Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in <i>Drosophila melanogaster</i>

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.当新合成的糖蛋白通过分泌通路转运时，其天冬酰胺连接聚糖（N-聚糖，N-glycan）会经历广泛的修饰过程，包括糖残基的依次移除与添加。这类修饰对于糖蛋白在生物合成过程中的正确组装、质量控制与运输至关重要。N-糖基化的重要性可通过日益增多的由N-连接聚糖生物合成与加工缺陷引发的疾病得到佐证。黑腹果蝇（Drosophila melanogaster）感光细胞中的主要视紫红质Rh1在糖蛋白中极具特殊性，其N-聚糖似乎在Rh1的生物合成与成熟阶段被完全移除。然而，目前Rh1的去糖基化通路仍有诸多环节尚未明确。为阐明体内Rh1去糖基化的关键步骤，我们对四种果蝇糖基水解酶的突变等位基因进行了表征，分别为α-甘露糖苷酶II（α-Man-II）、α-甘露糖苷酶IIb（α-Man-IIb）、名为融合叶（Fdl）的β-N-乙酰葡糖胺糖苷酶，以及己糖胺酶1（Hexo1）。我们证实，这四种酶在Rh1生物合成过程中的寡糖修剪高度协同通路中发挥着不可或缺且独一无二的作用。我们的研究结果显示，α-Man-II与α-Man-IIb并非如其哺乳动物同源蛋白那般为同工酶，而是在Rh1成熟的不同阶段分别行使功能。同样具有重要意义的是，我们的结果表明Hexo1在Rh1成熟过程中的N-聚糖加工环节具有生物合成功能。这一发现出人意料，因为在人类中，己糖胺酶通常为溶酶体酶，参与N-聚糖的分解代谢，目前已知其在蛋白质生物合成中并无相关功能。在此，我们呈现了果蝇糖蛋白加工过程的遗传解析，并揭示了Rh1生物合成过程中N-聚糖修剪的关键步骤。综上，我们的研究成果为理解体内复杂且高度调控的N-糖基化通路提供了基础性进展，同时为糖基水解酶在分泌通路中的功能提供了全新的认知。