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Journal of Bacteriology, February 2004, p. 638-645, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.638-645.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Streptococcus gordonii Platelet Binding Protein GspB Undergoes Glycosylation Independently of Export

Barbara A. Bensing,1 Bradford W. Gibson,2 and Paul M. Sullam1*

Division of Infectious Diseases, Veterans Affairs Medical Center, and Department of Medicine, University of California, San Francisco,1 Department of Pharmaceutical Chemistry, University of California, San Francisco, and the Buck Institute for Age Research, Novato,California2

Received 16 September 2003/ Accepted 23 October 2003

The binding of bacteria and platelets may play a central role in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii strain M99 is predominantly mediated by the 286-kDa cell wall-anchored protein GspB. This unusually large protein lacks a typical amino-terminal signal peptide and is translocated from the cytoplasm via a dedicated transport system. A 14-kb segment just downstream of gspB encodes SecA2 and SecY2, two components of the GspB-specific transport system. The downstream segment also encodes several putative glycosyl transferases that may be responsible for the posttranslational modification of GspB. In this study, we compared the abilities of M99 and two GspB- mutant strains to bind various lectins. GspB was found to have affinity for lectins that bind N-acetylglucosamine. We also examined variant forms of GspB that lack a carboxy-terminal cell wall-anchoring domain and thus are free of covalent linkage to cell wall peptidoglycan. Like native GspB, these truncated proteins appear to be heavily glycosylated, as evidenced by migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass >100 kDa in excess of the predicted mass, negligible staining with conventional protein stains, and reactivity with hydrazide following periodate oxidation. Furthermore, analysis of the carbohydrate associated with the GspB variants by high-pH anion-exchange chromatography revealed the presence of ~70 to 100 monosaccharide residues per GspB polypeptide (primarily N-acetylglucosamine and glucose). Analysis of GspB in protoplasts of secA2 or secY2 mutant strains, which do not export GspB, indicates that GspB is glycosylated in the cytoplasm of these strains. The combined data suggest that the native GspB is a glycoprotein and that it may be glycosylated prior to export.

* Corresponding author. Mailing address: Division of Infectious Diseases, VA Medical Center (111W), 4150 Clement St., San Francisco, CA 94121. Phone: (415) 221-4810, ext. 2550. Fax: (415) 750-0502. E-mail: sullam{at}itsa.ucsf.edu.

Journal of Bacteriology, February 2004, p. 638-645, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.638-645.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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