Mercurial activation of human polymorphonuclear leucocyte procollagenase

Abstract

The mechanism of human polymorphonuclear leucocyte (PMNL) procollagenase activation by HgCl, was investigated by kinetic and sequence analysis of the reaction products. HgClz activated PMNL procollagenase by intramolecular autoproteolytic cleavage of the Asn53 -Val54 peptide bond to generate a collagenase species of M , 65000, which was immediately converted into a second intermediate collagenase form by further autoproteolytic cleavage of the Asp64 -Met65 peptide bond within the propeptide domain. This intermediate form (Met65 N-terminus) reached maximum concentrations after 45 min and displayed only about 40% of the maximum available enzymatic activity. Final activation was obtained after autoproteolytic cleavage of either Phe79 -Met80 or Met80 -Leu81 peptide bonds. Furthermore, activation in the presence of TIMP-1 did not suppress the intramolecular autoproteolytic cleavage of the Asn53 -Val54 peptide bond. Complete inhibition of further autoproteolytic decay of the enzyme or generated peptides was observed, which was obviously due to complex formation between the intermediate collagenase form (Val54 N-terminus) and inhibitor, which was visualized using the Western blot technique. Thus PMNL procollagenase activation by HgCl, followed a three-step activation mechanism which is entirely different from the known activation mechanisms of the fibroblast matrix metalloproteinases.

Direct activation of human neutrophil procollagenase by recombinant stromelysin

“…Activation of the recombinant prostromelysin and activity assay Activation of recombinant stromelysin was achieved by heat treatment at 55°C for 45 min, as previously described (Koklitis et al, 1991 Met80 N-terminus following activation by trypsin; Met80 and Leu8' N-terminus following activation by HgCI2 (see Knauper et al, 1990a;Blaser et al, 1991); Phe79 N-terminus following activation by stromelysin.…”

“…Conversion of the procollagenase into the active enzyme is the key step in the initiation of collagenolysis during the connective tissue turnover caused by inflammatory processes mediated by neutrophils. Activation can be initiated by proteinases, mercurials and oxidative processes in vitro (Knauper et al, 1990a;Mookhtiar and Van Blaser et al, 1991;Michaelis et al, 1992). Activation of the proenzyme results in the removal of at least 80 or 81 N-terminal amino acid residues, thereby generating the active enzyme.…”

Fragmentation of human polymorphonuclear-leucocyte collagenase

“…The reaction was terminated by the addition of a 10-fold molar excess of trypsin kallikrein inhibitor (TKI) from bovine lungs (Knauper et al, 1990a). Alternatively, 500 ,ug of PMNL procollagenase was activated by incubation with 1 mM HgCl1 at 37 'C for 2 h as recently described (Blaser et al, 1991). The specific activities of trypsin and of HgCl2-activated PMNL collagenase were determined, and were 3004 and 3388 units/mg.…”

“…The proenzyme can be activated in vitro by proteinases or mercurials. Activation results in the removal of at least 79, 80 or 81 amino acid residues from the N-terminal part ofthe proenzyme (Knauper et al, 1990a;Mallya et al, 1990;Mookhtiar and Van Wart, 1990;Blaser et al, 1991). Once activated, the enzyme plays a major role in the connective-tissue turnover in inflammatory processes.…”

Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP‐8) to stability and collagenolytic activity by alanine scanning mutagenesis

“…Activation of the procollagenases involves proteolytic and autoproteolytic cleavages within the propeptide domain, which effects the removal of the 'Pro-Arg-Cys-Gly-ValPro-Asp' sequence motif, thereby expelling the free Cys residue of the propeptide from the co-ordination sphere of the catalytic zinc ion [5][6][7][8][9][10]. The active collagenases cleave the interstitial collagens type I III with different k cli JK M values into characteristic 3/4 and 1/4 fragments and slowly hydrolyse other substrates such as cartilage aggrecan, serpins, denatured collagen and small synthetic peptide substrates [11,12].…”