Lum L, Reid M S, Blobel C P
Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 1998 Oct 2;273(40):26236-47. doi: 10.1074/jbc.273.40.26236.
Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-alpha. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.
金属蛋白酶解整合素是一类膜锚定糖蛋白家族，在受精、成肌细胞融合、神经元发育以及膜锚定细胞因子肿瘤坏死因子-α的裂解过程中发挥作用。在此，我们报告小鼠金属蛋白酶解整合素MDC15的克隆及cDNA测序，并对其在分泌途径中的加工过程进行分析。小鼠MDC15（mMDC15）与其假定的人类同源物（hMDC15，metargidin）之间的一个显著差异是，在mMDC15的解整合素结构域中存在肽序列TDDC，而非hMDC15中发现的RGDC。在蛋白质印迹分析中，在所检测的所有小鼠组织中，大多数mMDC15被发现缺乏前结构域。在瞬时转染的COS-7细胞中进行的脉冲追踪实验表明，mMDC15是在高尔基体晚期区室中由一种前体蛋白转化酶进行加工的，因为：（i）添加布雷菲德菌素A或莫能菌素可阻断前结构域的去除；（ii）所有可检测到的加工后的mMDC15对内切糖苷酶H具有抗性；（iii）反式高尔基体网络前体蛋白转化酶弗林蛋白酶的重组可溶性形式可在体外模拟mMDC15的加工过程。细胞表面胰蛋白酶消化显示，超过一半的成熟mMDC15位于细胞内。免疫定位提供了证据，表明在类似于反式高尔基体网络和/或内体区室的区域有强烈的核周聚集。本研究首次对金属蛋白酶解整合素的细胞内加工过程进行了表征，并突出了前体蛋白转化酶在去除抑制性前结构域中的潜在作用。这些结果进一步表明，mMDC15除了在细胞表面蛋白水解或细胞黏附中可能发挥的作用外，在蛋白质成熟等方面可能具有细胞内功能。
