Collagenase Substrate

Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation < 15%). This improved protocol is fast and reproducible. Its linear range of almost two log scales permits assay of proteolytic activity in a wider range than current methods.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

In this report, we present a hypothesis on the mechanism used by interstitial collagenases to cleave their natural substrate, interstitial collagens. The hypothesis is based on the assumption that the proline hinge domain of interstitial collagenase adopts a collagen-like conformation. With a collagen-like domain, the enzyme is able to disturb the quaternary organization of the triple helix in the collagenase-susceptible site. A modeling analysis suggests that interaction between prolines of both collagen and collagenase forming a kind of "proline zipper" is involved in the destabilization step. This destabilization makes the three-collagen helix susceptible to the catalytic cleft of the catalytic core.