CLONING OF CDNA-ENCODING THE MEMBRANE-BOUND FORM OF BOVINE BETA-1,4-GALACTOSYLTRANSFERASE
CLONING OF CDNA-ENCODING THE MEMBRANE-BOUND FORM OF BOVINE BETA-1,4-GALACTOSYLTRANSFERASE
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DOI：
10.1111/j.1432-1033.1989.tb14915.x
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发表时间：
1989-07-15
期刊：
影响因子：
--
通讯作者：
TROPAK, M
中科院分区：
文献类型：
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作者：
DAGOSTARO, G;BENDIAK, B;TROPAK, M
UDP半乳糖：N-乙酰基-D-葡糖胺4-β- D-半乳糖基转移酶（EC 2.4.1.38）（GalT）是一种高尔基体膜结合酶，参与糖蛋白和糖脂寡糖结构的生物合成。合成的DNA寡聚体代表的部分公布的部分cDNA序列的牛半乳糖转移酶的片段被用作分子探针，从牛肝cDNA文库中分离重叠的cDNA克隆，跨越1728个核苷酸和潜在的代码的整个多肽链的牛半乳糖基转移酶。牛GalT的cDNA序列揭示了编码402个氨基酸的1206个碱基对的开放阅读框架，包括20个疏水氨基酸的假定N-末端膜锚定结构域。从可溶性形式的酶中分离的两个多肽的N-末端测序阳性鉴定的29个非重叠氨基酸残基的cDNA序列之间的共线性与翻译框架一致，并证实了cDNA克隆的真实性。N-末端疏水片段作为膜锚和信号序列的发现表明GalT多肽的C-末端区域在高尔基体膜的内腔内定向。这一结论与以前的生物化学研究一致，这些研究表明，51-kDa和42-kDa的可溶形式的酶，分别包含整个GalT多肽的C-末端324和297个氨基酸残基，包括催化位点。
UDPgalactose: N-acetyl-D-glucosamine 4-.beta.-D-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membrane-bound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids. Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated form the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones. The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal regions of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.