Van Leuven F, Marynen P, Cassiman J J, Van den Berghe H
Center for Human Genetics, University of Leuven, Belgium.
J Biol Chem. 1988 Jan 5;263(1):468-71.
Proteolysis of human alpha 2-macroglobulin (alpha 2M) in the bait region is the prerequisite and necessary trigger for the trapping of the proteinase by a massive conformational change of alpha 2M. This labilization of the native conformation of alpha 2M is mediated by activation of the internal thiolesters, but the underlying mechanism is unknown. We now describe observations on proteolysis of human alpha 2M without concomitant hydrolysis of the internal thiolesters or conformational change. This proteolysis was obtained with a novel bacterial proteinase we recently used to isolate the receptor-binding domain from alpha 2M (Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). This proteinase is not inhibited by alpha 2M, and therefore it was possible to study its effect on native alpha 2M at pH 4.5, conditions used previously to produce the receptor-binding domain (Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). The major observations are that despite extensive proteolysis, alpha 2M largely retained its native conformation as shown by rate electrophoresis, the absence of binding of monoclonal antibody F2B2, and the incorporation of [14C]methylamine into a 145-kDa fragment of alpha 2M. Moreover, the derivative still bound trypsin to 88% of control values. Treatment of the derivative with trypsin or methylamine produced the conformational change as with intact alpha 2M, and concomitantly released the receptor-binding domain. This indicated that proteolysis at Lys1313-Glu also proceeded in native alpha 2M. At least one more major proteolysis site was deduced from the observation of a 27-kDa heat-induced fragment, the 145-kDa [14C]methylamine-labeled fragment, and from the presence of the 20-kDa receptor-binding domain. These results demonstrate indirectly the particular relation of the bait region to the internal thiolesters and illustrate further the domain-structure of alpha 2M and the expression of the receptor-recognition site by activation of the internal thiolesters.
人α2-巨球蛋白（α2M）在诱饵区域的蛋白水解是α2M通过大规模构象变化捕获蛋白酶的前提条件和必要触发因素。α2M天然构象的这种不稳定是由内部硫酯的激活介导的，但其潜在机制尚不清楚。我们现在描述了关于人α2M蛋白水解的观察结果，在此过程中内部硫酯没有伴随水解或构象变化。这种蛋白水解是用我们最近用于从α2M分离受体结合结构域的一种新型细菌蛋白酶实现的（范·勒芬，F.，马里嫩，P.，索特鲁普-詹森，L.，卡西曼，J.-J.，和范·登·伯格，H.（1986年）《生物化学杂志》261，11369 - 11373）。这种蛋白酶不受α2M抑制，因此有可能在pH 4.5的条件下研究其对天然α2M的影响，此前曾用该条件制备受体结合结构域（范·勒芬，F.，马里嫩，P.，索特鲁普-詹森，L.，卡西曼，J.-J.，和范·登·伯格，H.（1986年）《生物化学杂志》261，11369 - 11373）。主要观察结果是，尽管发生了广泛的蛋白水解，但α2M在很大程度上保留了其天然构象，这通过速率电泳、单克隆抗体F2B2不结合以及[14C]甲胺掺入α2M的145 kDa片段得以证明。此外，该衍生物仍能将胰蛋白酶结合至对照值的88%。用胰蛋白酶或甲胺处理该衍生物会产生与完整α2M相同的构象变化，并同时释放受体结合结构域。这表明在Lys1313 - Glu处的蛋白水解在天然α2M中也会发生。从27 kDa热诱导片段、145 kDa [14C]甲胺标记片段的观察以及20 kDa受体结合结构域的存在推断，至少还有一个主要的蛋白水解位点。这些结果间接证明了诱饵区域与内部硫酯的特殊关系，并进一步说明了α2M的结构域结构以及通过内部硫酯激活来表达受体识别位点。
