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Infect Immun. 2005 Aug;73(8):4864-78.

Expression of Arg-Gingipain RgpB is required for correct glycosylation and stability of monomeric Arg-gingipain RgpA from Porphyromonas gingivalis W50.

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  • 1MRC Molecular Pathogenesis Group, Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom.


Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the alpha-catalytic chain and the beta-adhesin chain, the monomeric soluble Arg-gingipain comprising only the alpha-catalytic chain (RgpA(cat)), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the alpha-catalytic chain (mt-RgPA(cat)), are derived from rgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpA(cat). This work aims to confirm the role of RgpB in the posttranslational modification of RgpA(cat) and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA-derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpA(cat) from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpA(cat) from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived from rgpA and that this process is required for enzyme stabilization.

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