A novel matrix metalloprotease-like enzyme (karilysin) of the periodontal pathogen Tannerella forsythia ATCC 43037

Abstract

Proteases of Tannerella forsythia, a pathogen associated with periodontal disease, are implicated as virulence factors. Here, we characterized a matrix metalloprotease (MMP)-like enzyme of T. forsythia referred to as karilysin. Full length (without a signal peptide) recombinant karilysin (49.9 kDa) processed itself into the mature 18 kDa enzyme through a sequential autoproteolytic cleavages both at N-and C-terminal profragments. The first cleavage at the Asn14-Tyr15 peptide bond generated the fully active enzyme (47.9 kDa) and subsequent truncations at the C-termini did not affect proteolytic activity. Mutation of Tyr15 to Ala generated a prokarilysin variant that processed itself into the final 18 kDa form with greatly reduced kinetics. Inactive prokarilysin with the mutated catalytic Glu residue (E136A) was processed by active karilysin at the same sites as the active enzymes. The karilysin proteolytic activity and autoprocessing were inhibited by 1,10-phenanthroline and EDTA. Calcium ions were found to be important for both activity and thermal stability of karilysin. Using the CLiPS technology, specificity of karilysin was found to be similar to that of MMPs with preference for Leu/Tyr/Met at P1′ and Pro/Ala at P3. This specificity and the ability to degrade elastin, fibrinogen and fibronectin may contribute to the pathogenicity of periodontitis.

A Novel Mechanism of Latency in Matrix Metalloproteinases

“…1A) was cloned at BamHI and XhoI restriction sites into vector pGEX-6P-1 (GE Healthcare) as described elsewhere (30). The resulting vector, pKAR1 (see Table 1 for an overview of vectors and constructs used), confers resistance toward ampicillin and attaches an N-terminal glutathione S-transferase (GST) moiety followed by a human rhinovirus 3C proteinase (HR3CP) recognition site (L-E-V-L-F-Q-2-G-P; HR3CP cleavage leaves two extra residues, underlined, at the N terminus of the recombinant protein after digestion; three extra residues, L-G-S, are further present due to the cloning strategy).…”

“…The resulting vector, pKAR1 (see Table 1 for an overview of vectors and constructs used), confers resistance toward ampicillin and attaches an N-terminal glutathione S-transferase (GST) moiety followed by a human rhinovirus 3C proteinase (HR3CP) recognition site (L-E-V-L-F-Q-2-G-P; HR3CP cleavage leaves two extra residues, underlined, at the N terminus of the recombinant protein after digestion; three extra residues, L-G-S, are further present due to the cloning strategy). Single-residue point mutants pKly-Y35A and pKly-E156A (pKAR2 and pKAR3, respectively) were generated using the QuikChange Site-directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions as described (30). Double mutant pKly-D25A/Y35A (pKAR4) was similarly generated using pKAR2 as a template.…”

“…Cultures were induced at an A 600 of 0.8 with 0.2 mM isopropyl ␤-D-thiogalactopyranoside and incubated overnight at 18°C. Purification of wild-type and mutant pKly, and subsequent autolysis of the former to obtain Kly18, was achieved as described elsewhere (30). In turn, pKly18-E156A, Kly18-E156A, pKly18-Y35A, and pKly18-D25A/Y35A were purified as follows.…”

“…This is the primary activation cleavage and it releases an active 48-kDa form (Kly48 (30)). In recombinant protein production, subsequent cleavages within the C-terminal domain give rise to Kly38 and, finally, to a stable form of 18 kDa (Kly18), which corresponds to the isolated mature catalytic domain (CD) (5,28,30,33). These cleavages were shown to be autolytic as activation was repressed by general chelating MP inhibitors and in the inactive active-site variant, E156A, which ablated the catalytic glutamate of the CSBZ (1,5,30,79,80).…”

“…Similarly to vertebrate MMPs, karilysin showed preference for medium-sized to bulky hydrophobic residues (leucine, tyrosine and methionine) in the specificity pocket, S 1 Ј (Ref. 30; for active-site cleft subsite nomenclature, see Ref. 35).…”