title: Biosynthesis of alpha2(IV) and alpha1(IV) chains of collagen IV and interactions with matrix metalloproteinase-9.
abstract: In vitro binding studies with latent matrix metalloproteinase-9 (pro-MMP-9) have revealed the existence of nondisulfide-bonded alpha2(IV) chains on the cell surface capable of forming a high-affinity complex with the enzyme. Here we investigated the biosynthesis and cellular distribution of alpha2(IV) and alpha1(IV) chains in breast epithelial (MCF10A and MDA-MB-231) and fibrosarcoma (HT1080) cells by pulse-chase analysis followed by immunoprecipitation with chain-specific monoclonal antibodies (mAb). These studies showed that whereas the alpha1(IV) chain remained in the intracellular compartment, nondisulfide-bonded alpha2(IV) chains were secreted into the media in a stable form. Consistently, only alpha2(IV) was detected on the cell surface by surface biotinylation or indirect immunofluorescence. In agreement with the pulse-chase analysis, media subjected to co-precipitation experiments with pro-MMP-9 or pro-MMP-9-affinity chromatography followed by immunoblotting with chain-specific mAbs resulted in the detection of alpha2(IV). A preferential secretion of nondisulfide-bonded alpha2(IV) chains was also observed in CHO-K1 cells transiently transfected with full-length mouse alpha2(IV) or alpha (IV) cDNAs. However, a complex of mouse alpha1(IV) with pro-MMP-9 was coprecipitated with exogenous enzyme from lysates of CHO-K1 cells transfected with mouse alpha1(IV), suggesting that under overexpression conditions the enzyme can also interact with the alpha1 (IV) chain. Collectively, these studies further demonstrate the interactions of pro-MMP-9 with collagen IV chains and a unique processing and targeting of nondisulfide-bonded alpha2(IV) chains that may play a role in the surface/matrix association of pro-MMP-9.
authors.affiliation: Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: M
authors.initials: M
authors.lastname: Toth
authors.forename: Y
authors.initials: Y
authors.lastname: Sado
authors.forename: Y
authors.initials: Y
authors.lastname: Ninomiya
authors.forename: R
authors.initials: R
authors.lastname: Fridman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001940:Breast / Q000166:cytology; D016466:CHO Cells; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D024141:Collagen Type IV; D017364:Collagenases / Q000378:metabolism*; D006224:Cricetinae; D004792:Enzyme Precursors / Q000378:metabolism; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology; D000939:Epitopes / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D020780:Matrix Metalloproteinase 9; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D024141:Collagen Type IV; D004792:Enzyme Precursors; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C065384:collagen type IV alpha1 (531-543); D003094:Collagen; D017364:Collagenases; D020780:Matrix Metalloproteinase 9