Mádi A, Punyiczki M, di Rao M, Piacentini M, Fésüs L
Department of Biochemistry, University Medical School of Debrecen, Hungary.
Eur J Biochem. 1998 May 1;253(3):583-90. doi: 10.1046/j.1432-1327.1998.2530583.x.
Transglutaminase activity was characterized in extracts of the nematode Caenorhabditis elegans using a microtiter plate method, and found to be Ca2+-dependent, optimal at pH 8.0, and to be inhibited by EGTA, ammonia, iodoacetamide and GTP. Monoclonal and polyclonal antibodies raised against human tissue transglutaminase also inhibited the activity and detected a 61-kDa protein from the worm lysate. Constitutive expression of the enzyme in the wild-type intestinal cells was revealed by immunohistochemistry. Potential protein substrates for the enzyme were found in worm lysates using a biotin-labelled amine substrate. There is a basal level of protein-bound epsilon(gamma-glutamyl)lysine cross-links, characteristic of transglutaminase activity, formed in situ in adult wild-type animals. Developmental studies have revealed that the enzyme activity is highest in adult animals, and relatively higher in L1 larvae than in other larval stages. As compared to wild types, lower transglutaminase activity has been measured in lysates of ced-3, ced-4 and ced-9 mutants. Cross-link levels were also low in ced-4 and ced-9 mutants. By contrast, the crosslink content was high in several phagocytosis mutants. The highest concentration was found in the ced-5; ced-7 double phagocytosis mutants which carry an extra number of dead cells during their lifespan. In accordance with this finding, several transglutaminase-immunopositive cells were found in both the embryos and in the head of these double phagocytosis mutants. The results suggest that a transglutaminase is involved in, or related to, the death program of cells in C. elegans and the expression and crosslinking activity of the enzyme may be perturbed in some ced mutants.
利用微量滴定板法对线虫秀丽隐杆线虫提取物中的转谷氨酰胺酶活性进行了表征，发现其活性依赖于Ca2+，在pH 8.0时达到最佳，并且受到乙二醇双四乙酸（EGTA）、氨、碘乙酰胺和鸟苷三磷酸（GTP）的抑制。针对人组织转谷氨酰胺酶产生的单克隆抗体和多克隆抗体也抑制了该活性，并从线虫裂解物中检测到一种61 kDa的蛋白质。通过免疫组织化学揭示了该酶在野生型肠道细胞中的组成型表达。使用生物素标记的胺底物在线虫裂解物中发现了该酶的潜在蛋白质底物。在成年野生型动物体内原位形成了具有转谷氨酰胺酶活性特征的蛋白质结合ε（γ-谷氨酰）赖氨酸交联的基础水平。发育研究表明，该酶活性在成年动物中最高，在L1幼虫中相对高于其他幼虫阶段。与野生型相比，在ced-3、ced-4和ced-9突变体的裂解物中测得的转谷氨酰胺酶活性较低。ced-4和ced-9突变体中的交联水平也较低。相比之下，在几个吞噬作用突变体中交联含量较高。在ced-5；ced-7双吞噬作用突变体中发现浓度最高，这些突变体在其生命周期中携带额外数量的死亡细胞。根据这一发现，在这些双吞噬作用突变体的胚胎和头部都发现了几个转谷氨酰胺酶免疫阳性细胞。结果表明，转谷氨酰胺酶参与秀丽隐杆线虫细胞的死亡程序或与之相关，并且该酶的表达和交联活性在一些ced突变体中可能受到干扰。
