Abstract
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 α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpA
cat
), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-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.