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. 1989 Sep;77(3):378–383.

Identification, isolation and partial characterization of Mycobacterium tuberculosis glycoprotein antigens.

C Espitia 1, R Mancilla 1
PMCID: PMC1542043  PMID: 2478323

Abstract

In Mycobacterium tuberculosis culture filtrates, three concanavalin A (ConA)-binding bands of 55, 50 and 38 kilodaltons (kD) were identified by labelling blotted proteins with a ConA-peroxidase conjugate. Binding was inhibited by the competitor sugar alpha-methyl mannoside and by reduction with sodium m-periodate. Bands of 55, 50 and 38 kD stained with Coomasie blue were sensitive to digestion with proteases, thus indicating that they are proteins. Glycoproteins were isolated by lectin affinity chromatography or by elution from nitrocellulose membranes. On the isolated form, the 55-50 kD doublet glycoprotein was 65.4% protein and 34.6% sugar. The purified 38 kD molecule was 74.3% protein and 25.7% carbohydrate. By immunoblot, antibodies against mycobacterial glycoproteins were demonstrated in immunized rabbits and in patients with pulmonary tuberculosis, but not in healthy individuals. Treatment with sodium m-periodate abolished binding of rabbit antibodies to the 38 kD glycoprotein. Reactivity of the 55-50 kD doublet glycoprotein was not altered by reduction. By immunoblot with monoclonal antibodies TB71 and TB72, a carbohydrate-dependent and a carbohydrate-independent epitope could be identified on the 38 kD glycoprotein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander S., Elder J. H. Carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens. Science. 1984 Dec 14;226(4680):1328–1330. doi: 10.1126/science.6505693. [DOI] [PubMed] [Google Scholar]
  2. Alves M. J., Abuin G., Kuwajima V. Y., Colli W. Partial inhibition of trypomastigote entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi. Mol Biochem Parasitol. 1986 Oct;21(1):75–82. doi: 10.1016/0166-6851(86)90081-2. [DOI] [PubMed] [Google Scholar]
  3. Dalton J. P., Strand M. Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae. J Immunol. 1987 Oct 1;139(7):2474–2481. [PubMed] [Google Scholar]
  4. Espitia C., Cervera I., González R., Mancilla R. A 38-kD Mycobacterium tuberculosis antigen associated with infection. Its isolation and serologic evaluation. Clin Exp Immunol. 1989 Sep;77(3):373–377. [PMC free article] [PubMed] [Google Scholar]
  5. Gaylord H., Brennan P. J., Young D. B., Buchanan T. M. Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan. Infect Immun. 1987 Nov;55(11):2860–2863. doi: 10.1128/iai.55.11.2860-2863.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glass W. F., 2nd, Briggs R. C., Hnilica L. S. Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets. Anal Biochem. 1981 Jul 15;115(1):219–224. doi: 10.1016/0003-2697(81)90549-2. [DOI] [PubMed] [Google Scholar]
  7. Hamburger J., Lustigman S., Siongok T. K., Ouma J. H., Mahmoud A. A. Characterization of a purified glycoprotein from Schistosoma mansoni eggs: specificity, stability, and the involvement of carbohydrate and peptide moieties in its serologic activity. J Immunol. 1982 Apr;128(4):1864–1869. [PubMed] [Google Scholar]
  8. 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]
  9. Larraga V., Muñoz E. Molecular organization in bacterial cell membranes. Specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes. Eur J Biochem. 1975 May;54(1):207–218. doi: 10.1111/j.1432-1033.1975.tb04130.x. [DOI] [PubMed] [Google Scholar]
  10. Ma Y., Daniel T. M. Isolation, characterization, and specificity of a glycoprotein antigen from Mycobacterium kansasii. Am Rev Respir Dis. 1983 Dec;128(6):1059–1064. doi: 10.1164/arrd.1983.128.6.1059. [DOI] [PubMed] [Google Scholar]
  11. Maeba P. Y. Isolation of a surface glycoprotein from Myxococcus xanthus. J Bacteriol. 1986 May;166(2):644–650. doi: 10.1128/jb.166.2.644-650.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Marshall R. D. Glycoproteins. Annu Rev Biochem. 1972;41:673–702. doi: 10.1146/annurev.bi.41.070172.003325. [DOI] [PubMed] [Google Scholar]
  13. Mescher M. F., Strominger J. L. Purification and characterization of a prokaryotic glucoprotein from the cell envelope of Halobacterium salinarium. J Biol Chem. 1976 Apr 10;251(7):2005–2014. [PubMed] [Google Scholar]
  14. Mitchell C. G., Smith R., Lehner T. Recognition of carbohydrate and protein epitopes by monoclonal antibodies to a cell wall antigen from Streptococcus mutans. Infect Immun. 1987 Mar;55(3):810–815. doi: 10.1128/iai.55.3.810-815.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Morris E. J., Ganeshkumar N., Song M., McBride B. C. Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein. J Bacteriol. 1987 Jan;169(1):164–171. doi: 10.1128/jb.169.1.164-171.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moskophidis M., Müller F. Molecular characterization of glycoprotein antigens on surface of Treponema pallidum: comparison with nonpathogenic Treponema phagedenis biotype Reiter. Infect Immun. 1984 Dec;46(3):867–869. doi: 10.1128/iai.46.3.867-869.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nogueira N., Unkeless J., Cohn Z. Specific glycoprotein antigens on the surface of insect and mammalian stages of Trypanosoma cruzi. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1259–1263. doi: 10.1073/pnas.79.4.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Okuda S., Weinbaum G. An envelope-specific glycoprotein from Escherichia coli B. Biochemistry. 1968 Aug;7(8):2819–2825. doi: 10.1021/bi00848a018. [DOI] [PubMed] [Google Scholar]
  19. Parekh B. S., Mehta H. B., West M. D., Montelaro R. C. Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem. 1985 Jul;148(1):87–92. doi: 10.1016/0003-2697(85)90631-1. [DOI] [PubMed] [Google Scholar]
  20. Pfannenstiel M. A., Muthukumar G., Couche G. A., Nickerson K. W. Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1987 Feb;169(2):796–801. doi: 10.1128/jb.169.2.796-801.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ramasamy R., Reese R. T. A role for carbohydrate moieties in the immune response to malaria. J Immunol. 1985 Mar;134(3):1952–1955. [PubMed] [Google Scholar]
  22. Rapp V. J., Ross R. F. Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection. Infect Immun. 1986 Dec;54(3):751–760. doi: 10.1128/iai.54.3.751-760.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Robey W. G., Arthur L. O., Matthews T. J., Langlois A., Copeland T. D., Lerche N. W., Oroszlan S., Bolognesi D. P., Gilden R. V., Fischinger P. J. Prospect for prevention of human immunodeficiency virus infection: purified 120-kDa envelope glycoprotein induces neutralizing antibody. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7023–7027. doi: 10.1073/pnas.83.18.7023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Woodward M. P., Young W. W., Jr, Bloodgood R. A. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods. 1985 Apr 8;78(1):143–153. doi: 10.1016/0022-1759(85)90337-0. [DOI] [PubMed] [Google Scholar]
  26. Wu M. C., Heath E. C. Isolation and characterization of lipopolysaccharide protein from Escherichia coli. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2572–2576. doi: 10.1073/pnas.70.9.2572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Young D., Kent L., Rees A., Lamb J., Ivanyi J. Immunological activity of a 38-kilodalton protein purified from Mycobacterium tuberculosis. Infect Immun. 1986 Oct;54(1):177–183. doi: 10.1128/iai.54.1.177-183.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]

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