Protein N-glycosylation is sex-related in Nilaparvata lugens adults

Nilaparvata lugens, or the brown planthopper, is one of the most notorious pest insects of cultured rice, and a model for hemimetabolous development. Recently, the N-glycome of N. lugens was explored throughout post-embryonic development and reproductive stages, which revealed differential protein N-glycosylation events between adult sexes. Identifying the proteins carrying these differential carbohydrate structures would point to the functionality of sex-dependent N-glycosylation. Furthermore, potential effects of the adult wing type on protein N-glycosylation are of interest. Here, a comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes and wing forms at the glycopeptide level. N-glycopeptide enrichment using the N-glyco-FASP method with the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans led to the identification of over 1,300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes, while almost no differences were observed between wing types. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosites were shown to be derived from proteins with a putative role in insect reproduction. Transcript expression experiments with complete insects and insect tissues confirmed the sex-related N-glycosylation of proteins, and expression of glycoproteins in reproductive tissues. In conclusion, this study provides original data on N-glycosylation sites of N. lugens adults, providing novel insights into planthopper’s biology and revealing that protein N-glycosylation is sex-related in this insect.

褐飞虱（Nilaparvata lugens，俗称brown planthopper）是栽培水稻最具危害性的害虫之一，同时也是半变态发育（hemimetabolous development）研究的经典模式生物。近期，科研团队对褐飞虱整个胚后发育及生殖阶段的N-糖组（N-glycome）进行了系统性探究，揭示了成虫雌雄个体间存在蛋白质N-糖基化（N-glycosylation）水平的显著差异。鉴定携带这些差异碳水化合物结构的蛋白质，将为明确性别依赖型N-糖基化的生物学功能提供关键线索。此外，成虫翅型对蛋白质N-糖基化的潜在调控效应也亟待解析。

本研究针对褐飞虱成虫阶段的N-糖基化位点开展了全面调查，实现了在糖肽（glycopeptide）水平上对不同性别及翅型个体的定性与定量对比。研究采用N-糖基化过滤辅助样品制备法（N-glyco-FASP），分别结合可结合高甘露糖/寡甘露糖的伴刀豆球蛋白A（Concanavalin A），以及识别复合型N-糖链的立枯丝核菌凝集素（Rhizoctonia solani agglutinin）进行N-糖肽富集，最终从600余种糖蛋白（glycoprotein）中鉴定出超过1300个N-糖基化位点。

对上述N-糖肽的比较分析显示，雌雄个体间的蛋白质N-糖基化模式存在显著差异，而不同翅型间几乎未观察到明显差异。本研究鉴定出了雄性和雌性特异性的N-糖基化位点，其中部分性别特异性糖基化位点所在的蛋白质被推测在昆虫生殖过程中发挥重要功能。通过对完整昆虫及昆虫组织开展的转录表达（transcript expression）实验，研究团队验证了蛋白质的性别相关N-糖基化现象，以及糖蛋白在生殖组织中的特异性表达。

综上，本研究提供了褐飞虱成虫N-糖基化位点的原始数据，为解析飞虱的生物学特性提供了全新视角，并揭示了该昆虫体内蛋白质N-糖基化与性别密切相关的现象。