Disintegrin-like/cysteine-rich region of ADAM 12 is an active cell adhesion domain.
Disintegrin-like/cysteine-rich region of ADAM 12 is an active cell adhesion domain.
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DOI：
10.1006/excr.1999.4632
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发表时间：
1999-11
影响因子：
3.7
通讯作者：
A. Zolkiewska
中科院分区：
文献类型：
--
作者：
A. Zolkiewska
ADAM(一种去整合素和金属蛋白酶)蛋白含有与蛇毒金属蛋白酶(SVMP)的P-III类结构同源的蛋白质，通过类比这些SMVP，推测ADAM具有细胞黏附分子的功能。ADAM 12与成肌细胞的融合有关，但其作用机制尚不清楚。与SVMP去整合素中存在的RGD样细胞结合基序不同，ADAM 12的去整合素结构域包含一个独特的SNS序列，因此其粘附性一直存在争议。在这份报告中，我们证明了ADAM 12的去整合素样/富含半胱氨酸(DC)结构域构成了一个功能细胞粘附域。我们已经在昆虫细胞中表达了小鼠ADAM 12的DC结构域，并表明重组蛋白以二价阳离子依赖的方式支持C2C12成肌细胞和NIH3T3成纤维细胞的黏附。然而，巯基特异的生物素化试剂显示，ADAM 12 DC结构域的细胞结合区的整体构象和灵活性可能与SVMP去整合素的构象和灵活性有显著不同。此外，DC结构域的二硫键结构对其功能至关重要，因为重组蛋白与还原剂孵育后可消除随后的细胞黏附。重组DC与C2C12细胞具有高亲和力(K(D)约0.10微米，结合位点数n约4.6×10(5)个/细胞)。在C2C12细胞中表达并分泌到介质中的DC结构域与细胞表面的结合进一步证实了在昆虫细胞中产生的ADAM 12的DC结构域的粘附性，这与ADAM 12在细胞-细胞相互作用和成肌细胞融合中的作用一致。
ADAM (a disintegrin and metalloprotease) proteins contain structural homology to the P-III class of snake venom metalloproteases (SVMPs) and are postulated to function, by analogy to these SMVPs, as cell adhesion molecules. ADAM 12 has been implicated in fusion of myoblasts, but its mechanism of action is not known. Instead of the RGD-like cell-binding motif present in SVMP disintegrins, the disintegrin domain of ADAM 12 contains a unique SNS sequence and therefore its adhesive potential has been controversial. In this report we demonstrate that the disintegrin-like/cysteine-rich (DC) domain of ADAM 12 constitutes a functional cell adhesion domain. We have expressed the DC domain of mouse ADAM 12 in insect cells and shown that the recombinant protein supported adhesion of C2C12 myoblasts and NIH 3T3 fibroblasts in a divalent cation-dependent manner. A sulfhydryl-specific biotinylation reagent revealed, however, that the overall conformation and flexibility of the cell-binding region of ADAM 12 DC domain may be significantly different from those of the SVMP disintegrins. Moreover, the disulfide bond structure of the DC domain was critical for its function, as incubation of the recombinant protein with reducing agents abolished subsequent cell adhesion. Recombinant DC bound to C2C12 cells with high affinity (K(D) approximately 0.10 microM, total number of binding sites n approximately 4.6 x 10(5)/cell). Adhesive properties of the DC domain of ADAM 12 produced in insect cells were further confirmed by cell surface binding of the DC domain expressed in C2C12 cells and secreted to the medium, consistent with the role of ADAM 12 in cell-cell interactions and myoblast fusion.