Proteolytic and non-proteolytic activation of human neutrophil progelatinase B

Abstract

The activation of human neutrophil progelatinase B (pro-HNG) by a variety of proteolytic and non-proteolytic activators has been investigated. A quantitative comparison of the activation efficiencies of treatments previously reported to activate pro-HNG or the related gelatinase B species produced by other cells demonstrates that stromelysin and trypsin are good activators. HgC1 2 is a moderately effective activator, while p-chloromercuribenzoate and NaOCI are poor activators. It is also shown that human matrilysin and human fibroblast-type collagenase can activate pro-HNG by a mechanism that is very similar to that of stromelysin. Initially, these proteinases hydrolyze the Glu 40-Met 41 bond in the propeptide domain to generate an 88 kDa inactive HNG species. Collagenase also generates a 68 kDa HNG species through hydrolysis of the Ala 74-Met 75 bond. Ultimately, treatment with either matrilysin, collagenase or trypsin results in the production of a 65 kDa active form of HNG that arises from hydrolysis of the Arg 87-Phe 88 bond. This is the same active species produced on activation by stromelysin. This cleavage site is downstream of the ‘cysteine-switch’ residue located at position 80 and releases it, accounting for the permanent activation of the enzyme. These results suggest that matrilysin and collagenase may be physiologically relevant activators of pro-HNG and/or other progelatinase B species. Activation by HgCl 2 produces an active 68 kDa enzyme due to autolytic hydrolysis of the Ala 14-Met 75 bond. This species retains the cysteine switch residue; however, it is shown that it is only active in the continued presence of HgCl 2. Removal of the HgCl 2 restores latency, indicating that this species is reversibly activated by HgCl 2, which functions by complexing the sulfhydryl group of the cysteine switch residue and keeping it dissociated from the active site zinc atom. Thus, in spite of reports to the contrary, the cysteine switch mechanism can account for the latency and activation of pro-HNG.