The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal-domain family found in Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.

FIG. 1.

Alignment of the C termini of experimentally identified P. gingivalis proteins with homology to the RgpB C terminus. The sequences were initially aligned using Clustal V (22), with minor manual adjustment. Each sequence is described using TIGR ORF (http://www.tigr.org) annotation; RgpB is PG0506. Residues are grouped based on chemical character. Neutral nonpolar (hydrophobic) residues are A, V, I, L, G, M, P, and F; neutral polar residues are S, T, Y, W, N, Q, and C; acidic residues are D and E; and basic residues are K, H, and R. Motifs of interest are noted with the letters A to E above the alignment (see the text). A consensus is noted below the alignment, where x is any residue, h is hydrophobic residues, p is polar residues, and b is basic residues; individual residues with ≥50% conservation are specified.

FIG. 2.

Cellular localization of recombinant RgpB isoforms. (A) Western blots of outer membrane fractions separated by SDS-PAGE and probed with anti-rRgpA_cat antibodies. Lane 1, OPG44 (YH522KAB::rRgpB); lane 2, ECR7 (YH522AB::rRgpB); lane 3, OPG41 [YH522KAB::rRgpB(C244A)]; lane 4, OPG40 [YH522AB::rRgpB(C244A)]; lane 5, OPG37 (YH522KAB::pYH411); lane 6, ECR5 (YH522AB::pYH411); lane 7, ECR55 (YH522::pYH411); lane 8, ECR104 (YH522AB::rRgpB435). (B) Western blots of whole-cell lysate fractions and culture supernatant fractions separated by SDS-PAGE and probed with anti-RgpA_cat antibodies. Lanes 1 to 3, whole-cell lysate fractions. Lane 1, ECR7 (YH522AB::rRgpB); lane 2, ECR104 (YH522AB::rRgpB435); lane 3, ECR5 (YH522AB::pYH411). Lanes 4 to 6, culture supernatant fractions. Lane 4, ECR7 (YH522AB::rRgpB); lane 5, ECR104 (YH522AB::rRgpB435); lane 6, ECR5 (YH522AB::pYH411). Lanes 7 to 9, culture supernatant fractions after centrifugation at 40,000 × g. Lane 7, ECR7 (YH522AB::rRgpB); lane 8, ECR104 (YH522AB::rRgpB435); lane 9, ECR5 (YH522AB::pYH411). (C) Coomassie blue-stained SDS-PAGE gel of periplasmic fractions. Bands were identified using mass spectrometry. Lane 1, molecular mass markers; lane 2, ECR5 (YH522AB::pYH411); lane 3, ECR7 (YH522AB::rRgpB); lane 4, ECR104 (YH522AB::rRgpB435). The arrows indicate rRgpB435 in ECR104 (lane 4) and rRgpB in ECR7 (lane 3). Bands labeled 1 to 6 were shown to contain peptidase M16 (PG0196) (1), CPG70 carboxypeptidase precursor (PG0232) (2), prolyl oligopeptidase (PG1004) (3), conserved hypothetical protein (PG0491) (4), TPR domain protein (PG0449) (5), and TPR domain protein (PG1385) (6). (D) Western blots of whole-cell lysate fractions of YH522KAB recombinants separated by SDS-PAGE and probed with anti-RgpA_cat antibodies. Lane 1, ECR123 (YH522KAB::rRgpB435); lane 2, OPG37 (YH522KAB::pYH411); lane 3, OPG44 (YH522KAB::rRgpB). The amount of protein loaded onto the SDS-PAGE gels was standardized with the equivalent of protein from 6.0 × 10⁸ cells loaded onto each lane in A, B, and D and the equivalent of protein from 4.0 × 10⁹ cells loaded onto each lane in C. The identity of RgpB in the bands of the Western blots (A, B, and D) was confirmed by mass spectrometric analysis of Coomassie blue-stained replicate gels. Similarly, the identity of RgpA_cat in lane 7 of A was confirmed by the presence of RgpA_cat-specific peptides in the 45-kDa band of a replicate gel as described previously (59).