Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins.
The relationship of enzyme structure to substrate specificity for the matrix metalloproteinases interstitial collagenase and stromelysin-2 has been investigated by analysis of the cleavage specificity of recombinant human collagenase-stromelysin-2 hybrid proteins and C terminally truncated collagenase and stromelysin-2. Two series of chimeric proteins were devised by progressive substitution of exon-encoded domains. The recombinant proteins were expressed in COS-7 cells as protein A-fusion proteins and purified on an IgG affinity matrix. Treatment with 4-amino-phenylmercuric acetate released active metalloproteinase of the sizes predicted for the chimeric proteins. Active forms of both the chimeric protein series and the short form enzymes expressed both casein- and gelatin-degrading activities. Like stromelysin, the catalytic activity of stromelysin-2 was contained in the N-terminal domain (encoded by exons 1-5) and was apparently independent of the C-terminal domain (encoded by exons 6-10). Only full-length collagenase displayed a triple helicase (collagenolytic) activity; no combination of N- or C-terminal collagenase domains fused with stromelysin-2 domains had such activity. This suggests that the triple helicase activity is a composite of elements derived from both halves of the collagenase molecule. C terminally truncated collagenase (exons 1-5) and a hybrid of collagenase exons 1-5 and stromelysin-2 exons 6-10 cleaved denatured type I collagen (gelatin) to generate diagnostic peptides in gelatin fingerprint assays. When exon 5 (the exon encoding the zinc-binding domain) was derived from stromelysin-2, the enzyme specificity in the fingerprint assay changed to that of native stromelysin-2. In contrast, when exon 5 was derived from collagenase, the specificity reflected that of the parent enzyme. Our data also suggest that mismatching of exons 2 and 5 destabilizes the enzyme, presumably by altering the geometry of the propeptide-zinc-binding site interaction. We conclude that the loss of triple helicase collagenolytic activity is not accompanied by a shift to the broad specificity characteristic of stromelysin. Rather, the zinc-binding domain confers a distinct cleavage specificity on each metalloproteinase.
Sanchez-Lopez
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Laboratoire de Génétique Moléculaire des Eucaryotes du Centre de la Recherche Scientifique, Unité 184 Institut National de la Santé et de la Recherche Médicale, Strasbourg, France.
Alexander
C M
CM
Behrendtsen
O
O
Breathnach
R
R
Werb
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005780:Gelatin / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006466:Hemopexin / Q000378:metabolism*; D006801:Humans; D053506:Matrix Metalloproteinase 10; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
D002364:Caseins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C010024:4-aminophenylmercuriacetate; D005780:Gelatin; D003094:Collagen; D004247:DNA; D006466:Hemopexin; D017364:Collagenases; D008666:Metalloendopeptidases; D053506:Matrix Metalloproteinase 10; D015032:Zinc; D010662:Phenylmercuric Acetate