UK (1970) Cleavage of structural proteins during the assembly of the headof bacteriophase T4. Nature, 227:680-685Lathrop WF, Carmichael EP, Myles DG, Primakoff P (1990) eDNA Cloning Revealsthe Molecular Structure of A Sperm Surface Protein, P11-20, Involved inSperm-Egg Adhesion And the Wide Distribution of Its Gene among Mamm1s.3 of Cell Bio 22(6 pt2):2939-2949Lee C, Keefer M, Zhao ZW, Kroes R, Berg L, Liu XX (1989) Demonstration of therole of prostate-specific antigen in semen liquefaction by two-dimensionalelectrophoresis. J Androl, Nov-Dee; 10(6):432-438Lee CYG, Wong E, Richter DE, Menge AC (1984) Monoclonal antibodies to humansperm antigens II. 3 Repro Immun, 6:227-238Lee CYG, Wong E, Teh CZ (1984) Analysis of mouse sperm isoantigne usingspecific monoclonal antibodies. Am J Repro Immun, 6:27-33Lee CYG, Wong E, Menge AC (1984) Monoclonal antibodies to rabbits spermautoantigen. Fert Steril, 41:131-138Lee CYG, Wong E, Zhang H (1986) Inhibitory effects of monoclonal spermantibodies on the fertilization of mouse oocytes in vivo and in vitro. J ReproImmun, 9(4): 261-274Lee CYG, Wong E (1986) Developmental studies of sperm surface antigens usingsperm-specific monoclonal antibodies. J Repro Immun, 9:275-287Lee CYG, Liu MS, Su MW, Zhu JB (1990) Studies of sperm antigens reactive to HS11 and HS-63 monoclonal antibodies. In: Gamete Interaction, Prospects for60Immunocontraception (Alexander NJ, Griffm I), Spieler JM, Waites GMH,eds), New York, Wiley Liss, A John Wiley and Sons, Inc. Publications; 37-5 1Lee CYG, Hsaio L, Yang YY, Chow S, Chao HT, Ng HT, Wong E, Sun A, Hsu E(1993) Studies of a sperm/placenta cross-reacting antigen, STX-10. J ReproImmun 25:249-264Liang ZG, Shelton JA, Goldberg E (1986) Non-crossreactivity of antibodies to murineLDH-C4,with LDH-A4 and LDH-4. J Exp Zool, 240:377-384Lilja H, Oldbring 3, Rannevik 0, Laurell CB (1987) Seminal vesicle-secreted proteinsand their reactions during gelation and sequefaction of human semen. J ClinInvest, Aug; 80(2):281-85Lilja H, Abrahanisson PA, Lundwall A (1989) Semenogelin, the predominant proteinin human semen. Primary structure and identification of closely related proteinsin the male accessory sex glands and on the spermatozoa. J Bio Chem, Jan 25;264(3): 1894-1900Liu MS, Yang Y, Pan J, Liu HW, Menge A, Lee CYG (1989) Purification ofacrosome antigen reactive to a monoclonal antibody and assessment ofantifertility effect through active immunization, hit 3 Androl, 12:451-463Liu MS, Chan K, Lau YF, Lee CYG (1990) Molecular Cloning of an AcrosomalSperm Antigen Gene and the Production of Its Recombinant Protein forImmunocontraceptive Vaccine. Mol Reprod Dev 25:302-208Liu MS, Aebersold R, Fann CH, Lee CYG (1992) Molecular and DevelopmentalStudies of a Sperm Acrosome Antigen Recognized by HS-63 MonoclonalAntibody. Bio Reprod 46:937-94861Metchnikoff E (1899) Etudes stir la resorption de cellule. Ann Inst Pasteur, 13: 737-779Mitchison NA (1990) Lessons Learned and Future Needs. In: Gamete Interaction,Prospects for Immunocontraception (Alexander NJ, Griffm D, Spieler JM,Waites GMH, eds), New York, Wiley Liss, A John Wiley and Sons, Inc.Publications; 607-613Naz RK, Alexander NJ, Ishakia M, Hamilton MD (1984) Monoclonal antibodies to ahuman cell membrane gicoprotein that inhibits fertilization. Science, 225: 342-344Naz RK (1987) The fertilisation antigen (FA-1) causes reduction of fertility in activelyimmunized female rabbits. J Reprod Immun, 11: 117-133Naz R, Menge A (1990) Development of antisperm contraceptive vaccine for humans:why and how?. Human Repro, 5(5): 511-518Naz RK, Brazil C, Overstreet 3W (1992) Effects of antibodies to sperm surfacefertilization antigen-i on human sperm-zona pellucida interaction. FertSterility, 57(6): 1304-1310O’Rand MG, Irons OP (1984) Monoclonal antibodies to rabbit sperm autoantigens. II.Inhibition of human sperm penetration of zona-free hamster eggs. Bio Repro,30:731-736O’Rand MG, Widgren EE, Fisher SJ (1988) Characterization of the rabbit spermmembrane autoantigen, RSA, as a lectin-like zona binding protein. Dev Biol,129:231-24062O’Rand MG, Widgren EE (1989) Molecular biology of a sperm antigen: Identificationof the sequence of an autoantigenic epitope. In: Reproductive Immunology(MettletL, Billington WD, eds.) Amsterdam: Wlsevier, 61-67O’Rand MG, Widgren EE, Nikolajczyk BS, Richardson RT, Shabanowitz RB (1990)Receptors for Zona Pellucida on Human Spermatozoa. In: Gamete Interaction,Prospects for Immunocontraception (Alexander NJ, Griffin D, Spieler JM,Waites GMH, eds), New York, Wiley Liss, A John Wiley and Sons, Inc.Publications; 2 13-224Sambrook J, Fritsch ET, Maniatis T (1989): “Molecular Cloning: A LaboratoryManual” Second Ed, Cold Spring Harbor, NY: Cold Spring Harbor PressShaw G, Kamen R (1986) A conserved AU sequence from the 3’ untranslated regionof GM-CSF mRNA mediates selective mRNA degradation. Cell, Aug 29;46(5) :659-667Shulman S (1986) Sperm antigens and autoantibodies: effects on fertility. Am 3Reprod Immun Microbiol, 10: 86-89Towbin H, Srachelin T, Gordon JA (1979) Electrophoretic transfer of protein frompolyacrylamide gels to nitrocellulose sheets: procedure and some application.Proc Nat Acad Sci USA, 76:4350-4354Wheat TE, Goldberg E (1983) Sperm-specific lactate dehydrogenase C.: Antigenicstructure and imniuno-suppression of fertility. In: Isozymes: Current Topics inBiological and Medical Research, Volume 7: Molecular Structure andRegulation (Rattazzi MC, Scandalios JG, Whitt GS, eds.), New York, Alan R.Liss, Inc., 113-130Write RM, Flickinger CJ, Herr JC (1990) Cloning and sequencing of cDNAs codingfor the human intra-acrosomal antigen SP-1O. Bio Reprod 42: 693-70163Witkin SS (1988) Mechanisms of active suppression of the immune response tospermatozoa. Am J Repro Immun & MicrobiologyYoshiki T, McChesney P, Homyk M, Herr JC, Lee CYG, Characterization of asperm antigen recognized by two monoclonal antibodies, UNPUBLISHEDYoung RA, Davis RW (1983) Yeast RNA Polymerase II Genes: Isolation withAntibody Probes. Science, 222:778-78264Appendix AComputer search of peptide sequence databases. The deduced peptide sequence inFig. 6 was the subject of the query.65Date: Wed, 13 Apr 1994 12:34:01 +0500Subject: Results-B LAST SewerReply-To: ‘NCBI BLAST E-Mail Server” <blastncbLnim.nih.gov>From: NCBL BLAST E-Mail Sewer <blast@ncbi.nhn.nih.gov>Apparently-To: giee@ulam.generes.caContent-Length 58821=-+======= == == == ========= == == ========== === ========= =To Obtain Documentation: send an e-mail message to ‘blast@ncbi.nlm.nih.gov’with the word HELP in the body of the message. The documentation was lastmodified February 24th.=-+======= == ============= ======================= == == ======== == === ======== =Trying muncher... connectedNational Center for Biotechnology Information (NCBI)Experimental GENINFO(R) BLAST Network Service (Muncher)Wed Apr 13 12:33:32 EDT 1994, Up 7 days, 23:55, load: 20.58, 24.67, 24.65** *‘ 3***.*****1* ****t’* ***** *****‘$* *t***51****lt* ** *1*********1***l*Tomorrow, April 14th 1994, for computer maintenance reasons, the BLAST serverswill be shut down sequentially.Nevertheless, we should be able to manage the regular number of requests butthe service should be slower than usual.We apologize for any inconvenient.*****‘g****t* ******PEPTIDE SEQUENCE DATABASESnr Non-redundant PDB+SwissProt+PIR+SPUpdate-.GenPept+GPUpdate, updated dailyfor efficient, complete searches of the five component databases;pdb Brookhaven Protein Data Bank, October 1993 Releaseswissprot SWISS-PROT Release 28.0, March 1994pir FIR Release 39.0 (complete), December 31, 1993spupdate SWISS-PROT cumulative weekly update to the major releasegenpept CDS translations from GenBank(R) Release 82.0, April 9, 1994gpupdate cumulative daily updates to the major release of gipeptkabatpro Kabat Sequences of Proteins of Immunological Interest Release 5.0,August 1992tfd TFD transcription factor (protein) database Release 7.0, June 1993acr * Ancient Conserved Region subset of SWISS-PROT, Dec. 3, 1993pain * six-frame translations of representative human Alu repeatsNUCLEOTIDE SEQUENCE DATABASESnr Non-redundant PDB+GB IJpdate+GenBank+EmblUpdate+EMBL, updated dailyfor efficient, complete searches of the four component databases:pdb Brookhaven Protein Data Bank, October 1993 Releasegenbank GenBank(R) Release 82.0 (no daily updates), April 9, 1994gbupdate GenBank(R) cumulative daily updates to the major releaseembi EMBL Data Library, Release 38.0, April 1994emblu EMBL Data Library cumulative daily updates to the major releasevector Vector subset of GenBank(R), LANL, April 23,1992repbase Human and other primate Alu repeats, Dr. Jerry Ji.n-ka, Sept. 199366kabatnuc Kabat Sequences of Nucleic Acid of Immunological InterestRelease 5,0, August 1992epd Eukaryotic Promoter Database Release 35, June 1993dbest Database of Expressed Sequence Tags Release 2.5, April 1st, 1994‘Databases that are not accessible through the NCBI Retrieve E-mail sewer.‘ dbEST data are available from est_reportncbi.nlm.nih.gov. Send a HELPmessage to obtain instructions.For a free subscription to “NCBI News’, the NCBI newsletter, send a requestalong with your name and postal mailing address to: info@ncbi.nlm.nih.govGenBank(R) Release 82.0 is available via anonymous ftp on ncbi.nlm.nih.govAll direct submissions of sequences to the GenBank(R) database, includingthose composedwith Authorin, should be sent to the NCBI at any of thefollowing addresses. If data is submitted on diskette, please indicatewhether Mac or PC.Postal mail: GenBank SubmissionsNational Center for Biotechnology InformationBtnldmgS8A, Room SN-8038 600 Ro ckville PikeBethesda, Ml) 20894-0001Voioei 381-426-2475E-mail submissions of new sequences: gbsub@ncbi.nlm.nih.govE-mail submissions of updates: update@ncbi.nlm.nth.govGenBank is a registered trademark of the National Institutes of Health.The help document for the BLAST E-mail sewer was last modified Feb. 24th.BLASTP 1.3.11MP [29-Oct-93] [Build 14:35:03 Mar 3 1994]Reference: Altschul, Stephen F., Warren Gish, Webb Miller, Eugene V. Myers,and David J. Lipman (1990). Basic local alignment search tool. J. Mol. Biol.215:403-410.Query= hsag-59b6 frame +2(78 letters)Database: Non-redundant PDB+SwissProt+SPupdate+PIR+GenPept÷GPupdate, 5:04 AMEDT Apr 13, 1994114,708 seqnces; 32,528,034 total letters.Searching doneHighest-scoring Hit Extension vs. Count of Database SequencesHistogram units: = 130 Sequences : less than 130 sequencesHighestScoreI Count-->V0 25 INeighborhood word score threshold, T = 116711 88512 120013 1545 I===========14 170215 2354 I16 285517 3820 I18 4852 I19 6016 =============== === =======20 6544 I ===== === ====== === ======== === ====================21 7532 I22 8139 I23 7794 I =========================248947 I ==============—================25 7832 I =—=== =================== =============== =266909 I ==== === =========27 6138 I =========================================28 5152 I ===== ============================= =294300 ===303597 I ==== =====================31 297932 2321 I ============== =33 1717 I === =========34 1570 l===========351196 I..————...36 904 I======37787 I38 535Expect = 8.2e-i-02, Observed = 2037 -—---39433 I===40318 I==41 227 1=422051 =43152 I =441301 =451321 =— Expect=77.,Observed 440 -—46 119 I47791:48 481:49291:50 131:51 541:52 101:Expect = 7.3, Observed =82 ---53 141:54 151:>>>>>>>>>>>>> Expect = 3.7, Observed = 53 <<<<<<<<< Cutoff Score 5555201:56 51:57 31:58 31:—---- Expect = 0.94, Observed = 22 -—59 101:6Q 51:61 41:62 21:63 ii>63 0168SrLalltPoisorLH1L ProbabilitySequences producing High-scoring Segment Pairs: Score P(N) Nspl P06914! CSP_PLAYO CIRCUMSPOROZOITE PROTEIN (CS) PREC... 56 0.00095 2phi S22373 1522373 proline-rich protein - mouse >gp IX... 55 0.00 12 2pin S16907 S16907 collagen alpha WV) chain - bovin... 51 0.0015 3pir I S20500 I 520500 hydroxyproline-rich glycoprotein-... 49 0.0016 3spl P17656 I CC02_AEEL CUTICLE COLLAGEN 2. >sp I P17656! CCO... 50 0.0029 2gp I L05906 I PMMSGF_.1 major swface glycoprotein [Pneumo... 52 0.0032 2sp I P024541 CAll_RAT COLLAGEN ALPHA 1(1) CHAIN (FRAGMEN... 42 0.0060 3pin I A90559 I CGRTIS collagen alpha 1(1) chain - rat (f... 42 0.0060 3sp I P345451 YNJ1_CAEEL HYPOTHETICAL 222.4 KD PROTEIN alOE... 61 0.0062 2gp I X16711 I HSCOL2AIR_1 COL2A1 gene product [Homo sapierI 45 0.019 3pin S 18804 I S18804 collagen alpha 4(IV) chain - bovin... 44 0.019 3pirl B40333 I B40333 collagen alpha 1(11) chain precurs... 47 0.031 3gp! M63595 I XELCOL2A1_1 alpha-I type II collagen [Xenopus ... 47 0.03 1 3gp I M63596 I XELCOL2A1A_1 alpha-i type II’ collagen [Xeriopus... 45 0.031 3pin! A40333 I A40333 collagen alpha 1(11) chain precur... 45 0,031 3pirl A314271 A31427 collagen alpha 2(V) chain precurso... 49 0.037 2gp I M90464 I HUMCOL4ASX_1 alpha-S type IV collagen [Homo sap... 48 0.040 2gp I L31345 I CHKCOL3A1X_1 collagen type III [Gailus gallus] 47 0.046 4gp I L02918 I MUSCOL5A2X_1 procollagen type V alpha 2 [Mus mu... 53 0.066 2pin! S08012 I 508012 collagen-like protein, placental -... 55 0.069 2sp I P27393! CA24_ASCSU PROCOLLAGEN ALPHA 2(W) CHAIN PREC... 48 0.078 2gp I M25984 I CHKCOLL29_1 Chicken alpha-2 collagen gene type... 51 0.080 3gp I L15194 I MAUFEMRNA_1 Golden delicious apple fruit expre... 45 0.089 2gp I M20789 I HUMCIA1_1 alpha-I type I collagen [Horno sapi... 42 0.090 3pin A33848 I A33848 secreto]y protein 1- midge (Chino... 41 0.10 2gp I M63473 I HUMASCL1S_1 alpha-S type W collagen [Homo sap... 59 0.12 2pir I A37969 I A37969 collagen alpha 5(IV) chain - human... 59 0.12 2gp I M31115 I HUMCOL4A5_i COL4AS gene product [Hotno sapiens] 59 0.12 2sp I Q01493 I GP22_LITCA MAJOR MICROFILARIAL SHEATH PROTEIN... 48 0.12 2sp I P059971 CA2S_HUMAN PROCOLLAGEN ALPHA 2(V) CHAIN PRECU... 49 0.13 2pin! S20833I S20833 Collagen alpha 1(XIV) chain - Chic... 62 0.14 1sp I P350851 CBPJ)ICDI CALCIUM BINDING PROTEIN. >gp I U0341... 48 0.15 3pirIPQO612IPQO612 collagen alpha 1(XVDchain-huma... 55 0.19 2gpl U00697 I U00697_1 orphan receptor COUP-TFII [Gallus ... 45 0.22 2gp 1M13027 I MUSC4AI5_1 alpha-I type IV collagen [Mus musc... 38 0.22 2p1rIS22215IS22215 Collagenalphal(X)cbain-Mouse... 44 0.23 2sp I P12105! CA13_CHICK COLLAGEN ALPHA 1(111) CHAIN (FRAGM... 48 0.25 3pin I S23810 I S23810 collagen alpha 1(XV1) chain precur... 55 0.25 2gp I D140761 RATTESDYN_1 testicular dyrianiin[Rattus norv’egi... 47 0.25 2pin I 5128981512898 Collagen alpha 2(VIll) chain - Boy... 45 0.25 2gp I M58526 I HUMCOLA5IV_1 alpha-5 type IV collagen [Homo sap... 59 0.25 2sp I P02462! CAl4_HUMAN PROCOLLAGEN ALPHA 1(IV) CHAIN PREC... 47 0.26 2pir 15168761 CGHU4B collagen alpha 1(W) chain precurs... 47 0.26 2sp I P29400! CA54_HUMAN COLLAGEN ALPHA 5(W) CHAIN PRECURS... 59 0.26 2gp I Z22964 I CECOLA2W_1 a2(IV) collagen [Caenorhabditis el... 52 0.27 2sp I P081201 CA14_DROME PROCOLLAGEN ALPHA 1(IV) CHAIN PREC... 50 0.29 3pin! S17035 I SI 7035 Collagen alpha 1(XIV) chain - Chicken 54 0.32 2pin I S34665 1S34665 cuticular collagen- noot-kn’ot nem... 42 0.33 3pin I A30296 I A30296 collagen alpha (VII) chain - human... 49 0.33 2pin I A44309 I A44309 type II collagen alpha 1 chain, CO... 44 0.33 2pirl F304111F30411 synapsin lb -bovine 46 0.35 2gpIM2I811I BCVSIB_1 synapsin tb[Bostauru.1 46 0.35 269pin A36226 I A36226 collagen alpha-I chain - sea urchi... 63 0.56 1spl P175991 SYN1_BOVIN SYNAPSINS IA AND LB. 46 0.37 2phi E30411 I E30411 STL5fl Ia -bovine 460.37 2gp I M27810 I BOVSIA_1 synajin Ia [Bos taurus] 46 0.37 2pin A20982 I A20982 collagen short chain,, cartilage-sp... 44 0.38 2pir I A41 7521 A41752 hibernation-specific protein l-IP-27... 62 0.40 1pin 5136511 513651 collagen alpha 1(IV) chain homolog... 49 0.41 2gp I M91669 I HUIVIBP18OAA_l autoantigen [1-lomo sapiens] 49 041 2ph- I A03295 I PIHUSD proline-nich peptide P-D- human 60 0.42 1ph- I E38355 I E38355 basic proline-rich peptide LB-S - ... 60 0.42 1spi Q01149 I CA21_MOUSE PROCOLLAGEN ALPHA 2(1) CHAIN PRECIJ... 42 0.42 3gp I Z27078 I CEKO4H4_1 K04H4.1, a collagen [Caenorhabditi... 51 0.46 2spl P334791 1E18_PRVKA IMMEDIATE-EARLY PROTEIN 1E180. >pi... 41 0.46 3spl P17139 I CA14_CAEEL COLLAGEN ALPHA 1(W) CHAIN. >sp I P1... 51 0.46 2pin I S02624 I 502624 Collagen alpha 2(W) chain - Human... 40 0.49 2gp I L06863 I CRUCOLWLL_1 type VII collagen [Cricetulus gris... 48 0.50 3wIZ3O348I BMCOLGMR_1 Collagen [Bombyx mori] 50 0.51 2spl P13942 I CA2B_HUMAN COLLAGEN ALPHA 2(2(I) CHAIN (FRAGME... 45 0.51 3gp I X07882 I HSPRB 45_i Po protein [Homo sapiens] 61 0.54 1sp I P101631 PRP4_HUMAN SALIVARY PROLINE-RICH PROTEIN P0 P... 61 0.55pir I D25372 I D25372 proline-rich basic protein PRB4 pr... 61 0.55 1ph-I S319241S31924 Prol collagen type III - Rat (frag... 41 0.56 3gp I X70369 I RNPRO1C_1 prol collagen type m [Rattus nor... 41 0.56 3pir I B35363 I B 35363 synapsin lb -human 44 0.57 2ph-I B30411 I B30411 synapsin lb - rat >gp I M27924 I RATSI... 44 0.57 2gp I M58378 I HUMSYN1E13_2 synapsin lb [Homo sapiens] 44 0.57 2ph-I S28807IS28807 collagen alpha 1(X) chain precurso... 44 0.58 2ph-I S312161S31216 collagen alpha 1(X) chain precurso... 44 0.58 2spIPO9951ISYN1_RAT SYNAPSIMSIAAHDIB, 44 0.59 2ph-I A30411 I A30411 synapsin Ia - rat >gp I M27812 I RATSI... 44 0.59 2spl P176001 SYNUHUMAN SYNAPSINS IA AND TB (BRAIN PROTEIN... 44 0.59 2pir I A35363 I A 35363 synapsin Ia - human 44059 2gp I M58378 I HUMSYN1E13_1 synapsin I [Homo sapiens] 44 0.59 2ph-I JUO16O I JUO16O collagen RH - human (fragment) >gp... 45 0.60 2ph-I A41520 I A41520 chrornogranin A precursor - bovine ... 48 0.66 2spl P101611 PRPMJIUMAN SALIVARY PROLINE-RICH PROTEIN P0 (... 60 0.67ph-I A24661 I A24661 progesterone receptor - thicken (f... 51 0.67 1gp I M13972 I CHKPR_1 Chicken progesterone receptor (PR)... 51 0.67 1spl P101621 PRPL_HUMAN SALIVARY PROLINE-RICH PROTEIN PC (.. 60 0.68gp I X54412 I HSCOL9AL_3 Human mRNA for alphal(IX) collagen... 46 0.69 2spl P208491 CA19_HUMAN COLLAGEN ALPHA 1(IX) CHAIN PRECURS... 46 0.70 2ph- I G38355 I G38355 basic proline-nich peptide lB-Sb -... 36 0.71 2ph-I A34493 I A34493 collagen alpha 1(120 chain precurs... 41 0.72 2spl P16848 I UL31_HCMVA HYPOTHETICAL PROTEIN 11L31. >pirl SO... 60 0.72gp I M28658 I CHKCOLCOR_1 Chicken cornea alpha-1(IX) collage... 41 0.72 2ph-I A41 132 IA41132 collagen-related protein 1 precurs... 59 0.74 1ph-I A46053 I A46053 bullous pemphigoid antigen, BPAG2,... 53 0.80 3sp I P024571 CAll_CHICK PROOJLLAGEN ALPHA 1(0 CHAIN PRECU... 47 0.81 3WARNING: Descriptions of 108 database sequences were not reported due to theliniitingvalue ofparanieterV = 100.>spl P069141 CSP_PLAYO CIRCUMSPOROZOITE PROTEIN (CS) PRECURSOR.>ph- I A26271 I OZZQMY circumsponozoite protein precursor - Plasmodiurnyoelii >gp I J02695 I PFACSPH_1 circunisporozoite protein [Plasmodiumyoeliil70LerLgthL = 367Score =56(26.0 bits), Expect = 4.7, P = 0.99Idatities = 12135 (34%), Positives = 14/35 (40%)Query: 28 NIMYNPAQQEGPAFHI-IPGPLSQSPGTPGWEGDPQG 62M+ P +GP QPGP GPQ.GSbjct: 136 NVDQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQG 170Score = 52 (24.2 bits), Expect = 0.00095, Poisson P(2) = 0.00095Identities = 12/36(33%), Positives = 13/36 (36%)Query; 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLLGTDE 68P +GP QPGP GPQG ESbjct; 195 PGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQE 230>pirl S22373 S22373 proline-rich protein - mouse >gp I X63005 I MMPRP28_1proline-rich protein [Mus rnusculus] >gp I X63004 I MMPRP4628_1proline-rich protein [Mus musculus]Length = 260Score = 55(25.6 bits), Expect = 6.3, P = 1.0Identities = 10/38(26%), Positives = 18/38 (47%)Quet 24 GFTINIMYNPAQQEGPAPFIHPGPLSQSPGTPGWEGDPQ 61G++++P+ PPEGP+P +GP+Sbjct: 33 GHSTTVVSDPSPTPQPRPKHSGPPPKGPR.PGSTQGPPR 70Score = 51 (23.7 bits), Expect = 0.0012, Poisson P(2) = 0.0012Identities = 11/27(40%), Positives = 13/27 (48%)Query: 36 QEGPAPI-[HPGPLSQSPGTPGWEGDPQG 62Q+GPPPP S P G.i-QGSbjct: 74 QQGPPPPGPPPQGSSQQRPPQPGNQQG 100Score = 36 (16.7 bits), Expect = 0.12, Poisson P(3) = 0.11Identities = 9/27 (33%), Positives = 10/27 (37%)Query: 39 PAPHHPGPLSQSPGTPGWEGDPQGLAG 65PP G QPPG+P GSbjct: 60 PGPPPQGSSQQRPPQPGNQQGPPPQGG 106>pir I S16907 I S16907 collagen alpha 1(IV) chain - bovine (fragments)Length = 350Score = 51(23.7 bits), Expect = 26., P = 1.0Identities = 9/21 (42%), Positives = 10)21 (47%)Query: 52 GTPGWEGDPQGLAGTDEAGFQ 72GPGWGP +GFQSbjct: 251 GNPGWPGTPGAPGPXGDPGFQ 271Score = 42 (19.5 bits), Expect = 0.80, Poisson P(2) = 0.55Identities = 9/21 (42%), Positives = 11/21 (52%)Query: 51 PGTPGWEGDPQGLAGTDEAGF 71PG+PG GPG+ GF71Sbjct: 137 PGSPGPXGGPGGVGYPGLPGF 157Score = 41 (19.1 bits), Expect = 0.0015. Poisson P(3) = 0.0015Identities = 10/33 (30%), Positives = 12/33 (36%)Query: 38 GPAPHH PGPLSQSPGTPGWEGD PQGLAGTDEAG 70GPF + GPG GD +GSbjct: 65 GPGPXGVAGIPGPQGIPGLPGDXGAXGEXGQAG 97>pir I S20500 I S20500 hydroxyproline-rich glycoprotem - rice >gp I X61280 OSHPRGP_1hydraxyproline-rich glycoprotein [Oryza sativalLength =369Score = 49(22.8 bits), Expect = 51., P = 1.0Identities = 8/21 (38%), Positives = 11)21 (52%)Query: 31 YNPAQQEGPAPHFIPGPLSQSP 51YPA+ PP+PP+ PSbjct: 198 YKPAPKPTPTPYQPAPPTYKP 218Score = 45 (20.9 bits), Expect = 0.11, Poisson P(2) = 0.11Identities = 8/17 (47%), Positives = 10/17 (58%)Query: 30 MYNPAQQEGPAPHHPGP 46MY? + PAP+PPSbjct: 145 MYKPQPKPTPAPYTPTP 161Score = 41 (19.1 bits), Expect = 0.0016, Poisson P(3) = 0.0016Identities = 7/14 (50%), Positives = 9/14 (64%)Query: 41 PHHPGPLSQSPGTP 54?+H PS+PGPSbjct: 353 PYHKPPPSYTPGPP 366>sp I P176561 CCO2_CAEEL CUTICLE COLLAGEN 2. >sp I P176561 CCO2_CAEEL CUTICLECOLLAGEN 2. >pirI B31219 B31219 collagen 2- Caenorhabditis elegans>gp I V00 148 I CECOL2_1 Caenorhabditis elegans gene Col-2 cod±ng for acollagen. [Caenorhabditis elegansi >gp I J01048 I CELCOL2G_1 C.elegans(nematode) collagen 2 (col-2) gene, complete cds. [Caenorhabditisele gaxis]Length= )1Score = 50(23.2 bits), Expect = 0.0029, Poisson P(2) = 0.0029Identities = 11137 (29%), Positives = 17/37 (45%)Query: 38 GPAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQAQ 74GPPP +G?G+G+PG +G++Sbjct: 242 GPGPAGPKGPPGAAGAPGADGNPGGPGTAGKPGGEGE 278Score = 50(23.2 bits), Expect = 0.0029, Poisson P(2) = 0.0029Identities = 13/38 (34%), Positives = 16/38 (42%)Query; 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PQ P P + PGPG+G+P G GSbjct: 146 PCQPCPGGPPGPAGPAGPPGPPGPDGNP GSPAGPSGPG 183Score = 44 (20.5 bits), Expect = 0.17, Poisson P(2) = 0.1672Identities = 11/31 (35%), Positives = 13131 (41%)Query: 32 NPAQQEGPAPHHPGPLSQSPGTPGWEGDPQG 62+PA CYP? P + CY GGPGSbjct: 175 SPAGPSGPGPAGPPGPAGPAGNDGAPGAPGG 205>gp I L05906 I PMCMSGF_1 major surface glycoprotein [Pneurnocystis cariniilLangth = 1088Score = 52 (24.2 bits), Expect = 0.0032, Poisson P(2) = 0.0032Identities = 11/27 (40%), Positives = 12/27 (44%)Query; 39 PAPHHPGPLSQSPGTPGWEGDPQGLAG 65PPPP GTPGGG+GSbjct: 827 PPPGSPPPPPSQNGTPGTPGGETGASG 853Score = 52(24.2 bits), Expect = 0.0032, Poisson P(2) = 0.0032Identities = 10/29(34%), Positives = 16/29 (55%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQSFQD 78+PGTPG GPG+ +G +++DSbjct: 858 TPGTPGTPGTPGGMMKYAKLGLVKRTYVD 886Score = 45 (20.9 bits), Expect = 0.37, Poisson P(2) = 0.31Identities = 11/28 (39%), Positives = 11/28 (39%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDP 60PQG 0 SGTPGGPSbjct: 835 PPSQNGTPGTPGGETGASGGTPGTPGTP 862>spl P024541 CAll_RAT COLLAGEN ALPHA 1(0 CHAIN (FRAGMENTS),Length = 671Score = 42 (19.5 bits), Expect = 5.7e-i-02, P = 1.0Identities = 8/22 (36%), Positives = 10/22 (45%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60PP P + GPG+GPSbjct: 211 PGPPGPAGAAGPAGNPGADGQP 232Score = 41 (19.1 bits), Expect = 0.0060, Poisson P(3) = 0.0060Identities = 10/25 (40%), Positives = 11/25 (44%)Query: 48 SQSPGTPGWEGDPQGLAGTDEAGFQ 72SPGPG+Gt) AGQSbjct: 271 SGEPGAPGNKGDTGAKGEPGPAGVQ 295Score = 41 (19.1 bits), Expect = 0.0060, Poisson P(3) = 0.0060Identities = 9/26 (34%), Positives = 12126 (46%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEG 58?Q+PP L+GPG+GSbjct: 79 PGQRGPPGPQGARGLPGTAGLPGMKG 104Score = 40 (18.6 bits), Expect = 0.017, Poisson P(3) = 0.016Identities = 11/30 (36%), Positives = 12/30 (40%)73Query; 41 PHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PP SPGG+GP EAGSbjct: 342 PKGPAGERGSPGPAGPKGSPGEAGRPGEAG 371Score = 39 (18.1 bits), Expect = 13., Poisson P(2) = 1.0Identities = 9/24 (37%), Positives = 12/24 (50%)Query: 36 QEGPAPHHPGPLSQSPGTPGWEGD 59Q pp p ++PG G+GDSbjct: 526 QGPPGPAGPRGNNGAPGNDGAKGD 549Score =35 (16.3 bits), Expect = 0.020, Poisson P(4) = 0.020Identities = 7/23 (30%), Positives = 11/23 (47%)Query; 52 GTPGWEGDPQGLAGTDEAGFQAQ 74GPG+GPL +G÷+Sbjct; 494 GKPGZZGVPGDLGAPGPSGARGE 516>pir I A90559 I CGRTIS collagen alpha 1(1) chain - rat (fragments)Length= 671Score = 42 (19.5 bits), Expect = 5.7e+02, P = 1.0Identities = 8/22 (36%), Positives = 10/22 (45%)Query; 39 PAPHHPGPLSQSPGTPGWEGDP 60pp p + GPG+GPSbjct; 211 PGPPGPAGAAGPAGNPGADGQP 232Score = 41 (19.1 bits), Expect = 0.0060, Poisson P(3) = 0.0060Identities = 10125(40%), Positives = 11/25 (44%)Query; 48 SQSPGTPGWEGDPQGLAGTDEAGFQ 72SPGPG+GD AGQSbjct; 271 SGEPGAPGNKGDTGAKGEPGPAGVQ 295Score = 41 (19.1 bits), Expect = 0.0060, Poisson P(3) = 0.0060Identities = 9/26 (34%), Positives = 12/26 (46%)Query; 33 PAQQEGPAPHHPGPLSQSPGTPGWEG 58PQ+PP L+GPG+GSbjct; 79 PGQRGPPGPQGARGLPGTAGLPGMKG 104Score = 40 (18.6 bits), Expect = 0.017, Poisson P(3) = 0.016Identities = 11/30 (36%), Positives = 12/30 (40%)Query 41 PHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PP SPGG+GP EAGSbjct; 342 PKGPAGERGSPGPAGPKGSPGEAGRPGEAG 371Score = 37 (17.2 bits), Expect = 48., Poisson P(2) = 1.0Identities = 9/24 (37%), Positives = 12124 (50%)Query; 36 QEGPAPHHPGPLSQSPGTPGWEGD 59Q PP P ++PG G+GJ)Sbjct; 526 QGPPGPAGPRGNNGAPGBBGAKGD 549Score =35 (16.3 bits), Expect = 0.020, Poisson P(4) = 0.020Identities = 7/23(30%), Positives = 11/23(47%)74Query: 52 GTPGWEGDPQGLAGTDEAGFQAQ 74GPG+GPL +G+÷Sbjct; 494 GKPGZZGVPGDLGAPGPSGARGE 516>sp P34545 YNJ1_CAEEL HYPOTHETICAL 222.4 KD PROTEIN R1OEI1J IN CHROMOSOME HI.>spIP34S4SIYNJ1_CAEEL HYPOTHETICAL 222.4 KD PROTEIN R1OE11.1 INCHROMOSOME III. >gp Z290951 CER1OE11_1 R1OE11.1, contains chargedregions and a copy of the bromodomain [Caenorhabditis elegans]Length = 2815Score = 61 (28.4 bits), Expect = 0.93, P = 0.61Identities = 14/42(33%), Positives = 18/42 (42%)Query: 29 Hv[YNPAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70+M Q+ P H P +Q PG +GPQG G GSbj ct: 184 MMMGAQGQQFPGMMHRYPYAQGGPPPGAQGMPQGYPGVSRGG 225Score = 52(24.2 bits), Expect = 0.0062, Poisson P(2) = 0.0062Identities = 13143 (30%), Positives = 17/43 (39%)Query: 26 TINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAGTDE 68TN+M F+ PP GP PGP G+ D+Sbjct: 138 TPNMMSPPSMGRVPGPSPGGPQPPGPGQPQMRPGQPGMFQGIJQ 180>gp I X16711 I HSCOL2AIR1 COL2A1 gene product [Homo sapiens]Lngli = 1 10Score = 45 (20.9 bits), Expect 2.le+02, P = 1.0Identities = 13/36(36%), Positives = 14/36 (38%)Quet 35 QQEGPAPHHPGPLSQSPGTPGWEGDPQG[AGTDEAG 70+QPP L PGPGGP EAGSbjct: 586 EQGAPGPSGFQGLPGPPGPPGEGGKPGDQGVPGEAG 621Score = 44 (20.5 bits), Expect = 0.78, Poisson P(2) = 0.54Identities = 9/26 (34%), Positives = 14/26 (53%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQS 75+PGTPGGP T+G+++Sbjct: 997 APGTPGPPGSPGPAGPTGKQGDRGEA 1022Score =41(19.1 bits), Expect = 0.019, Poisson P(3) = 0.019Identities = 11/36(30%), Positives = 13/36 (36%)Quet 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQAQ 74PP G GPGGP+ EGF+Sbjct: 602 PGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGE 637>pir I S 18804 I S18804 collagen alpha 4(IV) chain - bovine (fragment)Length = 453Score =44 (20.5 bits), Expect = 2.8e+02, P = 1.0Identities = 9/2.3 (39%), Positives = 10/23 (43%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPG 5575P Q PP P + PGPGSbjct: 23 PGDQGPPGPDGPRGVPGPPGPPG 45Score = 41 (19.1 bits), Expect = 2.1, Poisson P(2) = 0.88Identities = 8/13 (61%), Positives = 9/13 (69%)Query: 47 LSQSPGTPGWEGD 59L SPGPG+GDSbjct: 185 LPGSPGPPGHKGD 197Score =39 (18.1 bits), Expect = 0.019, Poisson P(3) = 0.019Identities = 7/11 (63%), Positives = 8/11 (72%)Query: 50 SPGTPGWEGDP 60+PG PG GDPSbjct: 206 APGPPGPMGDP 216>pir I B40333 I B40333 collagen alpha 1(11) chain precursor - African clawed frogLength =1486Score = 47 (21.8 bits), Expect = 1.le+02, P = 1.0Idatities = 10/22(45%), Positives = 12/22 (54%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60PPPL÷ GPGEG+PSbjct: 1021 PGPVGPPGLTGPSGEPGREGNP 1042Score = 45 (20.9 bits), Expect = 0.52, Poisson P(Z) = GAOIdentities = 9126 (34%), Positwes = 14126 (53%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQS 75+PGPG GP + T+G++SSbjct: 1068 APGAPGAPGAPGSVGPTGKQGDRGES 1093Score = 41(19.1 bits), Expect = 0.032. Poisson P(3) = 0.031Identities = 9/27 (33%), Positives = 11)27 (40%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGD 59P PP P PGPG+G+Sbjct: 352 PGPAGPPGPVGPAGAPGFPGAPGSKGE 378>gp I M63595 I XELCOL2AL1 alpha-i type II collagen [Xenopus laevisjLangth = 1486Score = 47 (21.8 bits), Expect = 1.ie+02, P = 1.0Identities = 10/22 (45%), Positives = 12/22 (54%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60PPPL+ GPGEG+PSbjct: 1021 PGPVGPPGLTGPSGEPGREGNP 1042Score = 45 (20.9 bits), Expect = 0.52, Poisson P(2) = 0.40Identities = 9/26 (34%), Positives = 14126 (53%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQS 75+PGFG GP + T+G++SSbjct: 1068 APGAPGAPGAPGSVGPTGKQGDRGES 109376Score =41(19.1 bits), Expect = 0.032, Poisson P(3) = 0.031Identities = 9/27 (33%), Positives = 11/27 (40%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGD 59P F?? PGPG-i-G+Sbjct: 352 PGPAGPPGPVGPAGAPGFPGAPGSKGE 376>gp I M63596 T XELCOL2AIA...J alpha-i type II collagen [Xenopus laevis]Lnth = 1491Score = 45 (20.9 bits), Expect = 2.le+02, P = 1.0Identities = 13/36 (36%), Positives = 15/36 (41%)Query: 35 QQEGPAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70+QPP L PG+PGGP EAGSbjct: 660 EQGPPGPSGFQGLPGPPGSPGEGGKPGDQGVPGEAG 695Score = 43 (20.0 bits), Expect = 2.0, Poisson P(2) = 0.86Identities = 9(26(34%), Positives = 14/26 (53%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQS 75-i.PGPG OP + T+G++SSbjct: 1071 APGAPGSPGAPGPVGPTGKQGDRGES 1096Score = 41(19.1 bits), Expect = 0.032, PoissonP(3) = 0.031Identities = 9/27 (33%), Positives = 11/27 (40%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGD 59P PPP PGPG+G+Sbjct: 355 PGPAGPPGPVGPAGAPGFPGAPGSKGE 381>pir I A40333 I A40333 collagen alpha 1(11)’ chain precursor - African clawed frogLength = 1492Score = 45 (20.9 bits), Expect = 2.le-f-02, P = 1.0Identities = 13/36(36%), Positives = 15/36 (41%)Query: 35 QQEGPAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70+QPP L FG+PGGP EAGSbjct: 660 EQGPPGPSGFQGLPGPPGSPGEGGKPGDQGVPGEAG 695Score = 43 (20.0 bits), Expect = 2.0, Poisson P(2) = 0.86Identities = 9/26 (34%), Positives = 14)26 (53%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQS 75+PGPG GP + T+G++5Sbjct: 1071 APGAPGSPGAPGPVGPTGKQGDRGES 1096Score = 41(19.1 bits), Expect = 0.032, PoissonP(3) = 0.031Identities = 9/27 (33%), Positives = 11/27 (40%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGD 59P PP P FGPCT+G+Sbjct: 355 PGPAGPPGPVGPAGAPGFPGAPGSKGE 38177>pirl A31427 I A31427 collagen alpha 2(V) chain precursor - human (fragment)>gp I J04478 I HIJMC5A2B_1 COL5A2 gene product [Homo sapiensiLength 463Score = 49(22.8 bits), Expect = 52., P = 1.0Identities = 9/22 (40%), Positives = 10/22 (45%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60PP P + PGPG GPSbjct: 152 PGPRGPQGIDGEPGVPGQPGAP 173Score = 47(21.8 bits), Expect = 0.038, Poisson P(2) = 0.037Identities = 9/21 (42%), Positives = 11/21 (52%)Query: 51 PGTPGWEGDPQGLAGTDEAGF 71PGPG+G+P EGFSbjct: 260 PGKPGEDGEPGRNGNPGEVGF 280>gp I M90464 I HUMCOL4ASX_1 alpha-S type IV collagen [Homo sapiens]Length = 911Score =48 (22.3 bits), Expect = 0.040, Poisson P(2) = 0.040Identities = 9/20 (45%), Positives = 12/20 (60%)Query: 51 PGTPGWEGDPQGLAGTDEAG 70PGPG+G+PG+ EGSbjct: 584 PGLPGPKGEPGGITFKGERG 603Score =48(22.3 bits), Expect =0.040, Poisson P(2) = 0.040Identities = 9/22 (40%), Positives = 11/22 (50%)Query: 39 PAPF[FIPGPLSQSPGTPGWEGDP 60PPP+ PGPG+GPSbjct: 83 PGPPGPKGIRGPPGLPGFPGTP 104Score = 37(17.2 bits), Expect = 0.64, Poisson P(3) = 0.48Identities = 9/25 (36%), Positives = I 1125 (44%)Query: 41 PHHPGPLSQSPGTPGWEGDPQGLAG 65PP+ PGPGE +GGSbjct: 268 PPGPPGIRGPPGPPGGEKGEKGEQG 292>gp I L31345 I CHKCOL3AIX.J collagen type m [Gallus gallus]Length = 886Score = 47 (21.8 bits), Expect = 1.le+02, P = 1.0Identities = 9/17 (52%), Positives = 10/17 (58%)Query: 44 PGPLSQSPGTPGWEGDP 60PF+SPGPG GPSbjct: 168 PQPISGFPGPPGPSGPP 184Score =45(20.9 bits), Expect = 0.30, Poisson P(2) = 0.26Identities = 8/25 (32%), Positives = 12/25(48%)Query: 51 PGTPGWEGDPQGLAGTDEAGFQAQS 75PGPGG+PG G+++78Sbjct; 811 PGAPGQNGEPGGKGERGPPGLRGEA 835Score = 39 (18.1 bits), Expect = 0.081, PoissonP(3) = 0.078Identities = 8115(53%), Positives = 10/15 (66%)Query; 48 SQSPGTPGWEGDPQG 62SSPG G+G+PGSbjct; 733 SGSPGPKGDKGEPGG 747Score =39 (18.1 bits), Expect = 0.081, Poisson P(3) = 0.0 78Identities = 7/19 (36%), Positives = 12/19 (63%)Query; 41 PHHPGPLSQSPGTPGWEGD 59p p + PG+PG++G+Sbjct; 333 PPGPPGTAGFPGSPGFKGE 351Score =35 (16.3 bits), Expect = 0.04 7, Poisson P(4) = 0.046Identities = 8/20 (40%), Positives = 8/20 (40%)Query; 39 PAPFIHPGPLSQSPGTPGWEG 58PPP PGPGGSbjct: 298 PGPGGPTGERGRPGNPGGPG 317>gp 1 L02918 1 MUSCOL5A2X_1 procollagen type V alpha 2 [Mus niusculuslLength = 1497Score = 53(24.6 bits), Expect = 14., P = 1.0Identities = 10/21 (47%), Positives = 12/21 (57%)Query; 51 PGTPGWEGDPQGLAGTDEAGF 71PGPG+G+P TEGFSbjct; 258 PGECPGEDGEPGRNGNTGEVGF 278Score =48 (22.3 bits), Expect = 0.068, Poisson P(2) = 0.066Identities = 9/22 (40%), Positives = 10/22 (45%)Query; 39 PAPHHPGPLSQSPGTPGWEGDP 60pp p + PGPG GPSb.jct: 150 PGPRGPQGIDGEPGMPGQPGAP 171>pir I 5080121 S08012 collagen-like protein, placental - human (fragment)>gpl X15038 I HSCOLLHM_1 collagen-like protein [Homo sapiens]Length = 447Score = 55(25.6 bits), Expect = 6.7, P = 1.0Identities = 13134 (38%), Positives = 15/34 (44%)Query 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQ 72P PG PGG+GDP+TGFQSbjct; 83 PGKEGPGGKPGKPGVKGEKGDPCEVCPTLPEGFQ 116Score =46 (21.4 bits), Expect = 0.07 1, Poisson P(2) = 0.069Identities = 13/32 (40%), Positives = 14/32 (43%)Query; 34 AQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAG 65A+GP P PGTPGGPGGSbjct; 44 AGEPGPPGLPGPPGIGLPGTPGDPGGPPGPKG 7579>sp I P273931 CA24_ASCSU PROCOLLAGEN ALPHA 2(W) CHAIN PRECURSOR.>pir15163661 SI 6366 collagen alpha 2(W) chain precursor - pigroundworm >gp I M67507 I NEMA2C4A_1 alpha-2 (IV) collagen gene product[Ascaris swim]Length = 1763Score =48 (22.3 bits), Expect = 0.08 1, Poisson P(2) = 0.078Idaitities = 11131 (35%), Positives = 13/31 (41%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDPQGL 63P PPP PGFG+G+PLSbjct: 580 PGAPGRPGPPGPPGQDGLPGLPGQKGEPTQL 610Score =48 (22.3 bits), Expect = 0.08 1, Poisson P(2) = 0.078Identities = 12/32 (37%), Positives = 13/32 (40%)Query: 47 LSQSPGTPGWEGDPQGLAGTDEAGFQAQSFQD 78L PGPGEGP EAG +DSbjct: 1215 LKGEPGLPGLEGQPGPRGMKGEAGLPGAPGRD 1246Score = 47 (21.8 bits), Expect = 0.16, Poisson P(2) = 0.15Identities = 13/36 (36%), Positives = 13/36 (36%)Query 39 PAPHHPGPISQSPGTPGWEGDPQGLAGTDEAGFQAQ 74PPPLPGGGP EGFQSbjct: 1362 PGPQGPAGLPGLFGLKGEPGLPGFPGQKGETGF’PGQ 1397>gp I M25984 I CHKCOLA29_1 Chicken alpha-2 collagen gene type I gene, exon 52.[Gallus galh]Length = 896Score = 51(23.7 bits), Expect = 27., P = 1.0Identities = 11/27 (40%), Positives = 14/27 (51%)Query: 51 PGTPGWEGDPQGLAGTDEAGFQAQSFQ 77PGPGGP GEGFA++÷Sbjct: 624 PGPPGPPGPPGPNGGGYEVGFDAEVYR 650Score = 40 (18.6 bits), Expect = 8.8, Poisson P(2) = 1.0Identities = 11/35 (31%), Positives = 14/35 (40%)Query: 38 GPAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQ 72GPA SQPP PGG÷G+÷Sbjct: 607 GPAGVRGSHGSQGPAGPPGPPGPPGPPGPNGGGYE 641Score =39 (18.1 bits), Expect = 0.083, Poisson P(3) = 0.084)Identities = 8/26 (30%), Positives = 11)26 (42%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEG 58P÷+ PP P + PG G GSbjct: 243 PGERGEPGPVGPSGFAGPPGAAGQPG 268>gp I L15 194 I MAUFEMRNA_1 Golden delicious apple fruit expressed mRNA, completecds. [Malus domestica]Length= 11980Score = 45 (20.9 bits), Expect = 1.6e-’-02, P = 1.0Identities = 8/14 (57%), Positives = 9/14 (64%)Query: 47 LSQSPGTPGWEGDP 60+ SPGTPG GPSbjct: 48 MPMSPGTPGTPGTP 61Score =43 (20,0 bits), Expect = 0.093, Poisson P(2) = 0.089Identities = &18 (44%), Positives = 9/18 (50%)Query: SO SPGTPGWEGDPQGLAGTD 67+PGTPG GP DSbjct: 54 TPGTPGTPGTPASARAKD 71>gp I M20789 I HUMC1A1_i alpha-i type I collagen [Homx, sapiens]Length 589Score =42(19.5 bits), Expect = 5.7e+02, P = 1.0Identities = 8/22 (36%), Positives = 10/22 (45%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60PP P + GPG÷GPSbjct: 373 PGPPGPAGAAGPAGNPGADGQP 394Score = 40 (18.6 bits), Expect = 5.6, Poisson P(2) = 1.0Idatities = 11130(36%), Positives = 12/30 (40%)Quei 41 PHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PP SPGG+GP EAGSbjct: 504 PKGPAGERGSPGPAGPKGSPGEAGRPGEAG 533Score =38(17.7 bits), Expect = 0.094, Poisson P(3) = 0.090Identities = 8/26 (30%), Positives = 12/26 (46%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEG 58P++PP L+GPG+GSbjct: 241 PGERGPPGPQGARGLPGTAGLPGMKG 266Score = 35(16.3 bits), Expect = 1.9, Poisson P(3) = 0.85Identities = 8/21 (38%), Positives = 8i21 (38%)Query; 51 PGTPGWEGDPQGLAGTDEAGF 71PG G GP G GFSbjct: 475 PGPTGLPGPPGERGGPGSRGF 495>pir I A33848 I A33848 secretory protein 1 - rnidge (Chironoinus tentans) (fragment)>gp I M24276 I CHISPA_i C.tentans 140-kd secretory protein (spldO)mRNA, partial cds, clone pCtl4O.1. [Chironomns tentans]Length =64Score = 41(19.1 bits), Expect = 0.10, Poisson P(2) = 0.10Identities = 9/24 (37%), Positives = 12/24 (50%)Query: 32 NPAQQEGPAPHHPGPLSQSPGTPG 55J++EG.P+G PGGSbjct: 28 NGSKPEGKAPGNNGKSCSKPGGKG 5181Score = 41 (19.1 bits), Expect = 0.10, Poisson P(Z) = 0.10Identities = 10/32(31%), Positives = 15/32 (46%)Query: 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70P PG +S PG+G +G+G+ GSbjct: 32 PEGKAPGNNGKSCSKPGGKGPNKGKSGSKPEG 63>gp I M63473 HUMA5CLI&3 alpha-S type LV collagen [Homo sapiens]Length = 762Score =59(27.4 bits), Expect = 1.8, P = 0.83Identities = 12132 (37%), Positives = 15/32 (46%)Query 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PPP+SPGPGG+P+G GSbjct: 204 PGPPGPKGISGPPGNPGLPGEPGPVGGGGHPG 235Score = 46 (21.4 bits), Expect = 0.13, Poisson P(Z) = 0.12Identities = 11129(37%), Positives = 12/29(41%)Quer 32 NPAQQEGPAPHHPGPLSQSPGTPGWEGDP 60NP PP G PGPG+GPSbjct: 218 NPGLPGEPGPVGGGGHPGQPGPPGEKGKP 246Score = 46 (21.4 bits), Expect = 0.13, Poisson P(2) = 0.12Identities = 12/33 (36%), Positives = 14/33 (42%)Query: 28 TINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEG 58TIMPQ PP+PG+PGGSbjct: 105 TIGDMGFPGPQGVEGPPGPSGVPGQPGSPGLPG 137>pir I A37969 A37969 collagen alpha 5(W) chain - human (fragment)Length = 76Score =59(27.4 bits), Expect = 1.8, P = 0.83Identities = 12/52(37%), Positives = 15/32(46%)Quet 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PPP+SPGPGG+P+G GSbjct: 205 PGPPGPKGISGPPGNPGLPGEPGPVGGGGHPG 236Score =46 (21.4 bits), Expect = 0.13, Poisson P(Z) = 0.12Identities = 11/29(37%), Positives = 12/29 (41%)Query 32 NPAQQEGPAPHHPGPLSQSPGTPGWEGDP 60NP PP G PGPG+GPSbjct: 219 NPGLPGEPGPVGGGGHPGQPGPPGEKGKP 247Score =46(21.4 bits), Expect = 0.13, Poisson P(2) = 0.12Identities = 12/33(36%), Positives = 14/33 (42%)Query: 26 TINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEG 58TIIvIPQ PP+PG+FGGSbjct: 106 TIGDMGFPGPQGVEGPPGPSGVPGQPGSPGLPG 13882>gp I M31115 I HUMCOL4AS_1 COL4A5 gene product [Homo sapiens]Length = 772Score =59(27.4 bits), Expect = 1.8, P = 0.83Identities = 12132 (37%), Positives = 15/32 (46%)Quety: 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PPP+SPGPGG+P+G GSbjct: 214 PGPPGPKGISGPPGNPGLPGEPGPVGGGGHPG 245Score =46 (21.4 bits), Expect = 0.13, Poisson P(2) = 0.12Identities = 11129(37%), Positives = 12/29 (41%)Query 32 NPAQQEGPAPHHPGPLSQSPGTPGWEGDP 60NP PP 0 PGPG+GPSbjct: 228 NPGLPGEPGPVGGGGHPGQPGPPGE}CGKP 256Score =46(21.4 bits), Expect = 0.13, Poisson P(Z) = 0.12Identities = 12133(36%), Positives = 14/33 (42%)Query: 26 TINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEG 58TIMPQ PP+PG+PGGSbjct: 115 TIGDMGFPGPQGVEGPPGPSGVPGQPGSPGLPG 147>sp I Q01493 GP22_LITCA MAJOR MICROFILARIAL SHEATH PROTEIN PRECURSOR.>pir I JH0788 I JHO 788 sheath glycoprotein gp22 precursor - neiriatode(Litomosoides carinii) >gp I M96232 I LITGP22A_1 major microfilarialsheath protein [Litomoscides carinu]Length = 148Score =48(22.3 bits), Expect = 61., P = 1.0Identities = 10131 (32%), Positives = 14/31 (45%)Query: 35 QQEGPAPHHPGPLSQSPGTPGWEGDPQGLAG 65QGPPPPL P+P+ ++GSbjct: 57 QPMGPQPMEPQPLPMGPQSPQMQVPDRSCSG 87Score =43 (20.0 bits), Expect = 0.13, Poisson P(Z) = 0.12Identities = 8/19 (42%), Positives = 9/19 (47%)Query: 33 PAQQEGPAPHHPGPLSQSP 51PQ GPP PP+ PSbjct: 50 PQQPMGPQPMGPQPMEPQP 68>sp I P059971 CA25_HUMAN PROCOLLAGEN ALPHA 2(V) CHAIN PRECURSOR.Length = 16Score =49(22.8 bits), Expect = 55., P = 1.0Identities = 9/22 (40%), Positives = 10/22 (45%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60PP P + PGPG GPSbjct: 152 PGPRGPQGIDGEPGVPGQPGAP 173Score = 47 (21.8 bits), Expect = 0.13, Poisson P(2) = 0.13Identities = 9/21 (42%), Positives = 11)21 (52%)83Query: 51 PGTPGWEGIJPQGLAGTIJEAGF 71PGPG÷G+P EGFSbjct: 260 PGKPGEDGEPGRNGNPGEVGF 280>pir I S20833 I 520833 Collagen alpha 1(XIV) chain - Chicken (fragmenbs)Length = 56Score = 62(28.8 bits), Expect = 0.15, P 0.14Iditities = 14133(42%), Positives = 16/33 (48%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAG 65P +÷ GPA H P SQ P P DP AGSbjct: 23 PGEKGGPAGHTGPPGSQGPAGPPGYCDPSSCAG 55>sp I P350851 CBP_DICDI CALCIUM BINDING PROTEIN. >gp I U03413 I DDU03413..J calciumbindhgprotem [Dictyostelium cliscoideumJLength =Score =48(22.3 bits), Expect = 73., P = 1.0Iditities = 11125(44%), Positives = 11/25(44%)Query: 41 PHHPGPLSQSPGTPGWEGDPQGLAG 65PPGQPGPG QGGSbjct: 121 PGQPGYPPQQPGAPGQYPPQQGQPG 145Score = 39 (18.1 bits), Expect = 8.5, Poisson P(2) = 1.0Identities = sf23 (39%), Positives = 9,23 (39%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPG 55PQ PPGQPGGSbjct: 92 PGQYPPQQPGQPGYPPQQPGQSG 114Score =37(17.2 bits), Expect = 0.16, Poisson P(3) = 0.15Identities = 8118 (44%), Positives = 9/18 (50%)Query: 49 QSPGTPGWEGDPQGLAGT 66QPGPG QG+TSbjct: 180 QQPGQPGAYPPQQGVQNT 197>pirl PQ06121 PQ0612 collagen alpha 1(XVI) chain - human (fragment)>gp I S57 1321 557132_I type XVI collagen alpha I chairs [Homo sapiens]Length 1186Score = 55(25.6 bits), Expect = 7.1, P = 1.0Identities = 13/34(38%), Positives = 15/34(44%)Query: 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQ 72P PG PGG+GDP+TGFQSbjct: 67 PGKEGPGGKPGKPGVKGEKGDPCEVCPTLPEGFQ 100Score =46(21.4 bits), Expect = 0.21, Poisson P( 0.19Identities = 13132(40%), Positives = 14132(43%)Quet 34 AQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAG 65A+GP P PGTPGGPGGSbjct: 28 AGEPGPPGLPGPPGIGLPGTPGDPGGPPGPKG 5984Score = 44) (18.6 bits), Expect = 1.le+03, P = 1.0IdiUties = 10/28(35%). Positives = 11./28 (39%)Query; 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDP 60P + PP P L PG G GPSbjct; 806 PGLRG1JPGPAGPPGLMGPPGFKGKTGHP 833Score = 37 (17.2 bits), Expect = Li, Poisson P(3) = 0.67Identities = 8/24 (33%), Positives = 11/24 (45%)Query; 51 PGTPGWEGDPQGLAGTDEAGFQAQ 74PGPG+GP +GA+Sbjct; 500 PGVPGLQGVPGNNGLPGQPGLTAE 523>gp I U00697 I U00697_1 orphan receptor COUP-TFII [Gallus gallus]Length= 410Score =45(20.9 bits), Expect = 2.Oe+02, P = 1.0Identities = 12/36(33%), Positives = 16/36 (44%)Quet 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQAQ 74P P +P TPG G P A ++ A Q+QSbjct: 28 PVQGPPAGTPH9PQTPGPGGPPSTPAQSNAASQQSQ 63Score =44(20.5 bits), Expect = 0.25, Poisson P( = 022Identities = 10(23(43%), Positives = 12/23 (52%)Query; 36 QEGPAPHHPGPLSQSPGTPGWEG 58QPAP GP++PTP GSbjct; 22 QPAPAPPVQGPPAGTPHTPQTPG 44>gp I M13027 I MUSC4AI5_1 alpha-i type IV collagen IMus musculus]LrLgth =43Score =38(17.7 bits), Expect =0.25, Poisson P(2) = 022Identities = 7/10(70%), Positives = 7/10 (70%)Query: 51 PGTPGWEGDP 60PG PG EG PSbjct; 18 FGLPGPEGPP 27Score =38(17.7 bits), Expect = 0.25, Poisson P(2) = 022Identities = 8/18(44%), Positives = 8/18(44%)Query; 41 PHHPGPLSQSPGTPGWEG 58PPGP GPGGSbjct; 5 PGPPGPYDVIKGEPGLPG 22>pir I S22215 I S22215 Collagen alpha 1(X) chain - Mouse (fragment)>gp I X63013 I MMA1XCOL_1 collagen-alpha-I type X [Mtrs musculusiLength= 243Score =44 (20.5 bits), Expect = 2.7e+02, P = 1.0Identities = 7/11 (63%), Positives = 9/11 (81%)85Query: 50 SPGTPGWEGDP 60+PG PG+GDPSbjct: 18 NPGLPGQKGDP 28Scare =43 (20.0 bits), Expect =0.28, Poisson P() = 0.23Identities = 9/20 (45%), Positives = 10/20 (50%)Query: 51 PGTPGWEGDPQGLAGTDEAG 70PG PG +GDF AGSbjct; 76 PGFPGSKGOPGNPGAPGPAG 95>sp I P12105 I CA13_CHICK COLLAGEN ALPHA 1(111) CHAIN CFRAGMENTS).>pir I A05269 I A05269 collagen alpha 1(ffl) chain- chicken(fragments)Length = 615Score =48(22.3 bits), Expect = 75., P = 1.0Identities = 12139(30%), Positives = 14/39(35%)Query: 32 NPAQQEGPAPHHPGPLSQSPGTPGWEG13PQG[AAGTDEAG 70NP++PP GPGGPG EGSbjct: 100 NPGERGEPGPQGQAGPPGPQGPPGRAGSPGGKGEMGEPG 138Score =41 (19.1 bits), Expect = 3.0, Poisson P(Z) = 0.95Identities = 10/39(25%), Positives = 17/39 (43%)Query: 21 VVLGFTIN [MYNPAQQEGPAPHHPGPLSQSPGTPGWEGD 59+ LG + Q P F + PG+PG÷+G+Sbjct: 50 IPLGECCPVCPQTTPQPTKGPPGPPGTAGFPGSPGFKGE 88Score =37(17.2 bits), Expect =0.28, Poisson P(3) = 025Identities =9/22(40%), Positives = 1(W22 (45%)Query: 51 PGTPGWEGDPQGLAGTDEAGFQ 72PGPGG+ EGFQSbjct: 254 PGHPGPAGNNGAPGKAGERGFQ 275>pir I S23810 I S28810 collagen alpha 1(XVI) chain precursor - human>gp I M92642 I HTJMCOLI6A.J alpha-I type XVI collagen [Homo sapiensiLength = 13Score = 55(23.6 bits), Expect = 7.2, P = 1.0Identities = 13/34(38%), Positives = 15/34(44%)Query: 39 PAPHHPGPLSQSPGTPGWEGDPQGLAGTDEAGFQ 72P PG PGG+GDP+TGPQSbjct: 4.84 PGKEGPGGKPGKPGVKGEKGDPCEVCPTLPEGFQ 517Score =48(21.4 bits), Expect = 0.28, Poisson P(Z) = 025Identities = 13/32(40%), Positives = 14/32(43%)Query. 34 AQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAG 65A+GP P PGTPGGPGGSbjct: 445 AGEPGPPGLPGPPGIGLPGTPGDPGGPPGPKG 476Score = 40(18.6 bits) Expect = Lle+03, P = 1.0Identities = 10/28(35%), Positives = 11/28 (39%)86Query; 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDP 60P+ PP P L PGG GPSbjct; 1223 PGLRGDPGPAGPPGLMGPPGFKGKTGHP 1250Score = 37 (17.2 bits Expect = 2.0, Poisson P(3) = 0.87Identities 8/24(33%), Positives = 11/24 (45%)Query: 51 PGTPGWEGDPLAGTDEAGFQAQ 74PGPG+GP +GA+Sbjct; 917 PGVPGLQGVPGNNGLPGQPGLTAE 940>gp 1014076 I RATTESDYN_1 testicizlar dynamin [Rattus norvegicislLength 848Score = 47 (21.8 bits), Expect = 1.le÷02, P = 1.0Idenhtie = 10122(45%), Positives = 10/22(45%)Query: 33 PAQQEGPAPHHPGPLSQSPGTP 54PA GPAP PP S PSbjct: 779 PASSRGPAPAIPSPGPHSGAPP 800Score =45(20.9 bits), Expect = 0.28, Poisson P(Z) = 025Identities =9/22(40%), Positives = 10/22(45%)Query: 39 PAPHHPGPLSQSPGTPGWECDP 60PP PGPL P+ GPSbjct: 800 PVPFRPGPLPPFPNSSDSYGAP 821>pirl S12898 I S12898 Collagen alpha 2(Vfll) chain - Bcwin.e (fragment)Length = 459Score =45(20.9 bits), Expect = 2.Oe+02, P = LI)Identities = 10130(33%), Positives = 14/30(46%)Query; 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDPQG 62P++PHP + GPG+GPGSbjct: 337 PGERGLPGAHGPPGPTGPXGEPGFrGRPGG 366Score =44(20.5 bits), Expect = 0.), Poisson P(Z) = 025Identities = 10124(41%), Positives = 1 1i24 (45%)Query: 47 LSQSPGTPGWEGDPQGLAGTDEAG 70L+PGPGGP 1)EGSbjct; 446 LTGPPGPPGPPGPPGAPGAFDE1t 469>gp I M58526 I HUMCOLA5IV_1 alpha-S t3pe IV collagen [Homo sapienslLength = 1644Score =59(27.4 bits), Expect = 1.8, P = 0.84Identities = 12/32(37%), Positives = 15/32 (46%)Quet 39 PAPHHPGPLSQSPGTPGWEGDPQG[AGTDEAG 70PPP+SPGPGG+P+G GSbjct: 1046 PGPPGPKGISGPPGNPGLPGEPGPVGGGGHPG 107787Score = 46 (21.4 bits), Expect = 0.29, Poisson P(2) = 0.25Iditities = 11129 (37%), Positives = 12/29 (41%)Query 32 NPAQQEGPAPHHPGPLSQSPGTPGWEGDP 60NP PP G PGPG+GPSbjct: 1060 NPGLPGEPGPVGGGGHPGQPGPPGEKGKP 1088Score 46 (21.4 bits), Expect = 0.29, Poisson P(Z) = 0.25Identities = 12/33(36%), Positives = 14/35(42%)Query: 26 TINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEG 58TIM? Q PP + PG+PGGSbjct: 947 TIGDMGFPGPQGVEGPPGPSGVPGQPGSPGLPG 979>spl P024621 CA14_HUMAN PROCOLLAGEN ALPHA 1(IV) CHAIN PRECURSOR.Length = 1669Score =47(21.8 bits), Expect = 1.le+02, P = 1.0Identities = 8/15(53%), Positives = 10/15 (66%)Query: 51 PGTPGWEGDPQGLAG 65PG PG ÷GDP + GSbjct; 150 PGLPGMKGDPGEILG 164Score =46 (21.4 bits), Expect = 0.30, Poisson P(Z) = 026Identities =9/21(42%), Positives = 10/21 (47%)Query: 52 GTPGWEGDPQGLAGTDEAGFQ 72GPGWGP +GFQSbjct: 1269 GNPGWPGAPGVPGPKGDPGFQ 1289Score =45(20.9 bits), Expect = 0.58, Poisson P( = 044Identities = 9(17(52%), Positives = 10)17(58%)Query: 44 PGPLSQSPGTPGWEGDP 60PG+ PGPGEGPSbjct: 638 PGKIVPLPGPPGAEGLP 654Score = 44(20.5 bits), Expect = Li, Poisson P(2) = 0.68Identities = 10/21 (47%), Positives = 11/21 (52%)Query: 39 PAPHIIPGPLSQSPGTPGWEGD 59PP L SPGPG÷GDSbjct: 645 PGPPGAEGLPGSPGFPGPQGD 665>pirl SI6876 I CGHU4E collagen alpha 1(IV) chain precursor - human>gp1M26576 1 HUMCOLIA42_1 alpha-i type IV collagen [Homo sapiens]Length 1869Score = 47 (21.8 bits), Expect = Lle+02, P = 1.0Identities = 8/15(53%), Positives = 10/15(66%)Query: 51 PGTPGWEGDPQGLAG 65PGPG+Gt)P +GSbjct: 150 PGLPGMKGDPGEILG 164Score =46(21.4 bits), Expect = 0.30, Poisson P( = 02688Identities = 9/21 (42%), Positives = 10/21 (47%)Query: 52 GTPGWEGDPQGLAGTDEAGFQ 72GPGWGP +GFQSbjct: 1269 GNPGWPGAPGVPGPKGDPGFQ 1289Score =45(20.9 bits), Expect = 0.58, Poisson P(Z) = 0.44Identities = 9/17 (52%), Positives = 10/17 (58%)Query; 44 PGPLSQSPGTPGWEGDP 60PG+ PGPGEGPSbjct: 638 PGKIVPLPGPPGAEGLP 654Score =44 (20.5 bits), Expect = 1.1, Poisson P(2) = 0.68Identities = 10/21 (47%), Positives = 11121 (52%)Query: 39 PAPHHPGPLSQSPGTPGWEGI) 59PP LSPGPG+GOSbjct: 645 PGPPGAEGLPGSPGFPGPQGD 665>sp I P294001 CA54_HUMAN COLLAGEN ALPHA 5(W) CHAIN PRECURSOR. >pirIS229lT I S22917collagen alpha 5(W) chain prectu-sor - human>gp I U04520 I HS4COLSA51i COL4A5 gene product [Homo sapiens]>gp1U045201 HS4COL5A5LJ COL4A5geneprodtict [Homosapiens]Length 1685Score =59(27.4 bits), Expect = 1.8, P = 0.84Identities = 12(32(37%), Positives = 15/32(46%)Quet 39 PAPHHPGPISQSPGTPGWEGD PQGIAGTDEAG 70PPP+SPGPGG+P÷G GSbjct: 1127 PGPPGPKGISGPPGNPGLPGEPGPVGGGGHPG 1158Score =46(21.4 bits), Expect = 0.30, Poisson P(Z) = 026Identities = 11/29(37%), Positives = 12/29 (41%)Query: 32 NPAQQEGPAPHHPGPLSQSPGTPGWEGDP 60NP PP G PGPG+GPSbjct: 1141 NPGLPGEPGPVGGGGHPGQPGPPGEKGKP 1169Score =46(21.4 bits) Expect = 0.30, Poisson P(2) = 026Identities 12(33(36%), Positives = 14)33 (42%)Query: 26 TINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEG 58TI 1’1 P Q P P + PG+PG GSbjct: 1028 TIGDMGFPGPQGVEGPPGPSGVPGQPGSPGLPG 1060>gp I Z22964 I CECOLA2IV_1 a2(IV) collagen [Caenorhabditis elegans]Length = 1795Score = 52(24.2 bits), Expect 20., P = 1.0Identities = 12/28(42%), Positives = 13/28(46%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDP 60PQ P GL PGTPG+GPSbjct: 723 PGQPGFPGAKGL)GGLPGLPGTPGLQGMP 75089Score =46(21.4 bits), Expect = 0., Poisson P(2) = 0.27Idatities = 12132(37%), Positives = 16/32(50%)Queiy 47 LSQSPGTPGWEGDPQGLAGTDEAGFQAQSFQD 78L SPG PG +G P ++GF Q Q÷Sbjct: 1250 LKGSPGYPGQDGLPGIPGLKGDSGFPGQPGQE 1281Score = 36 (16.7 bits), Expect = 7.0, Poisson P(3) = 1.0Identities = 10130(33%), Positives = 1Z/30 (40%)Query 41 PHHPGPLSQSPGTPGWEGDPQGLAGTDEAG 70PPF÷Q P+ GLGTGSbjct: 750 PGEPAPENQVNPAPPGQPGLPGLPGTKGEG 779>sp I P081201 CA14_DROME PROCOLLAGEN ALPHA 1(W) CHAIN PRECURSOR.>pirI A31893 I A31893 collagen alpha WV) chath precursor - fruit fly(Drosophila melanogaster) >gp I M23704 I DROC4A1A_1 alpha-I type IVcollagen [Drosophila melanogasterl >gp I M96575 I DROCOL4G_1 type 1Vcollagen [Drosophila metanogaster] >gp J02?27 I DR(XOL1V_1 type Wpro-collagen [Drosophila melanogaster]Length 1775Score = 50(23.2 bits), Expect = 39., P = 1.0Identities = 10124(41%), Positives = 1324 (54%)Query: 47 LSQSPGTPGWEGDPQGLAGTDEAG 70L+PGPG+G+P TEGSbjctz 362 LNGLPGNPGQKGEPGRAGATGEPG 385Score =39(18.1 bits), Expect = 0.34, Poisson P(3) = 029Identities = S’20 (45%), Positives = 9,20(45%)Query: 39 PAPHHPGPLSQSPGTPGWEG 58PPPL GPGEGSbjct: 94 PGPLGPTGLKGEMGFPGMEG 113Score =39(18.1 bits), Expect = 0.34, Poisson P(3) = 029Identities = 7/21 (33%), Positives = 9/21 (42%)Query: 51 PGTPGWEGDPQGLAGTDEAGF TiPGPG+GP G+Sbjct: 1149 PGAPGMDGLPGAAGAPGAVGY 1169>pirl S170351 S17035 Collagen alpha 1(XIV) chain - ChickenLength = 196Score = 54 (.1 bits), Expect = 8.4, P = 1.0Identities = 13128(46%), Positives = 13,28 (46%)Query 38 GPAPHHPGP1SQSPGTPGWEGDPQGLAG 65GRH PSQP P DP AGSbjct: 168 GPAGHTGPPGSQGPAGPPGYCDPSSCAG 195Score =42(19.5 bits), Expect = 0.39, Poisson P(2) = 0.32Identities = 9/29 (31%), Positives = 12/29 (4 1%)Query: 32 NPAQQEGPAPHHPGFLSQSPGTPGWEGDP 6090+P PPP+PG+GG?Sbjct: 137 SPGPPGSPGPRGPAGHTGPPGSQGPAGPP 185>pir I S34665 1 S34665 cuticular collagen - root-knot nematode (Meloidogyneincognita) >gp I Z24734 MILEMMIA_l cuticular collagen [Meloidogyneincognita]Length 286Score = 42(19.5 bits), Expect = 5.3e+02, P = LI)Identities = 9/20 (45%), Positives = 9/20 (45%)Query: 39 PAPHHPGPLSQSPGTPGWEG 58PPP SPGPGGSbjct: 226 PGPVGPAGPSGKPGAPGQPG 245Score = 37(17.2 bits), Expect = 19., Poisson P(2) = 1.0Identities = 8(20 (40%), Positives = 9/20(45%)Queiy: 41 PHI-IPGPLSQSPGTPGWEGDP 60PP +PGPGGPSbjct: 231 PAGPSGKPGAPGQPGPHGPP 250Score =36 (16.7 bits), Expect = 4.0e-03, P = 1.0Identities = 8(20(40%), Positives = 9/21) (45%)Query: 51 PGTPGWEGDPQGLAGTDEAG 70PGPG GP +G GSbjct: 143 PGQPGRPGAPGKSSGAGPPG 162Score =35(16.5 bits), Expect = 0.40, Poisson F(S) = 0.33Identities = 7114 (50%), Positives = 7/14 (50%)Query: 33 PAQQEGPAPHHPGP 46P GFAHGPSbjct: 166 PQGPPGPAGKHGGP 179>pir I A30296 I A30296 collagen alpha (VII) chain - human (fragments)Lsngth =62Score = 49(22.8 bits), Expect = 25., P = 1.0Identities = 12126(46%), Positives = 12/26(46%)Query: 45 GPLSQSPGTPGWEGDPQGLAGTPEAG 70GLPGPG PGGEAGSbjct: 19 GELCGEPGKPGILPGPPGPPGPKEAG 44Score =39(18.1 bits), Expect = 0.40, Poisson P(Z) = 0.33Identities = 10129(34%), Positives = 12/29(41%)Query: 30 MYNPAQQEGPAPHHPGPLSQSPGTPGWEG 56+Y Q+G PG PGPGGSbjct: 11 VYXEKGQQGELCGEPGKPGILPGPPGPPG 39Score =35 (16.3 bits), Expect = 6,9, Poisson P(2) = 1.0Identities = 10/33(30%), Positives = 12/33(36%)Query: 33 PAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLAG 6591P+GP G+GPGGGGSbjct: 4 PRPEPGPVYXEKGQQGELCGEPGKPG[LPGPPG 35>pir I A44309 I A44309 type II co]iagn alpha I chain, COL2A1 - human (fragment)Length 346Score = 44 (20.5 bits), Expect = 2.Se1-02, P = 1.0Identities = 9/26 (34%), Positives = 14t26 (53%)Query: 50 SPGTPGWEGDPQGLAGTDEAGFQAQS 75+GT?GGP T--G÷+÷Sbjct: 315 APGTPGPPGSPGPAGPTGKQGDRGEA 340Score =43(20.0 bits), Expect = 0.40, Poisson P(Z) =1133Identities = 9122(40%), Positives = 11,22(50%)Query: 39 PAPHHPGPLSQSPGTPGWEGDP 60pp p L+ GPG+GPSbjct: 268 PGPVGPPGLTGPAGEPGIQGSP 289WARNING; lISPs involving 138 database sequences were not reported due to thelimiting value ofparameter B = 50.Parameters:E=10.,S=55(25.6bits), E2=014,S2=35W =3, T =11(5.1 bits), X =22(10.2 bits)M= BLOSUM62H = 1, V = 100, B =50-gapdecayrate 0.5 (the default)Statistics:Lambda = 0.322 nats/unit score, Lambda/1n2 = 0.465 bits/unit scoreK = 0.142, H = 0.667 bits,ositionExpectedlObserved high score =58(27.0 bits) /63 (.3 bits)# of letters in query: 78# of neighborhood words in quexy 2172t of exact wards scoringbelowT: 0Database: Non-redundant PDB+SwissProt+SPupdate+PIR+GenPept+GPupdate, 5:04 AMEDT pr 1, 1994# of letters in database: 32,528,034# of word hits against database: 6,536,751# of failed hit extensions: 4,809,644# of excluded hits: 1,722,483# of successful extensions; 4624# of overlapping HSPs discarded: 4162# of HSPs reportable: 462# of sequences in database: 114,708# of database sequences with at least one 1-ISP: 208No. of states in DFA: 462(46 KB)Total si of DFA; 70 KB (128 KB)Time to ganerate neighborhood: 0.Olu 0.OOs 0.Olt Real: 00:00:00No. ofprocessors used: 12Time to search database: 33.78u 3.34s 37.12t Real: 00:00:06Total cpu time: 33.87u 3.39s 37.26t Real: 00:00:06WARNINGS ISSUED: 292gregory lee TechMail-S Wed, 13 Apr 94Page 1Date: Wed, 13 Apr 94 08:17:54From: glee (gregory lee)To: blast@ncbi. nlm. nih. govPROGRAM blastpDATAI3IB nrBEGIN>HSAg-5/6 frame +2EFRISG IPFC TIKF IHNYKNVVLGFTINIMYNPAQQEGPAPHHPGPLSQSPGTPGWEGDPQGLATDEAGFQAQSFQD93

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0087540/manifest

Comment

Related Items