Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity
Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity
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DOI：
10.1021/bi982426k
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发表时间：
1999-02-23
期刊：
影响因子：
2.9
通讯作者：
Robson, SC
中科院分区：
文献类型：
--
作者：
Esch, JSA;Sévigny, J;Robson, SC
哺乳动物ATP二磷酸水解酶（atpase） CD39被认为包含两个跨膜结构域和五个“apyrase保守区域”（ACR），位于一个大的细胞外区域。为了研究这种外膜酶的结构，人类CD39通过在这些ACRs内的定向突变或在两个末端的顺序缺失进行修饰，atpase活性通过FLAG标记得到很好的保存，然后去除所示的C或n跨膜区域。然而，ACR-1 （aa 54-61）或-4 （aa 212-220）内的缺失，以及包括ACR-1、-4或-5 （aa 447-454）的截断突变体，导致了生化活性的实质性丧失。因此，CD39内完整的ACR-1、-4和-5是维持生化活性所必需的。缺乏TMR的CD39的原生和突变形式被观察到经历多聚化，与分子间二硫键的形成有关。有限的胰蛋白酶切割完整的CD39产生两个非共价膜相关片段（56和27 kDa），这大大增强了atpase的活性。糖基化变异在天然和突变形式的CD39中占很小的异质性，但不影响atpase功能。atp酶的酶活性可能受到某些与血管炎症相关的翻译后修饰的影响。
CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini, ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.