Department of Chemistry/Biochemistry, University of Gothenburg, P. O. Box 462, SE-405 30 Göteborg, Sweden.
J Biol Chem. 2011 Sep 9;286(36):31915-23. doi: 10.1074/jbc.M111.242677. Epub 2011 Jul 6.
Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.
人水通道蛋白 10（hAQP10）是一种在小肠中促进水和甘油运输的跨膜促进剂。这种水甘油通道蛋白位于肠上皮细胞的顶膜上，被认为有助于水和甘油通过这些肠道吸收细胞的传递。在这里，我们在毕赤酵母中过量表达了 hAQP10，并观察到该蛋白在细胞外环 C 的天冬酰胺 133 处发生糖基化。这一发现证实了 hAQP10 的三个预测糖基化位点之一，并且其糖基化对于在该宿主中过量表达的人水通道蛋白是独特的。使用聚糖亲和层析和将天冬酰胺 133 突变为谷氨酰胺两种方法均可分离出未糖基化的蛋白。所有三种形式的 hAQP10 都促进了水、甘油、赤藓糖醇和木糖醇的运输，糖基化对功能几乎没有影响。相比之下，糖基化的 hAQP10 与未糖基化的蛋白相比表现出 3-6°C 的热稳定性增加，表明 N-连接聚糖具有稳定作用。由于 hAQP10 只有三分之一被糖基化，但热稳定性滴定呈单峰模式，我们推测每个四聚体中至少存在一个糖基化蛋白足以赋予四聚体中其余 hAQP10 原聚体增强的结构稳定性。
