Abstract
SodC is one of two superoxide dismutases produced by Mycobacterium tuberculosis. This protein was previously shown to contribute to virulence and to act as a B-cell antigen. SodC is also a putative lipoprotein, and like other Sec-translocated mycobacterial proteins it was suggested to be modified with glycosyl units. To definitively define the glycosylation of SodC, we applied an approach that combined site-directed mutagenesis, lectin binding, and mass spectrometry. This resulted in identification of six O-glycosylated residues within a 13-amino-acid region near the N-terminus. Each residue was modified with one to three hexose units, and the most dominant SodC glycoform was modified with nine hexose units. In addition to O-glycosylation of threonine residues, this study provides the first evidence of serine O-glycosylation in mycobacteria. When combined with bioinformatic analyses, the clustering of O-glycosylation appeared to occur in a region of SodC with a disordered structure and not in regions important to the enzymatic activity of SodC. The use of recombinant amino acid substitutions to alter glycosylation sites provided further evidence that glycosylation influences proteolytic processing and ultimately positioning of cell wall proteins.