CHARACTERIZATION OF A 2ND CELL-ASSOCIATED ARG-SPECIFIC CYSTEINE PROTEINASE OF PORPHYROMONAS-GINGIVALIS AND IDENTIFICATION OF AN ADHESIN-BINDING MOTIF INVOLVED IN ASSOCIATION OF THE PRTR AND PRTK PROTEINASES AND ADHESINS INTO LARGE COMPLEXES

Porphyromonas gingivalis has been associated with the development of a dult periodontitis and cysteine proteinases with Arg-and Lys-specific activity have been implicated as major virulence factors. In a cell so nicate of P. gingivalis W50, a complex of non-covalently associated pr oteins has been previously characterized. This complex is composed of a 45 kDa Arg-specific, calcium-stabilized cysteine proteinase (PrtR45) , a 48 kDa Lys-specific cysteine proteinase (PrtK48) and seven sequenc e-related adhesins designated PrtR44, PrtR15, PrtR17, PrtR27, PrtK39, PrtK15 and PrtK44, with all proteins being encoded by the two genes pr tR and prtK. It has been proposed that these noncovalently associated complexes form extracellularly after autolytic processing of the PrtR and PrtK polyproteins, with the adhesins binding to the proteinases (P rtR45 and PrtK48) and autoaggregating. Another form of the cell-associ ated, Arg-specific, calcium-stabilized cysteine proteinase is describe d here. Designated PrtRII50, it is a discrete 50 kDa protein with no a dhesin-association and has enzymic characteristics and an inhibitor/ac tivator profile almost identical to PrtR45. The PrtRII50 proteinase is encoded as a preproprotein by a second gene, prtRII, with high sequen ce similarity to PrtR except that it lacks the C-terminal adhesin doma ins. A comparison of the deduced amino acid sequence of PrtRII50 with that of the adhesin-associated proteinases PrtR45 and PrtK48 revealed that PrtRII50 does not contain a C-terminal motif that is conserved in PrtR45 and PrtK48. Related motifs are also found in the adhesin domai ns of PrtR and PrtK. It is proposed that this conserved motif is an ad hesin-binding motif (ABM) involved in association of the PrtR and PrtK proteinases and adhesins into large complexes, as the PrtR-PrtK prote inase-adhesin complex inactivated by N-alpha-p-tosyl-L-lysine chlorome thyl ketone (TLCK) was shown to bind specifically to a synthetic pepti de corresponding to the conserved motif in a competitive binding assay .