Abstract：
Endo-β-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.
摘要：
内切-β-N-乙酰葡糖胺糖苷酶(ENGases)是水解N-连接的聚糖的酶。许多ENGases已被表征，但很少有人发现对多分支复合型N-聚糖具有水解活性。在这项研究中,根据数据库搜索和系统发育分析，从肠孢子菌中鉴定出三种候选ENGases。域搜索确定了所有三个候选中的NxE基序，表明它们是糖基水解酶家族85（GH85）的成员。三个候选人恩格斯，命名为Endo-BIN1，Endo-BIN2和Endo-BIN3，在大肠杆菌细胞中表达，通过高效液相色谱分析和SDS-PAGE分析测量它们对N-聚糖和糖蛋白的水解活性。所有的ENGases都显示出对糖蛋白的水解活性，但只有Endo-BIN2和Endo-BIN3对吡啶基胺化的N-聚糖表现出水解活性。Endo-BIN1、Endo-BIN2和End-BIN3的最适pH分别为pH6.5、4.0和7.0。我们测量了Endo-BIN2和Endo-BIN3对吡啶胺化N-聚糖的底物特异性，发现两种Endo-BIN酶表现出相似的底物特异性，优选在非还原末端具有半乳糖或α2,6-连接的唾液酸残基的双触角复合型N-聚糖。Endo-BIN2和Endo-BIN3也能够水解多分支复合型N-聚糖。SDS-PAGE分析显示，所有Endo-BIN酶都能够从糖蛋白如利妥昔单抗中释放复合型N-聚糖，转铁蛋白,还有Fetuin.我们期望肠型芽孢杆菌具有促进利用来自宿主细胞的复合型N-聚糖的ENGase。这些发现将在糖蛋白的N-聚糖重塑和药物开发中具有应用。
