Alfalah M, Parkin E T, Jacob R, Sturrock E D, Mentele R, Turner A J, Hooper N M, Naim H Y
Department of Physiological Chemistry, School of Veterinary Medicine, D-30559 Hannover, Germany.
J Biol Chem. 2001 Jun 15;276(24):21105-9. doi: 10.1074/jbc.M100339200. Epub 2001 Mar 23.
Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.
血管紧张素I转换酶（ACE）是众多整合膜蛋白之一，可被一种锌金属分泌酶从细胞表面进行蛋白水解切割。通过脉冲追踪分析确定，将ACE近膜柄区域的天冬酰胺（631）突变为谷氨酰胺会导致突变蛋白（ACE（NQ））更高效地分泌。与野生型ACE不同，ACE（NQ）的切割不受金属分泌酶抑制剂巴替司他的阻断，而是受丝氨酸蛋白酶抑制剂1,3-二氯异香豆素的阻断。在15℃下培养细胞发现，ACE（NQ）在内质网中被切割，对该蛋白分泌形式的质谱分析表明，它是在天冬酰胺（635）-丝氨酸（636）键处被切割的，该键位于正常分泌酶切割位点精氨酸（638）-丝氨酸（639）的N端三个残基处。这些数据清楚地表明，整合膜蛋白近膜区域的一个点突变可以引发一种机制和空间上不同的分泌酶的作用。鉴于这一观察结果，以前关于脱落蛋白近膜柄突变效应的数据，即由一种具有宽松特异性的单一分泌酶所适应，需要重新评估。
