γ谷氨酰环化转移酶对斑马鱼早期发育的影响

摘要

g谷氨酰环化转移酶(γ-Glutamyl cyclotransferase, GGCT)是谷胱甘肽循环过程中催化 L-γ-Glutamyl-L-amino acid 分解为L-amino acid和5-oxoprodline的关键酶,在防止细胞氧化性损伤中发挥重要作用。研究以斑马鱼为模型,利用吗啡啉(Morpholino, MO)敲降 GGCT,发现早期胚胎发育出现脑部发育缓慢、尾短弯曲和环心室水肿等异常表型,原位杂交检测发现脑发育相关标记基因shha、wnt8b与fgf8的表达发生改变。体外转录ggct-mRNA与GGCT-MO共注射,畸形率降低,部分脑部标记基因表达回复正常。此外,使用化学阻断剂阻断GGCT上游的g谷氨酰转肽酶(γ-Glutamyl transpeptidase, GGT)和下游的5羟脯氨酸酶,斑马鱼胚胎产生发育停滞和水肿表型。敲降GGCT和阻断GGT都降低了胚胎的谷胱甘肽(Glutathione, GSH)的含量,研究表明g谷氨酰环化转移酶可能通过谷胱甘肽循环在斑马鱼早期胚胎发育过程发挥重要作用。%γ-Glutamyl cyclotransferase (GGCT) is one of the key enzymes that contribute to the γ-glutamyl cycle and regulate the glutathione metabolism. Disturbance in the glutathione metabolism is usually associated with damages in central nervous system. Here we explored the role of GGCT in the early development of zebrafish. First we used Mor-pholino (MO) to reduce the expression of GGCT at early developmental stages, and we found that compared to the wild type the treated embryos showed retarded brain development, shorter body sizes and cardiac ventricle dropsy. The re-sults of whole mount in situ hybridization showed that the expression patterns of the brain development marker genes (shh, wnt8b, fgf8) were also changed. These abnormal phenotypes could be partially rescued by the co-injection of ggct-mRNA with GGCT-MO. Next we applied chemical inhibitors to antagonize other two important enzymes inγ-glutamyl cycle, γ-Glutamyl transpeptidase and 5 hydroxyproline enzyme. The zebrafish embryos injected with the inhibitors showed similar abnormalities that mimicked the GGCT-MO treatment. Finally we measured the concentration of GSH/GSSG in these embryos and observed a significant decrease in GSH concentration when the metabolic cycle was blocked. These results indicated that GGCT might play an important role in the early development of zebrafish through its function in theγ-glutamyl cycle.