β1,4-galactosyltransferase 4 (B4GalT4) is one of seven B4GalTs that belong to CAZy glycosyltransferase family 7 and transfer galactose to growing sugar moieties of proteins, glycolipids, glycosaminoglycans as well as single sugar for lactose synthesis. Herein, we identify two asparagine-linked glycosylation sites in B4GalT4. We found that mutation of one site (Asn220) had greater impact on enzymatic activity while another (Asn335) on Golgi localization and presence of N-glycans at both sites is required for production of stable and enzymatically active protein and its secretion. Additionally, we confirm B4GalT4 involvement in synthesis of keratan sulfate (KS) by generating A375 B4GalT4 knock-out cell lines that show drastic decrease in the amount of KS proteoglycans and no significant structural changes in N- and O-glycans. We show that KS decrease in A375 cells deficient in B4GalT4 activity can be rescued by overproduction of either partially or fully glycosylated B4GalT4 but not with N-glycan-depleted B4GalT4 version.

β1,4-半乳糖基转移酶 4 (B4GalT4) 是属于 CAZy 糖基转移酶家族 7 的七个 B4GalT 之一，并将半乳糖转移到蛋白质、糖脂、糖胺聚糖的生长糖部分以及用于乳糖合成的单糖。在此，我们确定了 B4GalT4 中两个天冬酰胺连接的糖基化位点。我们发现一个位点 (Asn220) 的突变对酶活性有更大的影响，而另一个位点 (Asn335) 对高尔基体的定位和 N-聚糖在两个位点的存在是稳定和酶活性蛋白的产生及其分泌所必需的。此外，我们通过生成 A375 B4GalT4 敲除细胞系来确认 B4GalT4 参与硫酸角质素 (KS) 的合成，这些细胞系显示 KS 蛋白聚糖的数量急剧减少，并且 N-和 O-聚糖的结构没有显着变化。