Itoh Kazuyoshi, Akimoto Yoshihiro, Kondo Shu, Ichimiya Tomomi, Aoki Kazuhiro, Tiemeyer Michael, Nishihara Shoko
Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
Dev Biol. 2018 Apr 15;436(2):108-124. doi: 10.1016/j.ydbio.2018.02.017. Epub 2018 Feb 27.
T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles.
T抗原（Galβ1-3GalNAcα1-Ser/Thr）是动物体内一种进化保守的粘蛋白型核心1聚糖结构，由核心1β1,3-半乳糖基转移酶1（C1GalT1）合成。先前的研究表明，果蝇C1GalT1（dC1GalT1）产生的T抗原在多种组织中表达，幼虫中dC1GalT1缺失会导致多种缺陷，包括循环血细胞数量减少、淋巴腺中造血干细胞过度分化、中枢神经系统畸形、神经肌肉接头（NMJ）轴突终扣定位错误以及NMJ和肌肉细胞的超微结构异常。尽管在果蝇中已鉴定出葡萄糖醛酸化的T抗原（GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr），但该结构的生理功能尚未阐明。在本研究中，我们首次揭示了葡萄糖醛酸化T抗原的生物学作用。我们的数据表明，在果蝇中，T抗原的葡萄糖醛酸化主要由果蝇β1,3-葡萄糖醛酸基转移酶-P（dGlcAT-P）进行。我们创建了dGlcAT-P基因敲除突变体，发现突变幼虫在肌肉和NMJ上的葡萄糖醛酸化T抗原表达较低。此外，在肌肉6/7边界处观察到NMJ轴突终扣定位错误以及基底膜成分胶原蛋白IV（Col IV）和巢蛋白（Ndg）部分缺失。这两种表型相互关联，且与先前描述的dC1GalT1突变幼虫的表型相同。此外，dGlcAT-P基因敲除突变体在肌肉6/7上的NMJ分支较少。而且，超微结构分析显示，缺乏Col IV和Ndg的基底膜明显变形。我们还发现dGlcAT-P表达缺失会导致NMJ轴突终扣出现超微结构缺陷。最后，我们展示了dGlcAT-P与dC1GalT1之间的遗传相互作用。因此，这些结果表明，由dGlcAT-P合成的葡萄糖醛酸化核心1聚糖是幼虫肌肉上NMJ轴突终扣定位、基底膜形成和NMJ分支形成的关键调节因子。
