Endo-β-N-acetylglucosaminidases are enzymes that hydrolyze the N,N′-diacetylchitobiose unit of N-glycans. Many endo-β-N-acetylglucosaminidases also exhibit transglycosylation activity, which corresponds to the reverse of the hydrolysis reaction. Because of these activities, some of these enzymes have recently been used as powerful tools for glycan remodeling of glycoproteins. Although many endo-β-N-acetylglucosaminidases have been identified and characterized to date, there are few enzymes that exhibit hydrolysis activity toward multibranched (tetra-antennary or more) complex-type N-glycans on glycoproteins. Therefore, we searched for novel endo-β-N-acetylglucosaminidases that exhibit hydrolysis activity toward multibranched complex-type N-glycans in this study. From database searches, we selected three candidate enzymes from Tannerella species—Endo-Tsp1006, Endo-Tsp1263 and Endo-Tsp1457—and prepared them as recombinant proteins. We analyzed the hydrolysis activity of these enzymes toward N-glycans on glycoproteins and found that Endo-Tsp1006 and Endo-Tsp1263 exhibited hydrolysis activity toward complex-type N-glycans, including multibranched N-glycans, preferentially, whereas Endo-Tsp1457 exhibited hydrolysis activity toward high-mannose-type N-glycans exclusively. We further analyzed substrate specificities of Endo-Tsp1006 and Endo-Tsp1263 using 18 defined glycopeptides as substrates, each having a different N-glycan structure. We found that Endo-Tsp1006 preferred N-glycans with galactose or α2,6-linked sialic acid residues in their nonreducing ends as substrates, whereas Endo-Tsp1263 preferred N-glycans with N-acetylglucosamine residues in their nonreducing ends as substrates.

中文翻译：

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来自 Tannerella 物种的新型内切-β-N-乙酰氨基葡萄糖苷酶水解具有不同特异性的多分支复合型 N-聚糖。

内切-β - N-乙酰氨基葡萄糖苷酶是水解N-聚糖的N,N'-二乙酰壳二糖单元的酶。许多内切-β- N-乙酰氨基葡萄糖苷酶也表现出转糖基化活性，这对应于水解反应的反向。由于这些活性，这些酶中的一些最近已被用作糖蛋白聚糖重塑的强大工具。尽管许多内切β- Ñ -acetylglucosaminidases已被鉴定和表征迄今为止，很少有酶朝向多支链（四触角或更多个）复合型表现出水解活性Ñ上糖蛋白-glycans。因此，我们寻找新的内-β- N-乙酰氨基葡萄糖苷酶在本研究中对多分支复合型N-聚糖表现出水解活性。从数据库搜索中，我们从Tannerella物种中选择了三种候选酶——Endo -Tsp1006、Endo-Tsp1263 和 Endo-Tsp1457——并将它们制备为重组蛋白。我们分析了这些酶对糖蛋白上N-聚糖的水解活性，发现 Endo-Tsp1006 和 Endo-Tsp1263 对复合型N-聚糖（包括多支链N-聚糖）表现出优先水解活性，而 Endo-Tsp1457 则表现出水解活性向高甘露糖型N- 专门的聚糖。我们使用 18 种定义的糖肽作为底物进一步分析了 Endo-Tsp1006 和 Endo-Tsp1263 的底物特异性，每种糖肽都​​具有不同的N-聚糖结构。我们发现，的Endo-Tsp1006优选Ñ在它们的非还原性末端为底物或半乳糖α2,6连接的唾液酸残基-glycans，而内切酶Tsp1263优选Ñ与-glycans Ñ乙酰氨基葡萄糖在其非还原性末端为底物的残基。