Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis
Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis
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DOI：
10.1074/jbc.m611107200
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发表时间：
2007-03-16
影响因子：
4.8
通讯作者：
Sugahara, Kazuyuki
中科院分区：
文献类型：
--
作者：
Kitagawa, Hiroshi;Izumikawa, Tomomi;Sugahara, Kazuyuki
5个克隆的人类EXT家族基因（EXT1、EXT2、EXTL1、EXTL2和EXTL3）编码的蛋白是生物合成硫酸肝素所需的糖基转移酶。在秀丽隐杆线虫基因组中，仅鉴定出两个与哺乳动物EXT基因同源的基因rib-1和rib-2。虽然rib-2编码的n -乙酰氨基葡萄糖转移酶参与启动硫酸肝素的生物合成和延伸，但rib-1编码的蛋白质参与硫酸肝素的生物合成尚不清楚。在这里，我们报道了RIB-1在生物合成和胚胎形态发生中是必不可少的。尽管RIB-1的个体糖基转移酶活性很小，但当RIB-1与RIB-2共表达时，在体外证明了硫酸肝素链的聚合。此外，RIB-1和RIB-2通过拉下实验证明了相互作用。为了研究RIB-1在体内的功能，我们通过缺失诱变来减少RIB-1的表达。无突变体蠕虫的硫酸肝素合成降低，胚胎致死率降低。值得注意的是，零突变体胚胎在原肠胚裂形成期或更晚出现异常，主要在1倍期停止。几乎100%的胚胎在L1期之前死亡，尽管一些神经元和肌肉细胞的分化正常进行。在rib-2零突变胚胎中也观察到类似的表型。因此，在秀丽隐杆线虫体内，除了RIB-2外，RIB-1也是硫酸肝素生物合成中不可缺少的，两者在体内协同合成硫酸肝素。这些发现还表明，硫酸肝素对胚胎细胞原肠胚形成后的形态发生运动是必不可少的，对保证分化组织和器官的正常空间组织是必不可少的。
The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pultdown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs.