Defining the minimal size of catalytically active primate alpha 1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme.
Defining the minimal size of catalytically active primate alpha 1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme.
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DOI：
10.1093/glycob/4.2.193
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发表时间：
1994-04
期刊：
影响因子：
4.3
通讯作者：
T. Henion;B. Macher;F. Anaraki;U. Galili
中科院分区：
文献类型：
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作者：
T. Henion;B. Macher;F. Anaraki;U. Galili
糖基化酶α1，3半乳糖基转移酶是合成糖类Galα1-3Galβ1-4GlcNAc-R的酶，在非灵长类哺乳动物、原猴和新大陆猴中具有活性，但在东半球猴、猿和人类中不活跃。在这项研究中，我们克隆和测序了在新世界猴子中表达的酶，确定了茎区域的确切大小，并评估了具有催化活性的α1，3半乳糖基转移酶(α1，3GT)的最小尺寸。在聚合酶链式反应中使用不同的引物组来产生编码在N-末端或C-末端区域缺失的α-1，3GT形式的cDNA。将该基因插入含有A蛋白编码序列的表达载体pPROTA中，并转入COS细胞。用免疫球蛋白-琼脂糖珠从细胞培养上清液中获得可溶性嵌合产物(截短酶和蛋白A)，并进行酶活性测定。在不影响其催化活性的情况下，可在酶的腔部分的氨基末端截断多达67个氨基酸。截短68、69和74个氨基酸分别导致体外催化活性损失50%、75%和95%。人类α-1，3GT基因和人猿特有的移码突变的引入导致酶活性完全丧失。此外，在α-1，3GT的羧基末端截断三个氨基酸会导致催化活性完全丧失。
The glycosylation enzyme alpha 1,3 galactosyltransferase, which synthesizes the carbohydrate Gal alpha 1-3Gal beta 1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active alpha 1,3 galactosyltransferase (alpha 1,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of alpha 1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG-Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and > 95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human alpha 1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of alpha 1,3GT resulted in complete loss of the catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)