Note (General)T antigen (Galβ1-3GalNAcα1-Ser/Thr), which is one of mucin-type O-glycans, is synthesized by Drosophila core 1 β1,3-galactosyltransferase 1 (dC1GalT1). Glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr), in which glucuronic acid is added in a β1,3-linkage to galactose of T antigen, has been identified in Drosophila. However, the physiological function of this glycan structure has not yet been clarified. In this study, to unravel the function of glucuronylated T antigen in Drosophila neuromuscular junctions (NMJs), we analyzed the mutant phenotypes of dC1GalT1 and Drosophila glucuronyltransferase-P (dGlcAT-P) that predominantly synthesizes glucuronylated T antigen.We firstly analyzed the phenotypes of dC1GalT1 mutants. The T antigen expression on the muscle surface and NMJs was decreased in dC1GalT1 mutant larvae. We found that the dC1GalT1 mutants showed the partial loss of basement membrane (BM) component and mislocalization of NMJ boutons at the muscle 6/7 boundary. Ultrastructural observation revealed that those mislocalized boutons connected the muscles 6 and 7. Moreover, dC1GalT1 mutants exhibited reduced number of NMJ branches. In Drosophila, three glucuronyltransferases (dGlcAT-I, dGlcAT-S, and dGlcAT-P) have been reported. By measurement of enzymatic activity and MS analysis of the enzymatic products, we found that dGlcAT-P is predominant glucuronyltransferase that produces glucuronylated T antigen. Then, we generated dGlcAT-P mutants by using CRISPR-Cas9 method. In dGlcAT-P mutant larvae, glucuronylated T antigen expression was reduced on the muscle surface and at NMJs. As is the case with the dC1GalT1 mutants, dGlcAT-P mutants showed the three phenotypes, i.e., the partial loss of BM component, mislocalization of NMJ boutons, reduced number of NMJ branches. Furthermore, ultrastructural analysis of muscle 6/7 boundary revealed that BMs underneath the mislocalized boutons were severely deformed in dGlcAT-P mutants. We found that there was a genetic interaction between dC1GalT1 and dGlcAT-P because double heterozygous mutants of dC1GalT1 and dGlcAT-P exhibited the BM and NMJ phenotypes. Finally, ultrastructural analysis of NMJ boutons showed the shorter length of postsynaptic density (PSD) in both dC1GalT1 and dGlcAT-P mutants.Taken together, these data demonstrated that glucuronylated T antigen synthesized sequentially by dC1GalT1 and dGlcAT-P is required for the normal formation of BMs, precise localization of NMJ boutons, arborization of NMJs, and organization of PSDs.
Collection (particular)国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文
Date Accepted (W3CDTF)2023-10-11T15:41:03+09:00
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