Panchenko M V, Stetler-Stevenson W G, Trubetskoy O V, Gacheru S N, Kagan H M
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 1996 Mar 22;271(12):7113-9. doi: 10.1074/jbc.271.12.7113.
Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.
赖氨酰氧化酶作为一种50 kDa的前体酶从成纤维细胞中分泌出来，该前体酶在细胞外空间经蛋白水解加工成为成熟酶。为了表征分泌的蛋白酶活性，制备了一种截短的重组赖氨酰氧化酶形式作为含有前肽切割区域序列的蛋白酶底物。培养的成纤维细胞分泌的加工蛋白酶活性对丝氨酸或天冬氨酸蛋白酶抑制剂以及基质金属蛋白酶-2（MMP-2）组织抑制剂具有抗性，但完全被金属离子螯合剂抑制。测试了已知金属蛋白酶对该底物的活性。羧基末端前胶原蛋白酶（C-蛋白酶）、MMP-2和条件性成纤维细胞培养基将赖氨酰氧化酶底物切割成成熟酶的大小。由动脉平滑肌条件培养基和C-蛋白酶产生的NH2末端序列，而不是MMP-2产生的，即天冬氨酸-天冬氨酸-脯氨酸-酪氨酸，与先前从结缔组织中分离出的成熟赖氨酰氧化酶中鉴定的序列相同。针对模型底物的C-蛋白酶活性被切割序列的合成寡肽模拟物（Ac-甲硫氨酸-缬氨酸-甘氨酸-天冬氨酸-天冬氨酸-脯氨酸-酪氨酸-天冬酰胺）抑制，而该肽也抑制培养的胎鼠肺成纤维细胞培养基中赖氨酰氧化酶活性的产生。总体而言，这些结果确定了一种参与前赖氨酰氧化酶激活的分泌金属蛋白酶活性，确定了加工活性的抑制剂，并表明前胶原C-蛋白酶在这一作用中发挥作用。
