Bombyx mori \u03b21,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

Hiroyuki Kajiura,Ryousuke Miyauchi,Ryo Misaki,Takao Ohashi,Kazuhito Fujiyama,Akemi Kakudo

doi:10.1038/s41598-021-84771-z

Hiroyuki Kajiura, Ryousuke Miyauchi + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-84771-z

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Abstract

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcβ1,2-R with β1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of β1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

Highlights

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells
One of the major GalNAcTs contributing to N-glycan biosynthesis are categorized as a Glycosyltransferase Family 7 (GT7) proteins in the Carbohydrate-Active enZYmes database and are mainly distributed in invertebrates, unlike β1,4-GALTs, which are distributed in vertebrates
These findings suggested that α1,6-Fuc, when close to the active site, interfered with the access of the acceptor N-glycan to BmGalNAcT, whereas the bisected GlcNAc residue prevented the access of donor substrates to BmGalNAcT

Summary

Introduction

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan Despite this tri-functional sugar transfer activity, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of β1,2-GlcNAc residue(s). Insect α2,6-STs prefer β-linked N-acetylgalactosamine (GalNAc) residue(s) to galactose (Gal) residue at the non-reducing terminus, and efficiently transfer N-acetylneuraminic acid (NeuAc) to arylglycosides substrates rather than N-glycans[13,14,15] These results suggested that insects might have specific GT(s), i.e., insect β1,4-N-ace tylgalactosaminyltransferase(s) (GalNAcT(s)) that are more suitable for sialylation of N-glycans than β1,4- and β1,3-galactosyltransferase (GALT). Its donor substrate preference, its subcellular localization and contribution to in vivo N-glycan biosynthesis were not extensively addressed

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