, 1999). As shown in Fig. 1b, wild-type W83 and 83K26 formed black-pigmented colonies, whereas 83K3 (Δsov), which is defective in the secretion of gingipains (Saiki & Konishi, 2007), formed selleck products white colonies. The mutants 83K8 and 83K25 produced pale gray colonies (Fig. 1b). Gingipain activity was then assessed in cell extract fractions or extracellular fractions from W83,

83K25, and 83K26 (Fig. 1c). The activities of Arg-gingipains and Lys-gingipain in both the cell extract and the extracellular fractions of 83K26 were comparable to those of W83 (64–84%). In 83K25, the activity of Lys-gingipain in the extracellular fraction was decreased GS-1101 in vitro to 22% of that of W83, while the activities of Arg-gingipains and Lys-gingipain in the cell extract fraction and the activity of Arg-gingipain in the extracellular fraction were decreased to 4–6% of those of W83. This indicates that PG534 is required for the generation of active gingipains. Porphyromonas gingivalis also secretes dipeptidyl aminopeptidases DPPIV and DPP-7, and tripeptidyl aminopeptidase PTP-A to the surface of this bacterium (Banbula et al., 1999, 2000, 2001). DPPIV, DPP-7, and PTP-A activities were

comparable between cell extracts from W83, 83K25, and 83K26 (71–107% of those of W83; Fig. 1c), indicating that the requirement for PG534 is specific to gingipains. Cell extract fractions, HSS fractions, and HSP fractions were prepared

from W83, 83K3, 83K10 [ΔPG0027; a secretion-defective mutant of gingipains (Ishiguro et al., 2009)], and 83K25, and subjected to a Western blot analysis using anti-RgpB antiserum (Ishiguro et al., 2009) to detect Arg-gingipains (Fig. 2a) or anti-Kgp antiserum (Saiki & Konishi, 2010) to detect Lys-gingipain (Fig. 2b). In W83, Arg-gingipains were detected as a 42-kDa catalytic domain form and 70–90-kDa glycosylated forms (Potempa et al., 1995; Nakayama, 1997; Seers et al., 2006) in the cell extract fraction (Fig. 2a, lane DAPT clinical trial 1) and the HSP fraction (lane 5). In the HSS fraction, neither Arg-gingipain protein bands (Fig. 2a, lane 9) nor Arg-gingipain activity (data not shown) were well detected in W83 (Vanterpool et al., 2005a). In contrast, cell extract fractions from 83K3, 83K10, and 83K25 showed similar Arg-gingipain band patterns including a 185-kDa unprocessed form of RgpA and a 76-kDa unprocessed form of RgpB (Vanterpool et al., 2005a), but no glycosylated forms of Arg-gingipains (Fig. 2a, lanes 2–4). In the cell extract fractions, 26–70-kDa protein bands were also detected (Fig. 2a, lanes 1–4), but may be degradation products of Arg-gingipains. In the HSS fractions, faint protein bands near 55 kDa were similarly detected in 83K3, 83K10, and 83K25 (Fig. 2a, lanes 10–12).