Núñez E, Aragón C
Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
J Biol Chem. 1994 Jun 17;269(24):16920-4.
The sodium- and chloride-coupled glycine transporter from pig brain stem has been shown to be a 100-kDa glycoprotein (López-Corcuera, B., Vázquez, J., and Aragón, C. (1991) J. Biol. Chem. 266, 24809-24814). To structurally identify the carbohydrate components of glycine transporter, the purified and radioiodinated protein was subjected to specific glycosidase treatments. When the glycine transporter was treated with peptide-N-glycosidase F (PNGaseF) to remove N-linked oligosaccharides, a significant reduction of the apparent molecular mass of the protein was observed. However, incubations with endoglycosidase F and O-glycanase did not affect the electrophoretic mobility of the protein, and neuraminidase produced a slight reduction of its apparent mass. The effect of PNGaseF indicates that sugar chains represent about 30% of the mass of this heavily glycosylated transporter. The deglycosylated form is recognized by previously characterized anti-100-kDa protein antiserum (López-Corcuera, B., Alcántara, R., Vázquez, J., and Aragón, C. (1993) J. Biol. Chem. 268, 2239-2243), suggesting that the epitopes are in the peptidic part of the glycoprotein. These and other results suggest that glycine transporter-linked carbohydrates are predominantly tri- or tetra- antennary complex N-linked oligosaccharides containing sialic acid residues. To investigate the functional role of the carbohydrate moiety, liposomes reconstituted with purified glycine transporter were subjected to PNGaseF and neuraminidase treatments, and the effect on specific glycine transport activity was tested. Whereas neuraminidase did not affect the activity of the transporter, PNGaseF treatment produced a drastic reduction of transport activity. This treatment produced two different deglycosylated glycine transporter species, suggesting that two N-glycosylation sites would be occupied in the native protein. These studies arise as a first evidence supporting the notion that N-linked carbohydrates play a relevant role in glycine transporter functionality.
来自猪脑干的钠和氯偶联甘氨酸转运体已被证明是一种100 kDa的糖蛋白（洛佩斯 - 科尔库埃拉，B.，巴斯克斯，J.，和阿拉贡，C.（1991年）《生物化学杂志》266，24809 - 24814）。为了从结构上鉴定甘氨酸转运体的碳水化合物成分，对纯化并经放射性碘化的蛋白质进行了特定糖苷酶处理。当用肽 - N - 糖苷酶F（PNGaseF）处理甘氨酸转运体以去除N - 连接寡糖时，观察到蛋白质的表观分子量显著降低。然而，用内切糖苷酶F和O - 聚糖酶孵育并未影响蛋白质的电泳迁移率，而神经氨酸酶使其表观分子量略有降低。PNGaseF的作用表明糖链占这种高度糖基化转运体质量的约30%。去糖基化形式能被先前鉴定的抗100 kDa蛋白抗血清识别（洛佩斯 - 科尔库埃拉，B.，阿尔坎塔拉，R.，巴斯克斯，J.，和阿拉贡，C.（1993年）《生物化学杂志》268，2239 - 2243），这表明表位位于糖蛋白的肽段部分。这些以及其他结果表明，与甘氨酸转运体相连的碳水化合物主要是含有唾液酸残基的三分支或四分支复杂N - 连接寡糖。为了研究碳水化合物部分的功能作用，用纯化的甘氨酸转运体重构的脂质体接受了PNGaseF和神经氨酸酶处理，并测试了对特定甘氨酸转运活性的影响。虽然神经氨酸酶不影响转运体的活性，但PNGaseF处理使转运活性急剧降低。这种处理产生了两种不同的去糖基化甘氨酸转运体物种，这表明在天然蛋白质中两个N - 糖基化位点会被占据。这些研究首次证明了N - 连接碳水化合物在甘氨酸转运体功能中起相关作用这一观点。
