昆虫糖基水解酶20家族β-N-乙酰己糖胺酶研究进展

doi:10.3864/j.issn.0578-1752.2014.07.007

摘要/Abstract

摘要： 糖基水解酶20家族β-N-乙酰己糖胺酶是N-乙酰己糖胺代谢过程中一个重要的酶，其可将位于非还原端以β糖苷键连接的N-乙酰己糖胺从寡糖及糖复合物中水解下来。昆虫糖基水解酶20家族β-N-乙酰己糖胺酶参与昆虫多个生理过程，包括表皮几丁质水解、蛋白质N糖基化修饰、糖复合物水解及精卵识别，因此可能成为生态农药设计的潜在靶标，受到国内外学者的普遍关注。根据昆虫20家族β-N-乙酰己糖胺酶的进化关系和生理功能，可以将其分为4个亚家族，分别为Hex1、Hex2、Hex3和Hex4。Hex1主要在昆虫蜕皮时期的表皮中表达，RNA干扰该基因能够导致昆虫在发育过程中不能正常蜕皮而死亡。通过酶学性质研究发现，Hex1能够专一性水解以β 1-4糖苷键连接的几丁质寡糖底物，而不能水解以其他形式连接的底物，是专一性的参与几丁质水解的β-N-乙酰己糖胺酶。目前获得的唯一一个昆虫β-N-乙酰己糖胺酶的晶体结构来自于亚洲玉米螟的OfHex1，其参与底物结合的“+1”位点存在一个特殊的三明治结构，使得Hex1对几丁质寡糖具有高度的选择性和水解活性。Hex2在双翅目昆虫中并不存在，与人类的β-N-乙酰己糖胺酶具有高度的相似性，RNA干扰该基因的表达能够导致幼虫化蛹时异常，成虫翅、附肢、触角等异常。通过酶学性质研究表明，Hex2能够水解释放几丁质寡糖，糖复合物等底物中的β-N-乙酰己糖胺，具有广泛的底物谱。Hex2的功能可能与人类Hex酶的功能类似，参与糖复合物的水解。Hex3是目前研究较少的一类β-N-乙酰己糖胺酶，RNA干扰该基因的表达能够导致幼虫蜕皮过程异常。Hex3存在于昆虫蜕皮液中，与Hex1存在相互作用，可能形成二聚体。通过酶学性质研究表明，Hex3能够水解几丁质寡糖底物，但活性较弱，说明其可能参与几丁质的水解。此外，Hex3、Hex1和Hex4被发现存在于双翅目昆虫精子的表面，可能参与精卵识别过程。Hex4又被称为FDL，是因为该基因的缺失能够导致果蝇左右大脑发生融合（fused lobe）。它是另外一种具有严格底物选择性的β-N-乙酰己糖胺酶，能够专一性地水解糖复合物GnGn中α1-3分支上以β 1-2糖苷键连接的GlcNAc β 1-2Man，通过细胞定位研究表明Hex4主要存在于高尔基体当中。以上表明Hex4的主要功能是参与细胞内的糖基化修饰过程。尽管目前对于昆虫β-N-乙酰己糖胺酶的研究取得了很大的进展。对Hex1的功能、酶学性质和晶体结构研究得较为透彻，对于设计环境友好的杀虫剂具有较强的指导意义。但其他3种β-N-乙酰己糖胺酶在生理条件下如何发挥功能，以及4种β-N-乙酰己糖胺酶活性差异的结构基础并不是十分清楚。作者从进化关系、晶体结构、酶学性质和生理功能等方面对昆虫糖基水解酶20家族β-N-乙酰己糖胺酶的研究进展进行综述。

关键词: &beta, -N-乙酰己糖胺酶 , 几丁质 , 糖基化修饰 , 晶体结构 , 农药靶标

Abstract: β-N-acetylhexosaminidase (Hex), a family 20 glycosyl hydrolyases (GH20), is an essential enzyme for the metabolism of N-acetylhexosamine by catalyzing the removal of β-linked N-acetylhexosamine from the non-reducing ends of glycans, glycoproteins and glycolipids. Insect GH20 β-N-acetylhexosaminidase involves in multiple physiological processes, including chitin degradation, protein N-glycan modification, glycoconjugates degradation and sperm-egg recognition. Because of its important functions, β-N-acetylhexosaminidase may serve as a potential target for designing eco-friendly pesticides. The insect family 20 β-N-acetylhexosaminidase could be grouped into 4 groups according to their phylogenetic relationship and physiological functions, that are Hex1, Hex2, Hex3 and Hex4. Hex1 is mainly expressed in epidermis during insect molting. The RNAi of Hex1 would cause lethal phenotype during insect molting. The old cuticle of the dsHex1 injected larvae could not be shed off. The study of the enzymatic properties of Hex1 demonstrates that it could efficiently hydrolyze β-1,4 linked chitin oligosaccharide with very high specificity but could not act on β-1,3 or β-1,2 linked substrates. It is a special enzyme for chitin degradation. So far, the only crystal structure of insect β-N-acetylhexosaminidase is OfHex1 from Ostrinia furnacalis. The structure at subsite “+1” in OfHex1 explains the high efficiency and specificity of OfHex1 towards chitin oligosaccharides. Hex2 could not be found in insects belong to diptera. It shows highly similarity towards human β-N-acetylhexosaminidase than the other insect β-N-acetylhexosaminidases. The RNAi of Hex2 will cause abnormalities of larval abdomen, pupa and adult appendages. The enzymatic properties of Hex2 show that it is an enzyme with broad substrate-spectrum by hydrolyzing β-N-acetylhexosamine from chitin oligosaccharides, N-glycan and glycolipids. Hex2 may be involved in the degradation of glycoconjugates like the function of human β-N-acetylhexosaminidase. Hex3 is not well studied so far. The RNAi of Hex3 could cause abnormalities during insect molting. It is proved to be in molting fluid and interact with Hex1. The enzymatic activity analysis indicates that OfHex3 is able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Besides, Hex3, together with Hex1 and Hex4, are also found to be in the plasma membrane of spermatozoa in dipteran insects, suggesting that it may be involved in sperm-egg recognition. Hex4 is also called FDL because the mutant of this gene will cause fused lobes phenotype of the mushroom body in Drosophila melanogaster. It is another β-N-acetylhexosaminidase with strict substrate specificity. It could exclusively hydrolyze the terminal β-1, 2-GlcNAc residue from the α-1,3 branch instead of the α-1,6 branch of the substrate GnGn. The subcellular localization of Hex4 indicates that it locates in the Golgi apparatus. All these suggest that Hex4 is a β-N-acetylhexosaminidase involved in the modification of N-glycans. So far, although big progress has been achieved on insect β-N-acetylhexosaminidase, only Hex1 has been well studied, including its physiological functions, enzymatic properties and crystal structures. It is proved to be a potential target for designing eco-friendly pesticides. How the other three β-N-acetylhexosaminidases function during insect development and the structure basis of the different enzymatic properties among the four groups of β-N-acetylhexosaminidases are still uncover. This review focuses on the recent progresses on phylogenetic relationship, crystal structure, enzymatic properties and physiological significance of insect β-N-acetylhexosaminidases.

Key words: β-N-acetylhexosaminidase , chitin , N-glycan modification , crystal structure , pesticide target

屈明博, 刘田, 陈磊, 陈琦, 杨青. 昆虫糖基水解酶20家族β-N-乙酰己糖胺酶研究进展[J]. 中国农业科学, 2014, 47(7): 1303-1312.

QU Ming-Bo, LIU Tian, CHEN Lei, CHEN Qi, YANG Qing. Research Progresses in Insect Glycosyl Hydrolyase Family 20 β-N-acetylhexosamindase[J]. Scientia Agricultura Sinica, 2014, 47(7): 1303-1312.

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