Protein O-mannosylation is necessary for normal embryonic development in zebrafish
Protein O-mannosylation is necessary for normal embryonic development in zebrafish
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DOI：
10.1093/glycob/cwq069
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发表时间：
2010-09-01
期刊：
影响因子：
4.3
通讯作者：
Tamaru, Yutaka
中科院分区：
文献类型：
--
作者：
Avsar-Ban, Eriko;Ishikawa, Hisayoshi;Tamaru, Yutaka
从早期发育的胚胎中克隆了两个不同的cDNA，分别对应于两个斑马鱼蛋白O-甘露糖基转移酶基因zPOMT 1和zPOMT 2。基因表达分析显示，zPOMT 1和zPOMT 2在胚胎发育早期和所有成人组织中以相似的模式表达。为了研究zPOMT 1和zPOMT 2 mRNA在斑马鱼胚胎发育过程中的分布规律，我们将增强型绿色荧光蛋白（EGFP）mRNA融合到zPOMT基因的3 '端非翻译区。由两种构建体产生的EGFP的分布是相似的。注射zPOMT 1和zPOMT 2的反义寡核苷酸导致几种严重的表型，包括弯曲的身体，水肿的心包和异常的眼睛色素沉着。使用抗糖基化α-肌营养不良聚糖抗体（IIH 6）的免疫组织化学和形态学分析表明，zPOMT 2敲除的表型比zPOMT 1敲除的表型更严重，尽管zPOMT 1和zPOMT 2 morphant中的IIH 6反应性均消失。最后，只有当zPOMT 1和zPOMT 2都在人胚肾293 T细胞中表达时，才检测到高水平的蛋白O-甘露糖基转移酶活性，表明zPOMT 1和zPOMT 2都是完全酶活性所必需的。此外，异源组合zPOMT 1和人POMT 2（hPOMT 2）或hPOMT 1和zPOMT 2在培养的细胞中产生酶活性。这些结果表明，蛋白质O-甘露糖基转移酶机制在斑马鱼和人类是保守的，并建议斑马鱼可能是有用的蛋白质O-甘露糖基化的功能研究。
Two distinct cDNAs corresponding to two zebrafish protein O-mannosyltransferase genes, zPOMT1 and zPOMT2, were cloned from early developmental embryos. Gene expression analysis revealed that zPOMT1 and zPOMT2 were expressed in similar patterns during early embryonic development and in all adult tissues. To study the regulation of zPOMT1 and zPOMT2 mRNA distribution during zebrafish embryogenesis, we injected enhanced green fluorescent protein (EGFP) mRNA fused to the 3'untranslated regions of each zPOMT gene. The distribution of EGFP resulting from the two constructs was similar. Injection of antisense morpholino oligonucleotides of zPOMT1 and zPOMT2 resulted in several severe phenotypes-including bended body, edematous pericaridium and abnormal eye pigmentation. Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6) and morphological analysis revealed that the phenotypes of zPOMT2 knockdown were more severe than those of zPOMT1 knockdown, even though the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants. Finally, only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity. Moreover, either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells. These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation.