Generation of Lys-gingipain protease activity in W50 is independent of Arg-gingipain protease activities Free

Abstract

, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, and , encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and , which encodes a Lys-X specific protease (KGP, Lys-gingipain). The and genes encode polyproteins comprising pro-peptide and catalytic domain with large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An double mutant (E8) of was constructed to study the role of RGPs in the processing of KGP. A mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for and , respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50% of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50% of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.

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2000-08-01
2024-03-29
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