Ando Y, Imamura S, Owada M K, Kannagi R
Department of Dermatology, Faculty of Medicine, Kyoto University, Japan.
J Biol Chem. 1991 Jan 15;266(2):1101-8.
Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.
研究表明，在用表皮生长因子或钙离子载体A23187处理的人表皮样癌A431细胞中存在共价交联的脂皮质素I多聚体。这种脂皮质素I的细胞内交联被认为是由组织转谷氨酰胺酶的作用介导的，组织转谷氨酰胺酶是一种Ca2(+)依赖性蛋白质交联酶。脂皮质素I的交联与该蛋白的蛋白水解消化相互竞争，用钙蛋白酶抑制剂（Ca2(+)依赖性细胞内蛋白酶）预处理细胞可显著增强脂皮质素I的交联。交联的脂皮质素I在A431细胞的可溶性部分以及颗粒部分均有存在；通过纤溶酶消化后者部分成功溶解了脂皮质素I的一个34 kDa片段。使用特异性抗脂皮质素-I抗体的免疫荧光显微镜检查显示，交联的脂皮质素I形成一种包膜样结构，该结构不能被[乙二胺双(氧乙基腈)]四乙酸（EGTA）或 Triton X-100提取。纯化的脂皮质素I与组织转谷氨酰胺酶在体外孵育导致形成共价交联的脂皮质素I二聚体、四聚体等。使用脂皮质素I及其N端截短衍生物的胺掺入和交联研究表明，交联位点位于脂皮质素I的纤溶酶敏感的N端29个氨基酸内。通过添加磷脂酰丝氨酸囊泡，脂皮质素I的交联加速了10倍以上，脂皮质素I分子最有可能以适合交联的构象排列在磷脂酰丝氨酸囊泡上。总的来说，这些发现表明细胞内钙浓度的增加导致脂皮质素I通过分子的C端结构域附着到质膜磷脂上，其中膜结合的脂皮质素I通过组织转谷氨酰胺酶的作用通过N端结构域发生交联。
