Krane S M, Byrne M H, Lemaître V, Henriet P, Jeffrey J J, Witter J P, Liu X, Wu H, Jaenisch R, Eeckhout Y
Department of Medicine, Harvard Medical School and the Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
J Biol Chem. 1996 Nov 8;271(45):28509-15. doi: 10.1074/jbc.271.45.28509.
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.
脊椎动物胶原酶，即基质金属蛋白酶（MMPs），在单螺旋位点切割I型胶原。我们在此表明，啮齿动物间质胶原酶（MMP - 13）而非人成纤维细胞胶原酶（MMP - 1），可在另一个氨基端肽位点切割I型胶原。将胶原酶cDNA和pET - 3d中的嵌合构建体在大肠杆菌中表达，这些构建体将催化结构域中活性位点氨基端的MMP - 13序列与羧基端的MMP - 1序列并列排列，反之亦然。实验使用来自野生型（+/ +）小鼠或携带靶向突变（r/r）小鼠的胶原，该突变在α1（I）链中编码替换，可防止胶原酶在螺旋位点切割。MMP - 13以及在活性位点氨基端包含MMP - 13序列的嵌合分子在螺旋位点切割（+/ +）胶原，并在氨基端肽（β成分转化为α链）中切割交联的（r/r）胶原。人MMP - 1以及在活性位点氨基端具有MMP - 1序列的嵌合MMP - 1/MMP - 13在螺旋位点切割胶原，但不在氨基端肽中切割。所有活性均被TIMP - 1、1,10 - 菲咯啉和EDTA抑制。催化结构域远端三分之二的序列决定了MMP - 13降解氨基端肽的能力。
