CD320 is a cell surface receptor that mediates vitamin B12 uptake in most tissues. To date, the mechanisms that regulate CD320 expression on the cell surface are not fully understood. In this work, we studied CD320 expression in transfected human embryonic kidney (HEK) 293 and hepatoma HepG2 cells. By glycosidase and trypsin digestion, monensin and brefeldin treatment, western blotting, flow cytometry, and lectin binding, we found that CD320 underwent N- and O-glycosylation and sialylation, resulting in a ∼70-kDa band that formed a high-molecular-weight complex on the cell surface. Site-directed mutagenesis altering Asn126, Asn195, and Asn213 residues, individually or together, abolished N-glycosylation in CD320 but did not block its intracellular trafficking and expression on the cell surface in HEK293 and HepG2 cells. In contrast, treatment of the cells with Ben-gal, a structural analog of GalNAc-α-1-O-Ser/Thr, inhibited O-glycosylation and cell surface expression of CD320 and decreased vitamin B12 uptake. Analysis of CD320 deletion mutants indicated that O-glycosylation sites in a Ser/Thr-rich region near the transmembrane domain were important for CD320 expression on the cell surface. These results reveal an important role of O-glycans, but not N-glycans, in the intracellular trafficking and cell surface expression of CD320, providing new insights into the cellular mechanisms in regulating CD320 function and vitamin B12 metabolism.

中文翻译：

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O-糖基化对于转钴胺受体 CD320 的细胞表面表达至关重要


CD320 是一种细胞表面受体，可介导大多数组织中维生素 B12 的摄取。迄今为止，调节细胞表面 CD320 表达的机制尚不完全清楚。在这项工作中，我们研究了转染的人胚胎肾 （HEK） 293 和肝癌 HepG2 细胞中 CD320 的表达。通过糖苷酶和胰蛋白酶消化、莫能菌素和布雷菲德菌素处理、蛋白质印迹、流式细胞术和凝集素结合，我们发现 CD320 经历了 N-和 O-糖基化和唾液酸化，产生一个 ∼70-kDa 条带，在细胞表面形成高分子量复合物。单独或一起改变 Asn126、Asn195 和 Asn213 残基的定点诱变消除了 CD320 中的 N-糖基化，但未阻断其在 HEK293 和 HepG2 细胞细胞表面的细胞内运输和表达。相比之下，用 Ben-gal（GalNAc-α-1-O-Ser/Thr 的结构类似物）处理细胞，抑制 O-糖基化和 CD320 的细胞表面表达，并降低维生素 B12 的摄取。CD320 缺失突变体的分析表明，跨膜结构域附近富含 Ser/Thr 区域的 O-糖基化位点对于细胞表面的 CD320 表达很重要。这些结果揭示了 O-聚糖（而非 N-聚糖）在 CD320 的细胞内运输和细胞表面表达中的重要作用，为调节 CD320 功能和维生素 B12 代谢的细胞机制提供了新的见解。