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. 1996 May;178(9):2498–2506. doi: 10.1128/jb.178.9.2498-2506.1996

Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.

K M Dobos 1, K H Khoo 1, K M Swiderek 1, P J Brennan 1, J T Belisle 1
PMCID: PMC177971  PMID: 8626314

Abstract

Chemical evidence for the true glycosylation of mycobacterial proteins was recently provided in the context of the 45-kDa MPT 32 secreted protein of Mycobacterium tuberculosis (K. Dobos, K. Swiderek, K.-H. Khoo, P. J. Brennan, and J. T. Belisle, Infect. Immun. 63:2846-2853, 1995). However, the full extent and nature of glycosylation as well as the location of glycosylated amino acids remained undefined. First, to examine the nature of the covalently attached sugars, the 45-kDa protein was obtained from cells metabolically labeled with D-[U-14C] glucose and subjected to compositional analysis, which revealed mannose as the only covalently bound sugar. Digestion of the protein with the endoproteinase subtilisin and analysis of products by liquid chromatography-electrospray-mass spectrometry on the basis of fragments demonstrating neutral losses of hexose (m/z 162) or pentose (m/z 132) revealed five glycopeptides, S7, S18, S22, S29, and S41 among a total of 50 peptides, all of which produced only m/z 162 fragmentation ion deletions. Fast atom bombardment-mass spectrometry, N-terminal amino acid sequencing, and alpha-mannosidase digestion demonstrated universal O glycosylation of Thr residues with a single alpha-D-Man, mannobiose, or mannotriose unit. Linkages within the mannobiose and mannotriose were all alpha 1-2, as proven by gas chromatography-mass spectrometry of oligosaccharides released by beta-elimination. Total sequences of many of the glycosylated and nonglycosylated peptides combined with published information on the deduced amino acid sequence of the entire 45-kDa protein demonstrated that the sites of glycosylation were located in Pro-rich domains near the N terminus and C terminus of the polypeptide backbone. Specifically, the Thr residues at positions 10 and 18 were substituted with alpha-D-Manp(1-->2)alpha-D-Manp, the Thr residue at position 27 was substituted with a single alpha-D-Manp, and Thr-277 was substituted with either alpha-D-Manp, alpha-D-Manp(1-->2)alpha-D-Manp, or alpha-D-Manp(1--> 2)alpha-D-Manp(1-->2)alpha-D-Manp. This report further corroborates the existence of true prokaryotic glycoproteins, defines the complete structure of a mycobacterial mannoprotein and the first complete structure of a mannosylated mycobacterial protein, and establishes the principles for the study of other mycobacterial glycoproteins.

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Selected References

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  1. Andersen P., Askgaard D., Ljungqvist L., Bennedsen J., Heron I. Proteins released from Mycobacterium tuberculosis during growth. Infect Immun. 1991 Jun;59(6):1905–1910. doi: 10.1128/iai.59.6.1905-1910.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bock K., Schuster-Kolbe J., Altman E., Allmaier G., Stahl B., Christian R., Sleytr U. B., Messner P. Primary structure of the O-glycosidically linked glycan chain of the crystalline surface layer glycoprotein of Thermoanaerobacter thermohydrosulfuricus L111-69. Galactosyl tyrosine as a novel linkage unit. J Biol Chem. 1994 Mar 11;269(10):7137–7144. [PubMed] [Google Scholar]
  3. Brennan P. J., Nikaido H. The envelope of mycobacteria. Annu Rev Biochem. 1995;64:29–63. doi: 10.1146/annurev.bi.64.070195.000333. [DOI] [PubMed] [Google Scholar]
  4. Chatterjee D., Aspinall G. O., Brennan P. J. The presence of novel glucuronic acid-containing, type-specific glycolipid antigens within Mycobacterium spp. Revision of earlier structures. J Biol Chem. 1987 Mar 15;262(8):3528–3533. [PubMed] [Google Scholar]
  5. Chatterjee D., Lowell K., Rivoire B., McNeil M. R., Brennan P. J. Lipoarabinomannan of Mycobacterium tuberculosis. Capping with mannosyl residues in some strains. J Biol Chem. 1992 Mar 25;267(9):6234–6239. [PubMed] [Google Scholar]
  6. Dobos K. M., Swiderek K., Khoo K. H., Brennan P. J., Belisle J. T. Evidence for glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium tuberculosis. Infect Immun. 1995 Aug;63(8):2846–2853. doi: 10.1128/iai.63.8.2846-2853.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Erickson P. R., Herzberg M. C. Evidence for the covalent linkage of carbohydrate polymers to a glycoprotein from Streptococcus sanguis. J Biol Chem. 1993 Nov 15;268(32):23780–23783. [PubMed] [Google Scholar]
  8. Espitia C., Espinosa R., Saavedra R., Mancilla R., Romain F., Laqueyrerie A., Moreno C. Antigenic and structural similarities between Mycobacterium tuberculosis 50- to 55-kilodalton and Mycobacterium bovis BCG 45- to 47-kilodalton antigens. Infect Immun. 1995 Feb;63(2):580–584. doi: 10.1128/iai.63.2.580-584.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Espitia C., Mancilla R. Identification, isolation and partial characterization of Mycobacterium tuberculosis glycoprotein antigens. Clin Exp Immunol. 1989 Sep;77(3):378–383. [PMC free article] [PubMed] [Google Scholar]
  10. Garbe T., Harris D., Vordermeier M., Lathigra R., Ivanyi J., Young D. Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation. Infect Immun. 1993 Jan;61(1):260–267. doi: 10.1128/iai.61.1.260-267.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerwig G. J., Kamerling J. P., Vliegenthart J. F., Morag E., Lamed R., Bayer E. A. The nature of the carbohydrate-peptide linkage region in glycoproteins from the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens. J Biol Chem. 1993 Dec 25;268(36):26956–26960. [PubMed] [Google Scholar]
  12. Goochee C. F., Gramer M. J., Andersen D. C., Bahr J. B., Rasmussen J. R. The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties. Biotechnology (N Y) 1991 Dec;9(12):1347–1355. doi: 10.1038/nbt1291-1347. [DOI] [PubMed] [Google Scholar]
  13. Gooley A. A., Classon B. J., Marschalek R., Williams K. L. Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1194–1201. doi: 10.1016/0006-291x(91)91019-9. [DOI] [PubMed] [Google Scholar]
  14. Gooley A. A., Williams K. L. Towards characterizing O-glycans: the relative merits of in vivo and in vitro approaches in seeking peptide motifs specifying O-glycosylation sites. Glycobiology. 1994 Aug;4(4):413–417. doi: 10.1093/glycob/4.4.413. [DOI] [PubMed] [Google Scholar]
  15. Hawke D. H., Harris D. C., Shively J. E. Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument. Anal Biochem. 1985 Jun;147(2):315–330. doi: 10.1016/0003-2697(85)90278-7. [DOI] [PubMed] [Google Scholar]
  16. Herscovics A., Orlean P. Glycoprotein biosynthesis in yeast. FASEB J. 1993 Apr 1;7(6):540–550. doi: 10.1096/fasebj.7.6.8472892. [DOI] [PubMed] [Google Scholar]
  17. Huddleston M. J., Bean M. F., Carr S. A. Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. Anal Chem. 1993 Apr 1;65(7):877–884. doi: 10.1021/ac00055a009. [DOI] [PubMed] [Google Scholar]
  18. Hunter S. W., Rivoire B., Mehra V., Bloom B. R., Brennan P. J. The major native proteins of the leprosy bacillus. J Biol Chem. 1990 Aug 25;265(24):14065–14068. [PubMed] [Google Scholar]
  19. Jacob G. S., Scudder P. Glycosidases in structural analysis. Methods Enzymol. 1994;230:280–299. doi: 10.1016/0076-6879(94)30019-4. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Laqueyrerie A., Militzer P., Romain F., Eiglmeier K., Cole S., Marchal G. Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex. Infect Immun. 1995 Oct;63(10):4003–4010. doi: 10.1128/iai.63.10.4003-4010.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lechner J., Wieland F. Structure and biosynthesis of prokaryotic glycoproteins. Annu Rev Biochem. 1989;58:173–194. doi: 10.1146/annurev.bi.58.070189.001133. [DOI] [PubMed] [Google Scholar]
  23. McNeil M., Chatterjee D., Hunter S. W., Brennan P. J. Mycobacterial glycolipids: isolation, structures, antigenicity, and synthesis of neoantigens. Methods Enzymol. 1989;179:215–242. doi: 10.1016/0076-6879(89)79123-0. [DOI] [PubMed] [Google Scholar]
  24. Messner P., Christian R., Kolbe J., Schulz G., Sleytr U. B. Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70. J Bacteriol. 1992 Apr;174(7):2236–2240. doi: 10.1128/jb.174.7.2236-2240.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Messner P., Christian R., Neuninger C., Schulz G. Similarity of "core" structures in two different glycans of tyrosine-linked eubacterial S-layer glycoproteins. J Bacteriol. 1995 Apr;177(8):2188–2193. doi: 10.1128/jb.177.8.2188-2193.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  27. Nagai S., Wiker H. G., Harboe M., Kinomoto M. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun. 1991 Jan;59(1):372–382. doi: 10.1128/iai.59.1.372-382.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Norman E., Dellagostin O. A., McFadden J., Dale J. W. Gene replacement by homologous recombination in Mycobacterium bovis BCG. Mol Microbiol. 1995 May;16(4):755–760. doi: 10.1111/j.1365-2958.1995.tb02436.x. [DOI] [PubMed] [Google Scholar]
  29. Parras F., Guerrero M. C., Bouza E., Blázquez M. J., Moreno S., Menarguez M. C., Cercenado E. Comparative study of mupirocin and oral co-trimoxazole plus topical fusidic acid in eradication of nasal carriage of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1995 Jan;39(1):175–179. doi: 10.1128/aac.39.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Plummer T. H., Jr, Tarentino A. L., Hauer C. R. Novel, specific O-glycosylation of secreted Flavobacterium meningosepticum proteins. Asp-Ser and Asp-Thr-Thr consensus sites. J Biol Chem. 1995 Jun 2;270(22):13192–13196. doi: 10.1074/jbc.270.22.13192. [DOI] [PubMed] [Google Scholar]
  31. Reinhold B. B., Hauer C. R., Plummer T. H., Reinhold V. N. Detailed structural analysis of a novel, specific O-linked glycan from the prokaryote Flavobacterium meningosepticum. J Biol Chem. 1995 Jun 2;270(22):13197–13203. doi: 10.1074/jbc.270.22.13197. [DOI] [PubMed] [Google Scholar]
  32. Romain F., Augier J., Pescher P., Marchal G. Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5322–5326. doi: 10.1073/pnas.90.11.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Romain F., Laqueyrerie A., Militzer P., Pescher P., Chavarot P., Lagranderie M., Auregan G., Gheorghiu M., Marchal G. Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria. Infect Immun. 1993 Feb;61(2):742–750. doi: 10.1128/iai.61.2.742-750.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schlesinger L. S., Hull S. R., Kaufman T. M. Binding of the terminal mannosyl units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to human macrophages. J Immunol. 1994 Apr 15;152(8):4070–4079. [PubMed] [Google Scholar]
  35. Schultz J. C., Takayama K. The role of mannosylphosphorylpolyisoprenol in glycoprotein biosynthesis in Mycobacterium smegmatis. Biochim Biophys Acta. 1975 Jan 13;381(1):175–184. doi: 10.1016/0304-4165(75)90199-3. [DOI] [PubMed] [Google Scholar]
  36. Shively J. E., Miller P., Ronk M. Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis. Anal Biochem. 1987 Jun;163(2):517–529. doi: 10.1016/0003-2697(87)90257-0. [DOI] [PubMed] [Google Scholar]
  37. Stimson E., Virji M., Makepeace K., Dell A., Morris H. R., Payne G., Saunders J. R., Jennings M. P., Barker S., Panico M. Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose. Mol Microbiol. 1995 Sep;17(6):1201–1214. doi: 10.1111/j.1365-2958.1995.mmi_17061201.x. [DOI] [PubMed] [Google Scholar]
  38. Takayama K., Schnoes H. K., Armstrong E. L., Boyle R. W. Site of inhibitory action of isoniazid in the synthesis of mycolic acids in Mycobacterium tuberculosis. J Lipid Res. 1975 Jul;16(4):308–317. [PubMed] [Google Scholar]
  39. Takayama K., Schnoes H. K., Semmler E. J. Characterization of the alkali-stable mannophospholipids of Mycobacterium smegmatis. Biochim Biophys Acta. 1973 Aug 23;316(2):212–221. doi: 10.1016/0005-2760(73)90011-8. [DOI] [PubMed] [Google Scholar]
  40. 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]

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