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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >PNAS Plus: Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway
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PNAS Plus: Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

机译：PNAS Plus：涉及哺乳动物分泌途径中Asn关联的聚糖成熟的GH47α-甘露糖苷酶对底物的识别和催化

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摘要

Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-mannosidases, including endoplasmic reticulum (ER) α-mannosidase I (ERManI) and Golgi α-mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme–substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.
机译：真核生物分泌途径中Asn连接的寡糖的成熟需要修整新生的聚糖链，以去除所有葡萄糖和一些甘露糖残基，然后再延伸到细胞表面的复杂类型结构和分泌的糖蛋白上。多个糖苷水解酶家族47（GH47）α-甘露糖苷酶，包括内质网（ER）α-甘露糖苷酶I（ERManI）和高尔基α-甘露糖苷酶IA（GMIA），负责切割末端α1,2-连接的甘露糖残基以产生ER糖蛋白质量控制和聚糖成熟所必需的独特修饰的低聚甘露糖异构体。 ERManI和GMIA具有相似的催化结构域结构，但每种酶均从三支寡甘露糖聚糖底物中切割出不同的残基。通过用镧（La 3 + ）离子代替必需的酶结合的Ca 2 + 离子，探索了ERManI和GMIA裂解特定分支的结构基础。这种离子交换导致酶失活，同时保留了高亲和力的底物相互作用。 La 3 + 结合酶与Man9GlcNAc2底物类似物的共结晶揭示了酶-底物复合物，其聚糖分支插入不同的酶活性位点的方式不同。两种酶都具有跨整个聚糖结构延伸的聚糖相互作用，但是每种酶都具有不同的聚糖分支，并使用不同的聚糖相互作用集。聚糖裂解的其他诱变和时程研究探讨了酶特异性的结构基础。结果提供了对酶催化机制的见解，并揭示了连续的聚糖裂解事件的结构快照。数据还表明，对聚糖底物的完全空间定位决定了控制哺乳动物细胞向复杂类型结构转化的甘露糖修饰反应的效率。

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