Fraij B M, Birckbichler P J, Patterson M K, Lee K N, Gonzales R A
Samuel Roberts Noble Foundation, Inc., Biomedical Division/Nutrition Section, Ardmore, Oklahoma 73402.
J Biol Chem. 1992 Nov 5;267(31):22616-23.
A 1.9-kilobase (kb) cDNA for a new transglutaminase protein has been cloned and sequenced from retinoic acid-induced human erythroleukemia (HEL) cells. Full-length cDNA analysis reveals an open reading frame coding for a polypeptide of 548 amino acid residues with a molecular weight of 61,740. The deduced amino acid sequence exhibited 98% identity to the human cellular transglutaminase sequence. The cysteine at position 277 in the active site and the putative Ca(2+)-binding pocket at residues 446-453 of cellular transglutaminase are conserved. Such evidence predicts that the encoded protein product is likely to be a transglutaminase homologue (TGase-H). Immunoprecipitation of the in vitro translation products from a synthetic TGase-H mRNA and from total protein of cultured erythroleukemia HEL cells revealed a protein with a molecular weight of 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis of HEL cells and normal human fibroblast cells WI-38 using a cellular TGase probe detected the 1.9- and 4.0-kb RNA species at a relative abundance of 1:3 and 1:7, respectively. The 3'-end of the human cellular transglutaminase mRNA was also cloned and sequenced to allow comparison to the 3'-end of TGase-H reported here. This new piece gives a full length of 4012 nucleotides (4.0 kb) for human cellular transglutaminase. Comparison of the 5'-end (bases 1-1747) of the 1.9- and 4.0-kb cDNA sequences revealed a very high degree of identity. Beginning with base 1748, the sequences diverge showing no homology. The divergence point correlates with known intron-exon consensus boundaries indicative of alternative splicing.
已从视黄酸诱导的人红白血病（HEL）细胞中克隆并测序了一种新的转谷氨酰胺酶蛋白的1.9千碱基（kb）互补DNA（cDNA）。全长cDNA分析显示一个开放阅读框，编码一个由548个氨基酸残基组成的多肽，分子量为61,740。推导的氨基酸序列与人类细胞转谷氨酰胺酶序列有98%的同一性。细胞转谷氨酰胺酶活性位点处第277位的半胱氨酸以及第446 - 453位残基处假定的钙结合口袋是保守的。这些证据预测所编码的蛋白质产物可能是一种转谷氨酰胺酶同系物（TGase - H）。对合成的TGase - H mRNA以及培养的红白血病HEL细胞总蛋白的体外翻译产物进行免疫沉淀，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示出一种分子量为63,000的蛋白质。使用细胞转谷氨酰胺酶探针，对HEL细胞和正常人成纤维细胞WI - 38进行Northern印迹分析，分别检测到相对丰度为1:3和1:7的1.9 kb和4.0 kb RNA种类。还克隆并测序了人类细胞转谷氨酰胺酶mRNA的3'端，以便与本文报道的TGase - H的3'端进行比较。这个新片段给出了人类细胞转谷氨酰胺酶4012个核苷酸（4.0 kb）的全长。对1.9 kb和4.0 kb cDNA序列的5'端（第1 - 1747位碱基）进行比较，发现同一性程度非常高。从第1748位碱基开始，序列出现分歧，没有同源性。分歧点与已知的内含子 - 外显子共有边界相关，表明存在可变剪接。
