Russo R N, Shaper N L, Shaper J H
Cell Structure and Function Laboratory, Johns Hopkins University, Baltimore, Maryland 21205.
J Biol Chem. 1990 Feb 25;265(6):3324-31.
We have used S1 and primer extension analysis to demonstrate that the gene for bovine beta 1----4-galactosyltransferase specifies two sets of mRNA transcripts of different lengths. The longer mRNA transcripts initiate upstream of two in-frame ATG codons and encode a protein of 402 amino acids (long form). The shorter mRNA transcripts initiate between the two in-frame ATG codons and encode a protein of 389 amino acids (short form). These two related forms of beta 1----4-galactosyltransferase have an identical large (358 amino acids), potentially glycosylated, COOH-terminal catalytic domain, and an identical single transmembrane domain. The only difference in primary structure between the two forms is that the long form contains an NH2-terminal extension of 13 amino acids. Thus, bovine beta 1----4-galactosyltransferase fits the pattern established for murine beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. L., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428). Inspection of the NH2-terminal domain suggests that the long form of the bovine enzyme, like its murine counterpart, has a functional cleavable signal sequence which would dictate that the two forms of the membrane-bound enzyme are oriented in opposite directions. We have tested this hypothesis by in vitro translation in the absence or presence of dog pancreas microsomes. In vitro translation of RNA transcripts for the long and short form of beta 1----4-galactosyltransferase in the absence of microsomes results in the synthesis of polypeptides with apparent Mr of 44,500 and 43,000, respectively. In vitro translation of each transcript in the presence of microsomes results in the synthesis of two glycosylated, endoglycosidase H-sensitive proteins with apparent Mr of 47,500 and 46,000. These experiments and additional protease protection experiments demonstrate that the COOH-terminal domain of both the short and the long form of bovine beta 1----4-galactosyltransferase are translocated into the microsomal lumen. By extrapolation, both forms of the enzyme are oriented in vivo as Type II membrane-bound glycoproteins.
我们利用S1核酸酶分析和引物延伸分析证明，牛β1----4-半乳糖基转移酶基因可产生两组长度不同的mRNA转录物。较长的mRNA转录物在两个读码框内的ATG密码子上游起始，编码一个含402个氨基酸的蛋白质（长形式）。较短的mRNA转录物在两个读码框内的ATG密码子之间起始，编码一个含389个氨基酸的蛋白质（短形式）。β1----4-半乳糖基转移酶的这两种相关形式具有相同的大的（358个氨基酸）、潜在糖基化的COOH末端催化结构域，以及相同的单个跨膜结构域。这两种形式在一级结构上的唯一区别是，长形式含有一个13个氨基酸的NH2末端延伸区。因此，牛β1----4-半乳糖基转移酶符合为鼠β1----4-半乳糖基转移酶所确立的模式（沙珀，N.L.，霍利斯，G.L.，道格拉斯，J.G.，基尔希，I.R.，和沙珀，J.H.（1988年）《生物化学杂志》263，10420 - 10428）。对NH2末端结构域的检查表明，牛酶的长形式与其鼠对应物一样，具有一个功能性的可切割信号序列，这表明膜结合酶的两种形式在体内的方向相反。我们通过在有无犬胰腺微粒体存在的情况下进行体外翻译来检验这一假设。在无微粒体存在的情况下，对β1----4-半乳糖基转移酶长形式和短形式的RNA转录物进行体外翻译，分别产生表观分子量为44,500和43,000的多肽。在有微粒体存在下对每个转录物进行体外翻译，产生两种糖基化的、对内切糖苷酶H敏感的蛋白质，表观分子量分别为47,500和46,000。这些实验以及另外的蛋白酶保护实验证明，牛β1----4-半乳糖基转移酶短形式和长形式的COOH末端结构域都被转运到微粒体腔中。由此推断，该酶的两种形式在体内均作为II型膜结合糖蛋白定向排列。
