Schwager S L, Chubb A J, Scholle R R, Brandt W F, Eckerskorn C, Sturrock E D, Ehlers M R
Department of Medical Biochemistry, University of Cape Town Medical School, South Africa.
Biochemistry. 1998 Nov 3;37(44):15449-56. doi: 10.1021/bi981260k.
Specialized proteases, referred to as sheddases, secretases, or membrane-protein-solubilizing proteases (MPSPs), solubilize the extracellular domains of diverse membrane proteins by catalyzing a specific cleavage in the juxtamembrane stalk regions of such proteins. A representative MPSP (tumor necrosis factor-alpha convertase) was cloned recently and shown to be a disintegrin metalloprotease that is inhibited by peptide hydroxamates including the compound TAPI. Substrate determinants that specify cleavage by MPSPs remain incompletely characterized, but may include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We constructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain (ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open, comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a typical MPSP activity. We found that ACE-JMEGF was solubilized inefficiently and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester and inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cleavage occurred at a Gly-Phe bond between the fifth and sixth cysteines within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell-associated via disulfide tethering. The mechanism for the release of ACE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is prevented by disulfide tethering to the TM domain.
一类特殊的蛋白酶，被称为脱落酶、分泌酶或膜蛋白溶解蛋白酶（MPSP），通过催化多种膜蛋白近膜柄区域的特定切割，使这些蛋白的细胞外结构域溶解。最近克隆出一种具有代表性的MPSP（肿瘤坏死因子-α转化酶），它是一种去整合素金属蛋白酶，可被包括化合物TAPI在内的肽羟肟酸抑制。决定MPSP切割作用的底物决定因素仍未完全明确，可能包括柄部的物理化学性质或柄部或细胞外结构域中未明确的识别基序。我们构建了一种突变型血管紧张素转换酶（ACE），其中柄部被表皮生长因子（EGF）样结构域取代（ACE-JMEGF），以检验MPSP切割需要开放、相对未折叠或伸展的柄部这一假说。野生型ACE是一种I型跨膜（TM）胞外蛋白，可被典型的MPSP活性有效溶解。我们发现ACE-JMEGF溶解效率低下，并以细胞相关形式积聚在转染的中国仓鼠卵巢（CHO）细胞上；佛波酯可刺激切割，TAPI可抑制切割，这些都是MPSP活性的典型特征。可溶性ACE-JMEGF C末端的测定结果显示，令人惊讶的是，切割发生在EGF样结构域第三个二硫键环内第五个和第六个半胱氨酸之间的Gly-Phe键处。用三丁基膦还原完整的CHO细胞会导致ACE-JMEGF（而非野生型ACE）迅速释放到培养基中，这表明一部分膜结合的ACE-JMEGF被切割，但通过二硫键连接仍与细胞相关。在没有化学还原的情况下，ACE-JMEGF释放的机制尚不清楚。我们得出结论，紧密的、二硫键桥接结构域的存在本身并不抑制MPSP活性的切割，但胞外结构域的释放会因与TM结构域的二硫键连接而受阻。
