The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family - An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell l ine, HD11. Biochemical and immunological characterization of the purified e nzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelat in cleavage. Incubation with an organomercurial compound (p-aminophenylmerc uric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelato rs and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase inc luding a cysteine switch prodomain, three fibronectin type II repeats, a ca talytic zinc binding region, and a hemopexin-like domain, The 75-kDa gelati nase is produced by phorbol ester-treated chicken bone marrow cells, monocy tes, and polymorphonuclear leukocytes, cell types that characteristically p roduce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-k Da gelatinase in these cell types indicates that the 75-kDa gelatinase is l ikely the avian counterpart of gelatinase B, However, the protein is only 5 9% identical to human gelatinase B, whereas all previously cloned chicken M MP homologues are 75-90% identical to their human counterparts. In addition , the new 75-kDa chicken gelatinase lacks the type V collagen domain that i s found in all mammalian gelatinase Bs, Furthermore, the secreted enzyme ap pears structurally distinct from known gelatinase Bs and the activated enzy me can cleave fibronectin, which is not a substrate for mammalian gelatinas e B, Thus the results of this study indicate that a second MMP gelatinase e xists in chickens, and although it is MIMP-9/gelatinase B-like in its overa ll domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.