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