The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.

中文翻译：

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布氏锥虫 β3 糖基转移酶超家族基因编码介导 N-聚糖和 GPI 锚定修饰的 β1-6 GlcNAc 转移酶。

寄生虫布氏锥虫以血流形式 (BSF) 和原环形式 (PCF) 存在，它们分别在 N 连接聚糖和糖基磷脂酰肌醇 (GPI) 锚上表现出较大的碳水化合物延伸。该寄生虫的糖复合物库表明至少有 38 种糖基转移酶 (GT) 活性，其中 16 种目前尚未表征。在这里，我们探讨了未表征的 GT67 糖基转移酶家族和 β3 糖基转移酶 (β3GT) 超家族基因 TbGT10 的功能。首次通过在 T. brucei 中应用 diCre/loxP 方法创建的 BSF 无效突变体显示出适应性成本，但在体外和体内均可行，并且可以分化为 PCF，这证明 TbGT10 不是必需的。TbGT10 的缺失分别损害了 BSF 和 PCF 寄生虫中 N-聚糖和 GPI 锚定侧链的加工。糖基化缺陷包括 BSF 糖蛋白与凝集素蓖麻素以及单克隆抗体 mAb139 和 mAbCB1 的结合减少。后者结合溶酶体糖蛋白 p67 上存在的碳水化合物表位，我们在此显示该表位由 (-6Galβ1-4GlcNAcβ1-)≥4 个聚 N-乙酰基乳糖胺重复序列组成。从 BSF 野生型和 TbGT10 无效寄生虫分离的链霉蛋白酶消化的糖肽的甲基化连锁分析显示 6-O-取代-和 3,6-二-O-取代-Gal 残基减少。这些数据将 TbGT10 定义为 UDP-GlcNAc:βGal β1-6 GlcNAc 转移酶。TbGT10 在 BSF N-聚糖和 PCF GPI-聚糖阐述中的双重作用是值得注意的，并且 β3GT 超家族基因产物的 β1-6 特异性是前所未有的。锥虫 TbGT10 和高等真核生物 I 分支酶 (EC 2.4.1.150)（分别属于糖基转移酶家族 GT67 和 GT14）在构建 N 连接聚糖方面具有相似的活性，是趋同进化的一个新例子。