Temporal analysis of N-acetylglucosamine extension of N-glycans in the middle silk gland of silkworm Bombyx mori.

N-glycosylation of proteins is an important post-translational modification in eukaryotic cells. One of the key modifications in protein N-glycosylation is N-acetylglucosamine (GlcNAc) extension mediated by N-acetylglucosaminyltransferase I (GNTI), which triggers N-glycan maturation from high-mannose-type to hybrid- and complex-type structures in Golgi. However, the temporal contributions of GNTI to GlcNAc extension and the resultant N-glycan structures in insects have not been analyzed. Here, focusing on GlcNAc extension of N-glycan in the silkworm Bombyx mori, we analyzed the temporal N-glycan alterations in the middle silk gland (MSG) and characterized the property of key enzyme for complex-type N-glycan biosynthesis, B. mori GNTI (BmGNTI). N-glycan analysis of N-glycoproteins in the MSG demonstrated that BmGNTI identified and characterized in this study consistently contributed to GlcNAc extension of N-glycans, which led to the accumulation of GlcNAc-extended N-glycans as predominant structures throughout the MSG development. The expression profile of GlcNAc extension-related genes revealed that the enzymes contributing to the hydrolysis of GlcNAc showed stage-specific expressions, thereby resulting in accumulations of the end product N-glycans of the enzyme. These results lead to the speculation that not BmGNTI but rather glycosylhydrolases critically influenced the structural formations and the changes in the ratio of N-glycans with GlcNAc residue(s) in MSG.