Structure-function relationships in the collagenase family member transin.
We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.
Sanchez-Lopez
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R
Laboratoire de Génétique Moléculaire des Eucaryotes, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine, Strasbourg, France.
Nicholson
R
R
Gesnel
M C
MC
Matrisian
L M
LM
Breathnach
R
R
D000595:Amino Acid Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D013205:Staphylococcal Protein A / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection; D014357:Trypsin
D004274:DNA, Recombinant; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C010024:4-aminophenylmercuriacetate; D004247:DNA; D014357:Trypsin; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D010662:Phenylmercuric Acetate