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

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-C-14]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, S-7, S-18, S-22, S-29, and S-41, 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 cy 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-n-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.