El-Battari Assou, Prorok Maëlle, Angata Kiyohiko, Mathieu Sylvie, Zerfaoui Mourad, Ong Edgar, Suzuki Misa, Lombardo Dominique, Fukuda Minoru
INSERM U-559/UEA-3289 Université de la Méditerranée, 27 Bd. J. Moulin, 13385 Marseille Cedex 5, France.
Glycobiology. 2003 Dec;13(12):941-53. doi: 10.1093/glycob/cwg117. Epub 2003 Sep 26.
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.
高尔基体糖基转移酶的修饰，如形成二硫键连接的二聚体以及以可溶形式从细胞中蛋白水解释放，是调节糖基转移酶活性的重要过程。为了更好地理解这些过程，基于供体糖选择了六种糖基转移酶，包括两种N - 乙酰葡糖胺基转移酶，核心1 β1,3 - N - 乙酰葡糖胺基转移酶（C1 - β3GnT）和核心2 β1,6 - N - 乙酰葡糖胺基转移酶（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 - 唾液酸转移酶 - I（ST3Gal - I）和α2,6 - 唾液酸转移酶 - I（ST6Gal - I）。这些酶与增强型绿色荧光蛋白融合，并在中国仓鼠卵巢细胞中稳定表达。荧光光谱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分泌到培养基中。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，所有酶在细胞中形成单体和二聚体的组合，而培养基中释放的分子要么完全是单体（C2GnT - I和ST6Gal - I）、二聚体（FucT - I）或两者的混合物（C1 - β3GnT）。这些结果表明二聚化并不总是导致高尔基体滞留。对这些酶的N - 糖基化状态分析表明，分泌的蛋白质通常比其膜相关对应物具有更重的N - 糖基化和唾液酸化，这表明蛋白水解切割发生在糖基化完成之前。以FucT - I和ST6Gal - I为模型，我们还表明这些糖基转移酶能够以二聚体形式进行自身糖基化。这些结果表明不同的糖基转移酶在二聚化、蛋白水解消化、分泌和自身糖基化方面存在显著差异。这些结果强烈表明二硫键连接的二聚化和分泌在高尔基体中不同糖基转移酶的加工和功能中发挥不同作用。
