Purification, characterization, and subunit structure of rat core 1 β1,3-galactosyltransferase
Purification, characterization, and subunit structure of rat core 1 β1,3-galactosyltransferase
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DOI：
10.1074/jbc.m109056200
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发表时间：
2002-01-04
影响因子：
4.8
通讯作者：
Canfield, WM
中科院分区：
文献类型：
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作者：
Ju, TZ;Cummings, RD;Canfield, WM
哺乳动物糖蛋白中的O-连接寡糖（O-聚糖）根据其核心结构进行分类。其中最常见的是由 Galbeta1-->3GalNAcalpha1-->Ser/ Thr 组成的核心 I 二糖结构，它也是许多扩展 O-聚糖结构的前体。从前体 GalNAc-α-Ser/Thr 生物合成核心 1 O-聚糖的关键酶是 UDP-Gal:GalNAc-α-Ser/Thr beta3-半乳糖基转移酶 (core1 beta3-Gal-T)。需要 Mn2+ 的核心 1 beta3-Gal-T 活性从大鼠肝膜中溶解，并通过 SP-Sepharose 上的离子交换层析、固定化脱唾液酸牛颌下粘蛋白上的亲和层析以及 Superose 12 上的凝胶过滤层析纯化 71,034 倍，达到表观同质性（>90% 纯度），收率 5.7%。纯化的酶不含污染的糖基转移酶。在 Superose 12 上的最后一步中鉴定出核心 1 beta3-Gal-T 活性的两个峰。一个活性峰在非还原 SDS-PAGE 上包含类似于 84-kDa 和类似于 86-kDa 二硫键连接二聚体的蛋白质条带，而第二个活性峰包含类似于 43-kDa 的单体。还原这些蛋白质的 SDS-PAGE 得到类似于 42-kDa 和类似于 43-kDa 的单体。 84/86-kDa 二聚体和 42/43-kDa 单体都具有相同的新型 N 端序列。纯化的酶非常稳定，使用 GalNAcalpha1-O-苯基作为受体时，UDP-Gal 的表观 K-m 为 630 mum，表观 V-max 为 206 mumol/mg/h 蛋白质。使用脱唾液酸牛颌下粘蛋白作为受体产生反应产物；用 O-糖苷酶处理产生了预期的二糖 Galbeta1-->3GalNAc。这些研究表明，来自大鼠肝脏的核心 1 beta1,3-Gal-T 的活性包含在一种新型二硫键二聚酶中。
The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core I disaccharide structure consisting of Galbeta1-->3GalNAcalpha1-->Ser/ Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 beta3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of similar to84- and similar to86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of similar to43-kDa. Reducing SDS-PAGE of these proteins gave similar to42- and similar to43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent K-m for UDP-Gal of 630 mum and an apparent V-max of 206 mumol/mg/h protein using GalNAcalpha1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.