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

Abstract

Initially, the occurrence of bona fide glycosylated proteins in eubacteria was difficult to accept. Now, as a result of the application of critical analytical techniques, the presence of such molecules in such eubacteria as Neisseria meningitidis (43), Flavobacterium meningosepticum (36, 37), Streptococcus sanguis (11), Bacillus alvei (32), Clostridium spp. (15, 31), Bacteroides cellulosolvens (15), Thermoanaerobacter thermohydrosulfuricus (3), and Mycobacterium tuberculosis (10) is recognized. In the case of M. tuberculosis, full definition of glycosylation sites and the nature and extent of glycosylation are lacking. Initial evidence for the presence of glycoproteins in M. tuberculosis was based on the observation of discrete concanavalin A-binding products upon polyacrylamide gel electrophoresis (PAGE) and electroblotting of protein preparations (13,14). However, since these patterns occurred in the midst of considerable quantities of mannose (Man)-containing lipoglycans and phospholipids (10), chemical proof of amino acid glycosylation was considered necessary.The 45-kDa culture filtrate protein of M. tuberculosis is one case in point. Recent antibody reactivity studies and N-terminal amino acid sequencing conducted by both Espitia et al. (12,13) and Dobos et al. (10) demonstrated that this 45-kDa protein is the same as MPT 32, a culture filtrate protein originally isolated by Nagai and colleagues (34). Also, more recently, the DNA sequence of a gene designated apa encoding a 45/47-kDa M. tuberculosis protein was elucidated by Laqueyrerie et al. (27), and the deduced amino acid sequence of this gene yielded 100% homology with the N-terminal sequence of the 45-kDa

Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

“…The resulting aqueous layer containing LAM, LM, and the polar PIMs was dialyzed and analyzed on commercial 10-20% gradient Tricine SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA). Blotting to nitrocellulose membrane was performed at 50 V for 1 h. Immunodetection was performed with monoclonal antibody CS-35 (21) and Con A conjugated to peroxidase (Sigma, St. Louis, MO) (34). The membranes probed with Con A-peroxi-…”

Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

“…Evidence for glycosylation of multiple, structurally related protein substrates has been reported in Bacteroides fragilis, although the nature of the glycan linkage involved remains unknown (11). Two surface lipoproteins of Mycobacterium tuberculosis have been proven to be O-glycosylated, and an implicated transferase shares some structural similarity to eukaryotic protein mannosyltransferases (12,13). However, whether this system is representative of bona fide general protein glycosylation has not been established.…”

Lipoarabinomannan of Mycobacterium : Mannose capping by a multifunctional terminal mannosyltransferase

“…tuberculosis ͉ glycosyltransferase M annose (Man) is a key component of several functionally important cell-wall and intracellular structures of mycobacteria, including the family of glycosylphosphoinositides consisting of phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM), a cytoplasmic methylmannose polysaccharide (MMP), and a few O-mannosylated glycoproteins (1)(2)(3). The diversity of their structures is afforded by an unparalleled range of glycosyltransferases (GTs) using either GDP-Manp or decaprenyl (C 50 )-P-Manp donors.…”