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-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.
Dobos
K M
KM
Department of Microbiology, Colorado State University, Fort Collins 80523, USA.
Khoo
K H
KH
Swiderek
K M
KM
Brennan
P J
PJ
Belisle
J T
JT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D013912:Threonine / Q000737:chemistry
C095715:45-47 kDa antigen complex, Mycobacterium; D001426:Bacterial Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D013912:Threonine; D008358:Mannose