Variable carbohydrate modifications to the catalytic chains of the RgpA and RgpB proteases of Porphyromonas gingivalis W50

Proteases of Porphyromonas gingivalis are considered to be important virule nce determinants of this periodontal bacterium. Several biochemical isoform s of arginine-specific proteases are derived from rgpA and rgpB. HRgpA is a heterodimer composed of the catalytic or chain noncovalently associated wi th a beta adhesin chain derived from the C terminus of the initial full-len gth translation product. The catalytic alpha chain is also present as a mon omer (RgpA) either free in solution or associated with membranes. rgpB lack s the coding region for the adhesin domain present in rgpA and yields only monomeric forms (RgpB) which again may be soluble or membrane associated. I n this study, the catalytic chains of this unusual group of enzymes are sho wn to be differentially modified by the posttranslational addition of carbo hydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did n ot react with the corresponding recombinant RgpA alpha chain expressed in E scherichia coli but was immunoreactive with P. gingivalis lipopolysaccharid e. MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB, RgpA tr eated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the. presence of carbohyd rate residues within the epitope. Chemical deglycosylation abolished immuno reactivity with MAb 1B5 and caused a similar to 30% reduction in the size o f the membrane-associated enzymes. Monosaccharide analysis of HRgpA and Rgp A demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of prote in. Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not onl y to different extents but also with different sugars. The variable nature of these additions may have a significant effect on the structure, stabilit y, and immune recognition of these protease glycoproteins.