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 fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin 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-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.
Steffensen
B
B
Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Bigg
H F
HF
Overall
C M
CM
D000906:Antibodies / Q000378:metabolism; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000502:physiology; D003094:Collagen / Q000737:chemistry* / Q000502:physiology; D017364:Collagenases / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D005347:Fibroblasts; D005353:Fibronectins / Q000737:chemistry*; D018093:Gelatinases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005881:Gingiva / Q000378:metabolism; D006493:Heparin / Q000502:physiology; D006801:Humans; D019012:Integrin beta1 / Q000737:chemistry / Q000276:immunology; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D000906:Antibodies; D004792:Enzyme Precursors; D005353:Fibronectins; D019012:Integrin beta1; D011994:Recombinant Proteins; D003208:Concanavalin A; D006493:Heparin; D003094:Collagen; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D020778:Matrix Metalloproteinase 2