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. 1987 Jul 1;166(1):63–76. doi: 10.1084/jem.166.1.63

Antigenic and immunogenic properties of cyanogen bromide peptides from gonococcal outer membrane protein IB. Evidence for the existence of a surface-exposed conserved epitope

PMCID: PMC2188640  PMID: 2439639

Abstract

Two distinct species of gonococcal porin proteins exist that differ with regard to surface exposure. Protein IB, expressed by strains of the WII/III serogroup, has both termini buried in the outer membrane, leaving a central region of the molecule exposed at the cell surface. We have attempted to define this region of protein IB in detail by studying the antigenic and immunogenic properties of peptides derived from protein IB. Treatment of gonococcal protein IB (serotype 5) with cyanogen bromide resulted in cleavage of protein IB into three major fragments of Mr of 15,000, 13,000, and 8,000. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8,000 Mr peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of protein IB revealed a cleavage site near one of the cyanogen bromide cleavage sites. Trypsin was found to cleave the protein, either in outer membranes complexes (OMC) or in detergent micelles, in the central CB2 fragment. These results suggest that CB2 is a part of the surface- exposed region of protein IB. Immunization of mice with purified protein IB (serotype 5) induced antibodies against all three CB- peptides. Absorption of the sera with homologous OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface-exposed nature. Antibodies against the 13,000 Mr peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane. Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface-exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.

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Selected References

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  1. Barrera O., Swanson J. Proteins IA and IB exhibit different surface exposures and orientations in the outer membranes of Neisseria gonorrhoeae. Infect Immun. 1984 Jun;44(3):565–568. doi: 10.1128/iai.44.3.565-568.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blake M. S., Gotschlich E. C. Gonococcal membrane proteins: speculation on their role in pathogenesis. Prog Allergy. 1983;33:298–313. [PubMed] [Google Scholar]
  3. Blake M. S., Gotschlich E. C. Purification and partial characterization of the major outer membrane protein of Neisseria gonorrhoeae. Infect Immun. 1982 Apr;36(1):277–283. doi: 10.1128/iai.36.1.277-283.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blake M. S., Gotschlich E. C. Purification and partial characterization of the opacity-associated proteins of Neisseria gonorrhoeae. J Exp Med. 1984 Feb 1;159(2):452–462. doi: 10.1084/jem.159.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blake M. S., Gotschlich E. C., Swanson J. Effects of proteolytic enzymes on the outer membrane proteins of Neisseria gonorrhoeae. Infect Immun. 1981 Jul;33(1):212–222. doi: 10.1128/iai.33.1.212-222.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buchanan T. M., Eschenbach D. A., Knapp J. S., Holmes K. K. Gonococcal salpingitis is less likely to recur with Neisseria gonorrhoeae of the same principal outer membrane protein antigenic type. Am J Obstet Gynecol. 1980 Dec 1;138(7 Pt 2):978–980. doi: 10.1016/0002-9378(80)91091-1. [DOI] [PubMed] [Google Scholar]
  7. Buckel P., Zehelein E. Expression of Pseudomonas fluorescens D-galactose dehydrogenase in E. coli. Gene. 1981 Dec;16(1-3):149–159. doi: 10.1016/0378-1119(81)90071-8. [DOI] [PubMed] [Google Scholar]
  8. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  9. Edwards M., McDade R. L., Schoolnik G., Rothbard J. B., Gotschlich E. C. Antigenic analysis of gonococcal pili using monoclonal antibodies. J Exp Med. 1984 Dec 1;160(6):1782–1791. doi: 10.1084/jem.160.6.1782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fletcher J. N., Zak K., Virji M., Heckels J. E. Monoclonal antibodies to gonococcal outer membrane protein I: location of a conserved epitope on protein IB. J Gen Microbiol. 1986 Jun;132(6):1611–1620. doi: 10.1099/00221287-132-6-1611. [DOI] [PubMed] [Google Scholar]
  11. Frantz I. D. Growth Requirements of the Meningococcus. J Bacteriol. 1942 Jun;43(6):757–761. doi: 10.1128/jb.43.6.757-761.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GROSS E., WITKOP B. Nonenzymatic cleavage of peptide bonds: the methionine residues in bovine pancreatic ribonuclease. J Biol Chem. 1962 Jun;237:1856–1860. [PubMed] [Google Scholar]
  13. Heckels J. E. The surface of Neisseria gonorrhoeae: isolation of the major components of the outer membrane. J Gen Microbiol. 1977 Apr;99(2):333–341. doi: 10.1099/00221287-99-2-333. [DOI] [PubMed] [Google Scholar]
  14. Heckels J. E. The surface properties of Neisseria gonorrhoeae: topographical distribution of the outer membrane protein antigens. J Gen Microbiol. 1978 Oct;108(2):213–219. doi: 10.1099/00221287-108-2-213. [DOI] [PubMed] [Google Scholar]
  15. Hook E. W., 3rd, Olsen D. A., Buchanan T. M. Analysis of the antigen specificity of the human serum immunoglobulin G immune response to complicated gonococcal infection. Infect Immun. 1984 Feb;43(2):706–709. doi: 10.1128/iai.43.2.706-709.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnston K. H., Gotschlich E. C. Isolation and characterization of the outer membrane of Neisseria gonorrhoeae. J Bacteriol. 1974 Jul;119(1):250–257. doi: 10.1128/jb.119.1.250-257.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnston K. H., Holmes K. K., Gotschlich E. C. The serological classification of Neisseria gonorrhoeae. I. Isolation of the outer membrane complex responsible for serotypic specificity. J Exp Med. 1976 Apr 1;143(4):741–758. doi: 10.1084/jem.143.4.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lynch E. C., Blake M. S., Gotschlich E. C., Mauro A. Studies of Porins: Spontaneously Transferred from Whole Cells and Reconstituted from Purified Proteins of Neisseria gonorrhoeae and Neisseria meningitidis. Biophys J. 1984 Jan;45(1):104–107. doi: 10.1016/S0006-3495(84)84127-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marshall T. Detection of protein in polyacrylamide gels using an improved silver stain. Anal Biochem. 1984 Feb;136(2):340–346. doi: 10.1016/0003-2697(84)90227-6. [DOI] [PubMed] [Google Scholar]
  22. Pepinsky R. B. Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a novel peptide-mapping technique. J Biol Chem. 1983 Sep 25;258(18):11229–11235. [PubMed] [Google Scholar]
  23. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  24. Sandstrom E. G., Knapp J. S., Buchanan T. B. Serology of Neisseria gonorrhoeae: W-antigen serogrouping by coagglutination and protein I serotyping by enzyme-linked immunosorbent assay both detect protein I antigens. Infect Immun. 1982 Jan;35(1):229–239. doi: 10.1128/iai.35.1.229-239.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schoolnik G. K., Fernandez R., Tai J. Y., Rothbard J., Gotschlich E. C. Gonococcal pili. Primary structure and receptor binding domain. J Exp Med. 1984 May 1;159(5):1351–1370. doi: 10.1084/jem.159.5.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  27. Swanson J. Studies on gonococcus infection. XVIII. 125I-labeled peptide mapping of the major protein of the gonococcal cell wall outer membrane. Infect Immun. 1979 Mar;23(3):799–810. doi: 10.1128/iai.23.3.799-810.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Virji M., Heckels J. E. Antigenic cross-reactivity of Neisseria pili: investigations with type- and species-specific monoclonal antibodies. J Gen Microbiol. 1983 Sep;129(9):2761–2768. doi: 10.1099/00221287-129-9-2761. [DOI] [PubMed] [Google Scholar]
  30. Virji M., Heckels J. E. Role of anti-pilus antibodies in host defense against gonococcal infection studied with monoclonal anti-pilus antibodies. Infect Immun. 1985 Sep;49(3):621–628. doi: 10.1128/iai.49.3.621-628.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Virji M., Zak K., Heckels J. E. Monoclonal antibodies to gonococcal outer membrane protein IB: use in investigation of the potential protective effect of antibodies directed against conserved and type-specific epitopes. J Gen Microbiol. 1986 Jun;132(6):1621–1629. doi: 10.1099/00221287-132-6-1621. [DOI] [PubMed] [Google Scholar]

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