Evidence for glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium tuberculosis

The occurrence of glycosylated proteins in Mycobacterium tuberculosis has been widely reported. However, unequivocal proof for the presence of true glycosylated amino acids within these proteins has not been demonstrated, and such evidence is essential because of the predominance of soluble lipoglycans and glycolipids in all mycobacterial extracts. We have confirmed the presence of several putative glycoproteins in subcellular fractions of M. tuberculosis by reaction with the lectin concanavalin A. One such product, with a molecular mass of 45 kDa, was purified from the culture filtrate. Compositional analysis demonstrated that the protein was rich in proline and that mannose, galactose, glucose, and arabinose together represented about 4% of the total mass. The 45-kDa glycoprotein was subjected to proteolytic digestion with either the Asp-N or the Glu-C endopeptidase or subtilisin, peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and glycopeptides were identified by reaction with concanavalin A. Peptides were further separated, and when they were analyzed by liquid chromatography-electrospray mass spectrometry for neutral losses of hexoses (162 mass units), four peptides were identified, indicating that these were glycosylated with hexose residues. One peptide, with an average molecular mass of 1,516 atomic mass units (AMU), exhibited a loss of two hexose units. The N-terminal sequence of the 1,516-AMU glycopeptide was determined to be DPEPAPPVP, which was identical to the sequence of the amino terminus of the mature protein, DPEPAP PVPXTA. Furthermore, analysis of the glycopeptide by secondary ion mass spectrometry demonstrated that the complete sequence of the glycopeptide was DPEPAPPVPTTA. From this, it was determined that the 10th amino acid, threonine, was O-glycosidically linked to a disaccharide composed of two hexose residues, probably mannose. This report establishes that true, O-glycosylated proteins exist in mycobacteria.

[1]  F. Romain,et al.  Antigenic and structural similarities between Mycobacterium tuberculosis 50- to 55-kilodalton and Mycobacterium bovis BCG 45- to 47-kilodalton antigens , 1995, Infection and immunity.

[2]  A. Hausladen,et al.  Purification and Characterization of Glutathione Reductase Isozymes Specific for the State of Cold Hardiness of Red Spruce , 1994, Plant physiology.

[3]  P. Brennan,et al.  Lipoarabinomannan from Mycobacterium tuberculosis modulates the generation of reactive nitrogen intermediates by gamma interferon-activated macrophages. , 1994, FEMS immunology and medical microbiology.

[4]  L. Schlesinger,et al.  Binding of the terminal mannosyl units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to human macrophages. , 1994, Journal of immunology.

[5]  D. Kilburn,et al.  Streptomyces lividans glycosylates the linker region of a beta-1,4-glycanase from Cellulomonas fimi , 1994, Journal of bacteriology.

[6]  A. Lemassu,et al.  Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis. , 1994, The Biochemical journal.

[7]  D. Maskell,et al.  Pilus‐facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin , 1993, Molecular microbiology.

[8]  M. Herzberg,et al.  Evidence for the covalent linkage of carbohydrate polymers to a glycoprotein from Streptococcus sanguis. , 1993, The Journal of biological chemistry.

[9]  P. Pescher,et al.  Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[10]  I. Ford,et al.  Mechanisms of platelet aggregation by Streptococcus sanguis, a causative organism in infective endocarditis , 1993, British journal of haematology.

[11]  M F Bean,et al.  Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. , 1993, Analytical chemistry.

[12]  P. Orlean,et al.  Glycoprotein biosynthesis in yeast , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  P. Pescher,et al.  Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria , 1993, Infection and immunity.

[14]  R. Lathigra,et al.  Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation , 1993, Infection and immunity.

[15]  J. Bennedsen,et al.  Identification of Immunodominant Antigens during Infection with Mycobacterium tuberculosis , 1992, Scandinavian journal of immunology.

[16]  W. Baumeister,et al.  Structural features of archaebacterial cell envelopes , 1992, Journal of bioenergetics and biomembranes.

[17]  A. Barbet,et al.  Surface epitope variation via mosaic gene formation is potential key to long-term survival of Trypanosoma brucei. , 1992, Molecular and biochemical parasitology.

[18]  P. Brennan,et al.  Characterization of the major membrane protein of virulent Mycobacterium tuberculosis , 1992, Infection and immunity.

[19]  H. Grey,et al.  MHC interaction and T cell recognition of carbohydrates and glycopeptides. , 1992, Journal of immunology.

[20]  U. Sleytr,et al.  Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 , 1992, Journal of bacteriology.

[21]  P. Brennan,et al.  Lipoarabinomannan. Multiglycosylated form of the mycobacterial mannosylphosphatidylinositols. , 1992, The Journal of biological chemistry.

[22]  S. Furney,et al.  T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. Evidence for different kinetics and recognition of a wide spectrum of protein antigens. , 1992, Journal of immunology.

[23]  C. Goochee,et al.  The Oligosaccharides of Glycoproteins: Bioprocess Factors Affecting Oligosaccharide Structure and their Effect on Glycoprotein Properties , 1991, Bio/Technology.

[24]  K L Williams,et al.  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. , 1991, Biochemical and biophysical research communications.

[25]  G von Heijne,et al.  Amino acid distributions around O-linked glycosylation sites. , 1991, The Biochemical journal.

[26]  A. Bacic,et al.  Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate , 1991, Infection and immunity.

[27]  D. Havlir,et al.  Human immune response to Mycobacterium tuberculosis antigens , 1991, Infection and immunity.

[28]  H. Wiker,et al.  Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis , 1991, Infection and immunity.

[29]  P. Brennan,et al.  The major native proteins of the leprosy bacillus. , 1990, The Journal of biological chemistry.

[30]  N Jentoft,et al.  Why are proteins O-glycosylated? , 1990, Trends in biochemical sciences.

[31]  P. Brennan,et al.  Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses. , 1990, The Journal of biological chemistry.

[32]  D. Kluepfel,et al.  Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. , 1990 .

[33]  P. Brennan,et al.  Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis , 1990, Journal of bacteriology.

[34]  W. Bessler,et al.  Activation of bone marrow‐derived mouse macrophages by bacterial lipopeptide: cytokine production, phagocytosis and Ia expression , 1990, European journal of immunology.

[35]  H. Rammensee,et al.  In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine , 1989, Nature.

[36]  T. M. Daniel.  The chemical composition of immunoaffinity-purified Mycobacterium tuberculosis antigen 5. , 1989, American Review of Respiratory Disease.

[37]  J. Shively,et al.  Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis. , 1987, Analytical biochemistry.

[38]  A. Sanson,et al.  Characterization of Mycobacterium tuberculosis antigen 5 epitopes by using a panel of 19 monoclonal antibodies , 1987, Journal of clinical microbiology.

[39]  W. Bessler,et al.  Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes. , 1985, Journal of immunology.

[40]  D. C. Harris,et al.  Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument. , 1985, Analytical biochemistry.

[41]  J. Katzmann,et al.  Specificity of Mycobacterium tuberculosis antigen 5 determined with mouse monoclonal antibodies , 1984, Infection and immunity.

[42]  T. Kawamura,et al.  Purification and some properties of the endogenous, autolytic N-acetylmuramoylhydrolase of Streptococcus faecium, a bacterial glycoenzyme. , 1983, The Journal of biological chemistry.

[43]  J. Morrissey,et al.  Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. , 1981, Analytical biochemistry.

[44]  L. Hood,et al.  A gas-liquid solid phase peptide and protein sequenator. , 1981, The Journal of biological chemistry.

[45]  L. Hnilica,et al.  Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets. , 1981, Analytical biochemistry.

[46]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[47]  E. L. Armstrong,et al.  Site of inhibitory action of isoniazid in the synthesis of mycolic acids in Mycobacterium tuberculosis. , 1975, Journal of lipid research.

[48]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[49]  T. Lee,et al.  Trace structural analysis of proteins. , 1996, Methods in enzymology.

[50]  Estimates of future global tuberculosis morbidity and mortality. , 1994, Canada communicable disease report = Releve des maladies transmissibles au Canada.

[51]  E. Ong.  Streptomyces lividans Glycosylates theLinker Region ofa 3-1,4-Glycanase fromCellulomonas fimi , 1994 .

[52]  Christian P. Kubicek,et al.  The cellulase proteins of Trichoderma reesei: Structure, multiplicity, mode of action and regulation of formation , 1992 .

[53]  R. Hill,et al.  Identification of O-Glycosylation Sites with a Gas Phase Sequencer , 1992 .

[54]  D. Young,et al.  Lipoprotein antigens ofMycobacterium tuberculosis , 1991 .

[55]  A. Kochi,et al.  The global tuberculosis situation and the new control strategy of the World Health Organization. , 1991, Tubercle.

[56]  C. Espitia,et al.  Identification, isolation and partial characterization of Mycobacterium tuberculosis glycoprotein antigens. , 1989, Clinical and experimental immunology.

[57]  M. Fukuda.  Characterization of O-linked saccharides from cell surface glycoproteins. , 1989, Methods in enzymology.

[58]  F. Wieland,et al.  Structure and biosynthesis of prokaryotic glycoproteins. , 1988, Biochimie.

[59]  M. Tokunaga,et al.  Biogenesis of lipoproteins in bacteria. , 1986, Current topics in microbiology and immunology.

[60]  Alan Wisemann.  Practical Protein Chemistry-A Handbook , 1986 .