Abstract
We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.
Highlights
SV40-transformed Human Lung Fibroblasts Secretea 92-kDa Type IV Collagenase Which Is Identical to ThatSecreted by Normal Human Macrophages4‘
SV40-transformed human lung fibroblasts secrete an additional gelatinolytic enzyme of 92-kDa which wasnot detected in the parental cell line
We have purified this enzyme from SV40-transformedfetal lung fibroblasts and the gelatinolytic metalloprotease of identical molecular weight from the conditioned medium of TPA-differentiated U937 cells
Summary
To the GenBankTM/EMIILDataBankwith accession number(s) Cell Culture-SV40-transformed fetal lung fibroblasts andthe ‘505070. Fractions containing 92-kDa gelatinolytic activity werepooled, adjusted to 0.5 M NaCl and 0.01% Brij-35 and chromatographed on a 1.0 X 10-cm column of gelatin-Sepharose (Sigma) as previously described [10, 37] except that the enzyme was eluted using a 0-10% MeZSOgradient. Enzyme Assays-Samples of purified 92-kDa U937 proenzyme in 0.05 M Tris-HC1,pH 7.5, containing 0.005 M CaCl,, 0.05 M NaCl, and 0.01%Brij-35 were activated by the addition of 0.01 M stock solutions of the organomercurial compounds p-aminophenylmercuric acetate or PMC dissolved in 0.05 N NaOH to a final concentration of 0.5 M for up to 16 h at 37 "C. Oligonucleotide synthesis, Northern blot analysis, and hybridization were performed as described for human fibroblast interstitial collagenase [3],human type IV collagenase (IO), and human stromelysin [6], withoutsignificant modification
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