THE INVOLVEMENT OF THE FIBRONECTIN TYPE II-LIKE MODULES OF HUMAN GELATINASE-A IN CELL-SURFACE LOCALIZATION AND ACTIVATION

Recombinant collagen-binding domain (rCBD) comprising the three fibron ectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surfac e of normal human fibroblasts in culture. Upon concanavalin A treatmen t of cells, the induced cellular activation of gelatinase A was marked ly elevated in the presence of the rCBD. Therefore, the mechanistic as pects of gelatinase A binding to cells by this domain were further stu died using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, bloc king antibodies to the beta(1)-integrin subunit but not the cu,integri n subunit, and bacterial collagenase treatment. Addition of soluble co llagen rescued the attachment of collagenase-treated cells to the rCBD . As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solub ilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bac terial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen arl(I) and al pha 2(I) chains. A rCBD mutant protein (Lys(263) --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-bind ing deficient mutant (Lys(357) --> Ala), heparinase treatment, or hepa rin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta(1)-integrin forms as a result of interactions inv olving the collagen-binding domain of the enzyme. Moreover, this disti nct pool of cell collagen-bound proenzyme appears recalcitrant to cell ular activation.