Different collagenase gene products have different roles in degradation of type I collagen.
Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus. We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus. Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli. Assays utilized collagen from wild type (+/+) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in alpha1(I) chains that prevent collagenase cleavage at the helical locus. MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (+/+) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (beta components converted to alpha chains). Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences amino-terminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA. Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.
Krane
S M
SM
Department of Medicine, Harvard Medical School and the Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. kranes@A1.mgh.harvard.edu
Byrne
M H
MH
Lemaître
V
V
Henriet
P
P
Jeffrey
J J
JJ
Witter
J P
JP
Liu
X
X
Wu
H
H
Jaenisch
R
R
Eeckhout
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D003094:Collagen / Q000378:metabolism*; D024042:Collagen Type I; D017364:Collagenases / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D020781:Matrix Metalloproteinase 1; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D051381:Rats; D019714:Tissue Inhibitor of Metalloproteinases
D024042:Collagen Type I; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D010455:Peptides; D019714:Tissue Inhibitor of Metalloproteinases; C052929:collagen type I trimeric cross-linked peptide; D003094:Collagen; D017364:Collagenases; D020781:Matrix Metalloproteinase 1