Schlomann Uwe, Wildeboer Dirk, Webster Ailsa, Antropova Olga, Zeuschner Dagmar, Knight C Graham, Docherty Andrew J P, Lambert Marc, Skelton Lisa, Jockusch Harald, Bartsch Jörg W
Developmental Biology and Molecular Pathology, W7, Bielefeld University, Germany.
J Biol Chem. 2002 Dec 13;277(50):48210-9. doi: 10.1074/jbc.M203355200. Epub 2002 Oct 7.
ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.
ADAMs（解聚素和金属蛋白酶结构域）是一类含金属蛋白酶和解聚素结构域的跨膜糖蛋白，具有蛋白水解、细胞黏附、细胞融合和细胞信号传导特性。ADAM8最初是从单核细胞中克隆出来的，其独特的表达模式表明它在免疫学和神经病理学中可能发挥作用。在此，我们描述了对其生化特性的分析。在转染的COS-7细胞中，ADAM8定位于质膜，并被加工成两种形式，一种是通过去除前结构域形成的，另一种是作为残余蛋白形成的，该残余蛋白包含细胞外区域，其N端有解聚素结构域。在锌离子结合基序（HE(330)LGHNLGMSHD）中发生谷氨酸（330）到谷氨酰胺交换的突变型ADAM8（EQ-A8）中，ADAM8前肽的蛋白水解去除被完全阻断，这表明前结构域的去除是自催化的。在共转染实验中，ADAM8的胞外结构域而非整个MP结构域能够从EQ-ADAM8中去除前结构域。在无血清条件下，表达ADAM8的细胞能与底物结合的重组ADAM8解聚素/富含半胱氨酸结构域发生细胞黏附，这种黏附被针对ADAM8解聚素结构域的抗体所阻断。由金属蛋白酶结构域或完整胞外结构域组成的可溶性ADAM8蛋白酶可切割髓鞘碱性蛋白和一种荧光肽底物，并被batimastat（BB-94，IC(50)约为50 nm）抑制，但不被重组基质金属蛋白酶组织抑制剂1、2、3和4抑制。我们的研究结果表明，ADAM8通过自催化加工产生一种潜在的脱落酶以及一种具有细胞黏附功能的ADAM8形式。
