Recombinant Chinese hamster ovary cell matrix metalloprotease-3 (MMP-3, stromelysin-1). Role of calcium in promatrix metalloprotease-3 (pro-MMP-3, prostromelysin-1) activation and thermostability of the low mass catalytic domain of MMP-3.
Recombinant human fibroblast pro-MMP-3 (prostromelysin-1) expressed in Chinese hamster ovary cells and the zymogen from cultured human dermal fibroblasts have been purified by monoclonal antibody immunoaffinity chromatography, and the role of Ca2+ in proenzyme activation and thermostability of the low mass catalytic domain of MMP-3 has been investigated. In the presence of high Ca2+ (5.0 mM), the organomercurial aminophenylmercuric acetate (APMA) initiated the stepwise removal of both NH2- and COOH-terminal domains from both recombinant and dermal fibroblast proenzymes, resulting in the generation of a heterogeneous family of nonglycosylated low mass truncated active enzyme species beginning at Phe83. However, in the presence of low Ca2+ (0.1 mM), incubation of recombinant pro-MMP-3 with or without APMA did not result in formation of either the high or low mass forms of active MMP-3 but resulted in complete autolysis of both enzyme species. The concentration of Ca2+ required for optimal pro-MMP-3 activation and stability of the low mass catalytic domain was 2.0 mM. The low mass truncated enzyme species containing the catalytic domain were remarkably heat-stable (90 min at 60 degrees C) in high Ca2+ (5.0 mM) but rapidly autolyzed when heated at 60 degrees C in low Ca2+ (0.1 mM). The thermostability properties of MMP-3 appeared to be specific for Ca2+, since no other divalent metal ions tested were able to confer thermostability to the low mass catalytic domain of MMP-3. From homology to the thermostable bacterial metalloprotease, thermolysin, two putative Ca2+ binding sites were found in the catalytic domain of MMP-3 and several other members of the MMP gene family. These putative Ca2+ binding sites are postulated to play an important role in stabilizing active MMP-3 and other members of the matrix metalloprotease gene family by protecting them against autolysis.
Housley
T J
TJ
Institute for Bone and Cartilage Metabolism, Miles Research Center, West Haven, Connecticut 06516.
Baumann
A P
AP
Braun
I D
ID
Davis
G
G
Seperack
P K
PK
Wilhelm
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D006224:Cricetinae; D004789:Enzyme Activation; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000201:enzymology
D000911:Antibodies, Monoclonal; D008670:Metals; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D002118:Calcium