title: 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.
authors.affiliation: Cooperative Research Centre for Oral Health Science, School of Dental Science, University of Melbourne, 720 Swanston Street, Victoria, Australia.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Seers
authors.forename: Nada
authors.initials: N
authors.lastname: Slakeski
authors.forename: Paul D
authors.initials: PD
authors.lastname: Veith
authors.forename: Todd
authors.initials: T
authors.lastname: Nikolof
authors.forename: Yu-Yen
authors.initials: YY
authors.lastname: Chen
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Dashper
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000502:physiology*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C117151:KGP-381 protein, Porphyromonas gingivalis