Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.
Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.
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DOI：
10.1371/journal.pgen.1004349
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发表时间：
2014-05
期刊：
影响因子：
4.5
通讯作者：
Colley NJ
中科院分区：
文献类型：
--
作者：
Rosenbaum EE;Vasiljevic E;Brehm KS;Colley NJ
随着新合成的糖蛋白通过分泌途径移动，天冬酰胺连接的聚糖（N-聚糖）经历广泛的修饰，包括糖残基的顺序去除和添加。这些修饰对于生物合成过程中糖蛋白的正确组装、质量控制和转运至关重要。N-糖基化的重要性通过越来越多的疾病列表来说明，这些疾病是由N-连接聚糖的生物合成和加工缺陷引起的。果蝇感光细胞中的主要视紫红质Rh 1在糖蛋白中是非常独特的，因为在Rh 1的生物合成和成熟过程中N-聚糖似乎被完全去除。然而，Rh 1的大部分去糖基化途径仍然未知。为了阐明Rh 1体内去糖基化的关键步骤，我们表征了四种果蝇糖基水解酶的突变等位基因，即α-甘露糖苷酶-II（α-Man-II）、α-甘露糖苷酶-IIb（α-Man-IIb）、称为融合叶（Fdl）的β-N-乙酰氨基葡萄糖苷酶和己糖胺酶1（Hexo 1）。我们已经证明，这四种酶在Rh 1生物合成过程中寡糖修剪的高度协调的途径中发挥着重要和独特的作用。我们的研究结果表明，α-Man-II和α-Man-IIb不是同工酶，像他们的哺乳动物同行，而是在不同的阶段，在Rh 1成熟的功能。同样重要的是，我们的研究结果表明，Hexo 1在Rh 1成熟过程中的N-聚糖加工中具有生物合成作用。这是出乎意料的，因为在人类中，氨基己糖苷酶通常是参与N-聚糖催化的溶酶体酶，在蛋白质生物合成中没有已知的作用。在这里，我们提出了一个基因解剖的糖蛋白加工在果蝇和揭示的关键步骤，N-聚糖修剪在Rh 1生物合成。两者合计，我们的研究结果提供了根本性的进展，了解复杂的和高度调节的N-糖基化在体内的途径，并揭示了新的见解糖基水解酶的分泌途径中的功能。随着新合成的糖蛋白通过分泌途径移动，天冬酰胺连接的聚糖（N-聚糖）经历广泛的修饰，包括糖残基的顺序去除和添加。这些修饰对于生物合成过程中糖蛋白的正确组装、质量控制和转运至关重要。N-糖基化的重要性通过越来越多的疾病列表来说明，这些疾病是由N-连接聚糖的生物合成和加工缺陷引起的。果蝇眼睛中的主要视紫红质Rh 1在糖蛋白中是非常独特的，因为在Rh 1的生物合成和成熟过程中N-聚糖似乎被完全去除。然而，Rh 1的大部分去糖基化途径仍然未知。为了阐明Rh 1去糖基化的关键步骤，我们进行了体内糖蛋白加工的遗传解剖。我们已经证明，四个糖基水解酶发挥重要的和独特的作用，在一个高度协调的途径，N-聚糖修剪在Rh 1生物合成。我们的研究结果揭示了糖基水解酶在分泌途径中的功能的新见解，并为了解体内N-糖基化提供了根本性的进展。
As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway. As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in the Drosophila (fruit fly) eye, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation, we conducted a genetic dissection of glycoprotein processing in vivo. We have demonstrated that four glycosyl hydrolases play essential and unique roles in a highly coordinated pathway for N-glycan trimming during Rh1 biosynthesis. Our results reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway and provide fundamental advances towards understanding N-glycosylation in vivo.
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