Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50

Abstract

Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis. We have undertaken a proteomic study of the outer membrane of P. gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteins were identified by reference to the pre-release genomic sequence of P. gingivalis available from The Institute for Genomic Research. Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins. Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins. The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors. Several domains were found to be C-terminally truncated 16–57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine. This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp. Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing. The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13–42kDa higher than that calculated from their gene sequences. The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.

Defining the role ofPorphyromonas gingivalispeptidylarginine deiminase (PPAD) in rheumatoid arthritis through the study of PPAD biology

“…As such, glycosylation of cPPAD for membrane attachment may be sufficient to explain the striking difference in molecular weight to sPPAD,29 as previously shown for arginine gingipain 41. The truncation sites identified in this study may indicate specific processing by P gingivalis envelope-associated cysteine proteases with arginine-X (at R43) and lysine-X activity (at K459) 42. Truncation at R43 has previously been shown for sPPAD by N-terminal sequencing9 and MS 27.…”

“…MS identified both as a truncated form of PPAD missing the first 43 N-terminal and 97 C-terminal amino acids. Full-length PPAD was not detected in our analysis, suggesting that similar to proforms of other P gingivalis virulence factors, this proenzyme is rapidly processed by truncation and concomitantly translocated to the bacterial cell surface 27 39 40. As such, glycosylation of cPPAD for membrane attachment may be sufficient to explain the striking difference in molecular weight to sPPAD,29 as previously shown for arginine gingipain 41.…”

The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function

“…SDS-PAGE analysis of OM-associated proteins produced diffuse bands about 20 kDa larger than that predicted from the primary structure of T9SS-secreted proteins (Veith et al, 2002). The difference is due to the presence of an A-LPS (Paramonov et al, 2005; Rangarajan et al, 2008) covalently attached to the cargo proteins imbedded into the OM, as indicated by western blot using specific antibodies (Abs).…”

“…Then, the OM-anchored gingipains activate themselves by cleaving off the NTP. For RgpB, this is the end of processing, but the polypeptide chains of RgpA and Kgp are further fragmented to form a large, non-covalent complex of catalytic and hemagglutinin domains on the bacterial surface (Bhogal et al, 1997; Veith et al, 2002; Sztukowska et al, 2012). The activation and further processing are still not well-understood, and, in addition to trans- and cis-autoproteolysis, they also involve the removal of the C-terminal Arg and Lys residues by the Arg/Lys-specific carboxypeptidase CPG70 (PG0232/PGN_0335; Chen et al, 2002).…”

Iron and heme utilization inPorphyromonas gingivalis

“…The arginine-specific gingipains (Arg-gingipains; cleave polypeptide chains after arginine residues) are encoded by two related genes rgpA and rgpB, while the lysine-specific gingipain (Lys-gingipain; cleaves polypeptide chains after lysine residues) is encoded by the single kgp locus [102,[110][111][112][113]. 4) [102,[110][111][112][113][114][115][116][117][118]. 4) [102,[110][111][112][113][114][115][116][117][118].…”