N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Gal (Gb3 and the P1 antigen) and Galα1→4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N₁₂₁ and N₂₀₃. Intriguingly, we found that while the N-glycan at N₂₀₃ is essential for activity and correct subcellular localization, the N-glycan at N₁₂₁ is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N₁₂₁ glycan correctly localized in the Golgi, whereas a glycoform without the N₂₀₃ site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants resulted from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

中文翻译：

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错过了最佳点：人类 Gb3/CD77 合酶上的两个 N-聚糖之一是可消耗的

N-糖基化是一种普遍存在的翻译后修饰，可能影响蛋白质的折叠、亚细胞定位、分泌、溶解度和寡聚化。在这项研究中，我们检查了 N-聚糖对人 Gb3/CD77 合酶活性的影响，该酶催化具有末端 Galα1→4Gal（Gb3 和 P1 抗原）和 Galα1→4GalNAc 二糖（NOR 抗原）的鞘糖脂的合成. _{人 Gb3/CD77 合酶在 N 121}和 N ₂₀₃位置包含两个占据的 N-糖基化位点。有趣的是，我们发现虽然 N ₂₀₃上的 N-聚糖对于活性和正确的亚细胞定位至关重要，但 N _{121上的 N-聚糖}是可有可无的，它的缺失并没有降低，但令人惊讶的是，它甚至增加了酶的活性。完全 N-糖基化的人 Gb3/CD77 合酶及其糖型缺少 N ₁₂₁聚糖正确定位于高尔基体，而没有 N _203的糖型位点部分错误定位在内质网中。缺少两个 N-聚糖的双突变蛋白是无活性的并在内质网中积累。我们的研究结果表明，人类 Gb3/CD77 合酶糖变体的比活性降低是由于它们不正确的亚细胞定位，并且在较小程度上是酶溶解度的降低。总之，我们的研究结果表明，人类 Gb3/CD77 合酶的两个 N-聚糖对其特性具有相反的影响，揭示了 N-糖基化的双重性质和潜在的控制蛋白质生物活性的新调节机制。