Human fibroblast stromelysin catalytic domain: expression, purification, and characterization of a C-terminally truncated form

Abstract

Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.

Fragmentation of human polymorphonuclear-leucocyte collagenase

“…Since inhibition of PMNL collagenase and Mr,40000 fragment with TIMP-1 or rTIMP-2 demonstrated similar Kd values, it may be concluded that the C-terminal domain of PMNL collagenase does not contribute to the binding reaction of both matrix metalloproteinase inhibitors. (Chin et al, 1985;Okada et al 1986;Clark and Cawston, 1989;Marcy et al, 1991;. Autoproteolysis of all these enzymes, including the PMNL collagenase, takes place in a proline-rich region, which is possibly a hinge region between the catalytic and C-terminal domains.…”

The Hemopexin-like Domain (C Domain) of Human Gelatinase A (Matrix Metalloproteinase-2) Requires Ca2+ for Fibronectin and Heparin Binding

“…Indeed, the C domain alone of collagenase-1 binds native type I collagen (32)(33)(34), as can the C domain of stromelysin-1 (33,34), even though collagen is not cleaved by stromelysin-1. In contrast, neither the activity nor the substrate specificity of the stromelysins is dramatically modified by the removal of the C domain (35)(36)(37)(38). Likewise, the gelatinases degrade gelatin and native type IV collagen in a manner that appears independent of the presence of the C domain (39).…”

Formation of a covalent Hg‐Cys‐bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine‐switch mechanism

“…They all consist of a propeptide and a catalytic domain followed by a hemopexin-like domain, which is coupled by a hinge region to the first two domains [15]. PMNL coilageaase contains three eysteine residues° with two of them believed to be disulfide bridged in the hemopexinlike domain and one unbridged in the propeptide domain.…”