Studies on the biosynthetic mechanism and regulation of the expression of sulfated glycosaminoglycans

硫酸糖胺聚糖包括肝素/肝素硫酸盐和软骨素/皮肤素硫酸盐，与脊椎动物和无脊椎动物的许多病理生理现象有关。在本研究中，我们发现了以下新的功能：1)我们鉴定了编码软骨素6-O-磺基转移酶-1(C6ST-1)的致病基因为CHST3。C6ST-1通过将硫酸盐转移到软骨素GalNAc的C-6位来催化硫酸软骨素合成的修饰步骤。结果表明，CHST3基因突变导致硫酸软骨素链硫化的特异性缺陷，导致软骨发育不良，主要累及脊柱。2)克隆了线虫软骨素聚合因子(CChPF)。RNAi去除cChPF后观察到的蠕虫表型，包括胞质分裂逆转，与线虫软骨蛋白合成酶(CChSy)-RNAi phen…非常相似更多的体型。因此，除了cChSy外，cChPF对于软骨素的生物合成和胚胎细胞的分裂是必不可少的。3)我们发现以E-二糖单位为特征的硫酸软骨素是一种有效的单纯疱疹病毒感染性抑制剂，并提供了gro2C细胞上的病毒结合部位。了解硫酸软骨素-E的抗病毒特性决定因素将有助于开发人类单纯疱疹病毒感染的抑制剂。4)硫酸乙酰肝素(HS)链的形成是由EXT(遗传性多个外骨骼蛋白基因)家族成员编码的糖基转移酶催化的。在果蝇中，已鉴定出三个EXT家族基因，分别为Tout-velu(TTV)、Tout-velu(SOTV)和Tout-velu(Botv)兄弟，它们分别编码人类EXT1、EXT2和EXTL3的同源基因。在这里，我们证明了果蝇中的所有三个ExT成员，TTV，SOTV和BOTV，都是果蝇合成全长HS所必需的。5)所有生物中的所有硫酸盐反应都需要活性硫酸盐，PAPS作为通用供体。我们克隆了线虫的PPS-1基因。RNAi干扰PPS-1基因导致肌肉图案的多向性发育缺陷和上皮细胞形状的改变，糖胺多聚糖的硫酸盐化减少，这表明硫酸盐化对线虫表皮的完整性是必不可少的。较少

Sulfated glycosaminoglycans including heparin/heparan sulfate and chondroitin/dermatan sulfate have been implicated in numerous pathophysiological phenomena of vertebrates and invertebrates. In this study, we found novel functions of sulfated glycosaminoglycans as follows.1)We identified the causal gene for the spondylepiphyseal dysplasia Omani type as CHST3 that encodes chondroitin 6-O-sulfotransferase-1 (C6ST-1). C6ST-1 catalyzes the modifying step of chondroitin sulfate synthesis by transferring sulfate to the C-6 position of the GalNAc of chondroitin. The findings indicate that the mutation in CHST3 causes a specific but generalized defect of chondroitin sulfate chain sulfation resulting in chondrodysplasia with major involvement of the spine.2)We cloned C.elegans chondroitin polymerizing factor (cChPF). The worm phenotypes including the reversion of cytokinesis, observed after the depletion of cChPF by RNAi, were very similar to the C.elegans chondroitin synthase (cChSy)-RNAi phen … More otypes. Thus, cChPF in addition to cChSy is indispensable for the biosynthesis of chondroitin and embryonic cell division in C.elegans.3)We found that chondroitin sulfate characterized by the E-disaccharide unit was a potent inhibitor of herpes simplex virus infectivity and provided the virus binding sites on gro2C cells. Knowledge of the determinants of antiviral properties of chondroitin sulfate-E will help in the development of inhibitors of herpes simplex virus infections in humans.4)The formation of heparan sulfate (HS) chains is catalyzed by glycosyltransferases encoded by EXT (hereditary multiple exostosin gene) family members. In Drosophila, three EXT family genes named tout-velu (ttv), sister of tout-velu (sotv) and brother of tout-velu (botv), which encode homologues of human EXT1, EXT2 and EXTL3, respectively, have been identified. Here, we demonstrated that all three EXT members in Drosophila, TTV, SOTV and BOTV, are required for the biosynthesis of full-length HS in Drosophila.5)All sulfation reactions in all organisms require activated sulfate, PAPS as a universal donor. We cloned a C.elegans PAPS-synthase gene pps-1. Disruption of the pps-1 gene by RNAi caused pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with decrease in glycosaminoglycan sulfation, suggesting that sulfation is essential for integrity of epidermis in C.elegans. Less