Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.

中文翻译：

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高尔基体中富含 GlcNAc 的 N-和 O-聚糖的生物合成不需要核苷酸糖转运蛋白 SLC35A3。

由 SLC35 基因家族编码的核苷酸糖转运蛋白将核苷酸糖输送到整个细胞，用于各种糖基转移酶催化的糖基化反应。UDP-GlcNAc 形式的 GlcNAc 和 UDP-Gal 形式的半乳糖分别通过 SLC35A3 和 SLC35A2 转运蛋白输送到高尔基体中。然而，尽管 SLC35A2 的 UDP-Gal 运输活动已被清楚地证明，但 SLC35A3 的 UDP-GlcNAc 传递尚未完全了解。因此，我们分析了一组 CHO、HEK293T 和 HepG2 细胞系，包括 WT 细胞、SLC35A2 敲除、SLC35A3 敲除和双敲除细胞。缺乏 SLC35A2 的细胞在 N-和 O-聚糖合成方面表现出显着变化。然而，在敲除 SLC35A3 的 CHO 细胞中，仅观察到有限的变化；GlcNAc 仍然结合到 N-聚糖中，但复合型 N-聚糖分支受损，尽管 UDP-GlcNAc 向高尔基体囊泡的转运并未减少。在敲除 SLC35A3 的 HEK293T 细胞中，UDP-GlcNAc 运输显着减少但并未完全消除。然而，这些细胞中的 N-聚糖分支并未受损。在 CHO 和 HEK293T 细胞中，SLC35A3 缺乏对 N-聚糖分支的影响在 SLC35A2 不存在的情况下增强。此外，在敲除 SLC35A3 的 HEK293T 和 HepG2 细胞中，GlcNAc 仍被掺入 O-聚糖中。然而，在 HepG2 细胞的情况下，在 WT 和 SLC35A3 敲除细胞之间以及 SLC35A2 敲除和双敲除细胞之间没有观察到 N-聚糖的质的变化。