Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.
To elucidate structure-function relationships of stromelysin-3, a putative matrix metalloproteinase originally identified at the tumor-stromal cell interface in breast carcinomas, the human cDNA was expressed in mammalian cells, and its products were isolated and characterized. In stably transfected cells, stromelysin-3 was recovered as a complex mixture of species ranging in size from approximately 20 to 65 kDa. Among these products, a major 45-kDa species with an N terminus of Phe98 and an intact C-terminal domain was identified as a true endopeptidase on the basis of its ability to cleave the bait region of alpha 2-macroglobulin between Phe684 and Tyr685, a site identical to that recognized by stromelysin-1. However, unlike stromelysin-1 or other members of the matrix metalloproteinase family, the mature form of stromelysin-3 was unable to hydrolyze a range of extracellular matrix molecules associated with either the basement membrane or interstitium. To probe for alternate substrates among tumor cell-derived products, purified stromelysin-3 was incubated with [35S]methionine-labeled medium conditioned by the breast cancer cell line, MCF-7. Under these conditions, a single, tumor cell-derived protein was hydrolyzed as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Following anion-exchange chromatography and preparative gel electrophoresis, the stromelysin-3 substrate was identified by N-terminal sequencing as the serine proteinase inhibitor, alpha 1-proteinase inhibitor. Further studies demonstrated that stromelysin-3 rapidly destroyed the antiproteolytic function of alpha 1-proteinase inhibitor by cleaving the antiproteinase at a distinct site between Ala350 and Met351 within the reactive-site loop. Together, these data not only demonstrate that human stromelysin-3 acts as a powerful endopeptidase with a restricted substrate specificity distinct from all other matrix metalloproteinases, but also serve to identify serine proteinase inhibitors as potential physiologic targets at sites of extracellular matrix remodeling.
Pei
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D
Department of Internal Medicine, University of Michigan, Ann Arbor 48109.
Majmudar
G
G
Weiss
S J
SJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology*; D002277:Carcinoma / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000145:classification; D005260:Female; D006801:Humans; D006868:Hydrolysis; D019272:Leukocyte Elastase; D053507:Matrix Metalloproteinase 11; D008666:Metalloendopeptidases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D015843:Serpins / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
D002364:Caseins; D016326:Extracellular Matrix Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D015843:Serpins; D000515:alpha 1-Antitrypsin; C037741:alpha 1-antitrypsin-leukocyte elastase complex; D000511:alpha-Macroglobulins; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D053507:Matrix Metalloproteinase 11; D008666:Metalloendopeptidases