Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation
Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation
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DOI：
10.1093/glycob/cwg117
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发表时间：
2003-12-01
期刊：
影响因子：
4.3
通讯作者：
Fukuda, M
中科院分区：
文献类型：
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作者：
El-Battari, A;Prorok, M;Fukuda, M
高尔基糖基转移酶的修饰是调节糖基转移酶活性的重要过程，如形成二硫键二聚体和以可溶性形式从细胞中释放蛋白质。为了更好地了解这些过程，根据供体糖选择了六种糖基转移酶，包括两种N-乙酰氨基葡萄糖转移酶，核心1β1，3-N-乙酰氨基葡萄糖转移酶(C1-Beta3GnT)和核心2β1，6-N-乙酰氨基葡萄糖转移酶(C2GnT-I)；两种岩藻糖基转移酶，α1，2-岩藻糖基转移酶-I(Fuct-I)和α1，3-岩藻糖基转移酶-VII(Fuct-VII)；以及两种唾液酸转移酶，α2，3-唾液酸基转移酶-I(ST3Gal-I)和2，6-唾液酸基转移酶-I(ST6Gal-I)。将这些酶与增强型绿色荧光蛋白融合，在中国仓鼠卵巢细胞中稳定表达。荧光检测和免疫印迹分析表明，除Fuct-VII外，所有这些糖基转移酶都在培养液中分泌。通过检测细胞和培养基中形成的二聚体，我们发现，除ST3Gal-I外，所有的酶在细胞中形成单体和二聚体的组合，而在培养基中释放的分子要么是单体(C2GnT-I和ST6Gal-I)，要么是二聚体(Fuct-I)，或者是两者的混合物(C1-beta3GnT)。这些结果表明，二聚化并不总是导致高尔基保留。对这些酶的N-糖基化状态的分析表明，分泌的蛋白质通常比它们的膜相关蛋白更严重的N-糖基化和唾液酸化，这表明蛋白水解性切割发生在糖基化完成之前。以Fuct-I和ST6Gal-I为模型，我们还证明了这些糖基转移酶能够以二聚体的形式进行自糖化。这些结果表明，不同的糖基转移酶在二聚化、蛋白水解性消化和分泌以及自糖化方面存在显著差异。这些结果有力地表明，二硫键二聚和分泌在高尔基体中不同糖基转移酶的加工和功能中起着不同的作用。
Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.