LEUKEMIC-CELLS (HL-60) PRODUCE A NOVEL EXTRACELLULAR MATRIX-DEGRADINGPROTEINASE THAT IS NOT INHIBITED BY TISSUE INHIBITORS OF MATRIX METALLOPROTEINASES (TIMPS)

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hitherto undescribed 45-kd met alloproteinase into the culture medium. This enzyme cleaves the synthe tic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cl eavage site for collagenases in collagen type I not between isoleucine and alanine-the typical cleavage site for collagenases-but between al anine and glycine. The enzymatic activity was purified through a combi nation of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Micros equence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could no t be found in other proteins when searched in several protein data bas es. Incubation of the enzyme immobilized on nitrocellulose membranes w ith polyclonal antibodies to collagenase and stromelysin or gelatinase s revealed no cross-reactivity. The proteolytic activity was not incre ased by treatment with trypsin, 8M urea, acid, or organomercurials. Th e proteinase, which was inhibited by chemical inhibitors of-metallopro teinases, such as phenanthrolene or EDTA, is able to degrade several m atrix constituents, such as collagen type IV, fibronectin, gelatin, an d proteoglycans. In contrast to all known MMPs, the proteolytic activi ty of the 45-kd enzyme was not abolished upon incubation with recombin ant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus , the novel enzyme may influence extracellular matrix (ECM) turnover i n vivo because its activity is not influenced by specific inhibitors o f MMPs.