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 13-42 kDa 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.

Identification of a New Membrane-associated Protein That Influences Transport/Maturation of Gingipains and Adhesins of Porphyromonas gingivalis

“…N-terminal sequencing of these proteins and the kgpЈ-ЈrgpB chimera gene product in the porT mutant produced no results, indicating a blocked N terminus, consistent with previous predictions that the pro domains of these proteins have N-terminal pyroglutamate (43).…”

Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis

“…The pattern of protein bands obtained by SDS-PAGE analysis of the RgpA-Kgp complexes purified by Pike et al (1994) and Bhogal et al (1997) were the same as those obtained in this study with the Triton-wt complex, where PMF analysis confirmed the absence of the C-terminal domains of both RgpA and Kgp in the purified complex. Veith et al (2002) showed using 2D-PAGE analysis of P. gingivalis W50 outer membranes that the C-terminal domain of RgpA, RgpA A4 was present as a highly post-translationally modified form that reacted with mAb 1B5 whereas the other RgpA and Kgp domains obtained did not react with the mAb. This mAb has recently been reported to react with a phosphorylated branched mannan that has been suggested to be part of the outermost layer of the P. gingivalis cell wall (Paramonov et al, 2005).…”

“…The N-terminal sequencing and PMF data shown in the present study demonstrate that the Kgp proteinase is involved in processing of the RgpA A3 adhesin domain and the majority of the RgpA and Kgp domains are processed by either the RgpA or RgpB proteinases. Using proteinase inhibitors to eliminate proteolytic processing during extraction, Veith et al (2002) showed that the RgpA and Kgp proteinase and adhesins are fully processed domains on the cell surface of P. gingivalis. Our data suggest that processed domains of the three polyproteins RgpA, Kgp and HagA combine noncovalently to form complexes either on the cell surface or immediately upon release from the cell surface.…”

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

“…Interestingly, most of the prevalent T. forsythia glycoproteins identified in this study (the two S-layer glycoproteins TfsA (TF2261-2262) and TfsB (TF2663), TF2339, and its paralog TF1259) are located in the outer membrane of the bacterium, whereas the others (TF1056 and TF0091) are predicted lipoproteins. TF1259 and TF2339 exhibit C-terminal sequence similarity to the CTD family of P. gingivalis (51). TF1056 and TF0091 are predicted non-CTD lipoproteins, showing similarity to TonB-dependent receptor-associated proteins, which are generally known to be important for signal transmission to the cytoplasm and activation of target gene transcription (52).…”