N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase
N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase
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DOI：
10.1042/bj20020946
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发表时间：
2002-11-15
影响因子：
4.1
通讯作者：
Gieselmann, V
中科院分区：
文献类型：
--
作者：
Eckhardt, M;Fewou, SN;Gieselmann, V
3-O-硫代半乳糖基神经酰胺(硫脂)是髓鞘膜的主要脂质成分，是形成正常髓鞘所必需的。磺胺在高尔基体中由半乳糖神经酰胺硫转移酶(CST；EC 2.8.2.11)合成。鼠和人的CSTs含有两个假定的N-糖基化位点(ASN-66和ASN-312)。在迄今克隆的所有半乳糖3-O-硫代转移酶中，第二个位点是保守的。为了研究N-糖基化的功能相关性，我们分别或联合构建了两个N-糖基化位点缺失的表位标记的CST和可溶性蛋白A-CST融合蛋白。我们的结果表明，当CST在中国仓鼠卵巢(CHO)或COS细胞中表达时，这两个位点都发生了糖基化。此外，转导缺乏两个N-糖基化位点或仅有ASN-312的CST突变体，显著减少硫脂的合成量，而用谷氨酰胺残基取代ASN-66则不显著。相比之下，体外活性降低了约1/4。在ASN-66-->；Gln(N66Q)突变体中检测到50%，在N312Q和N66/312Q中几乎检测不到。此外，在衣霉素存在下表达的可溶性蛋白A-CST几乎没有活性，并在转基因细胞中积累。在缺乏N-乙酰氨基葡萄糖基转移酶的CHO糖基化突变体中表达完全活性的CST表明，N-连接的五氨基酚核心结构的缩合足以形成完全活性的酶。
3-O-Sulphogalactosylceramide (sulphatide) is a major lipid component of myelin membranes, and is required for proper myelin formation. Sulphatide is synthesized in the Golgi apparatus by galactosylceramide sulphotransferase (CST; EC 2.8.2.11). Murine and human CSTs contain two putative N-glycosylation sites (Asn-66 and Asn-312). The second site is conserved among all galactose 3-O-sulphotransferases cloned to date. In order to study the functional relevance of N-glycosylation, we generated epitope-tagged CST and soluble Protein A-CST fusion proteins lacking both N-glycosylation sites, separately or in combination. Our results show that both sites are glycosylated when CST is expressed in Chinese hamster ovary (CHO) or COS cells. Moreover, transfecting CST mutants lacking both N-glycosylation sites, or only Asn-312, reduced significantly the amount of sulphatide synthesized, whereas substituting Asn-66 with a glutamine residue did not. In contrast, activity in vitro was reduced by approx. 50 % in the Asn-66-->Gln (N66Q) mutant, and was almost undetectable in N312Q and N66/312Q transfectants. Furthermore, soluble Protein A-CST expressed in the presence of tunicamycin was almost inactive, and accumulated in transfected cells. Expression of fully active CST in a CHO-glycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrated that condensation of the N-linked pentamannosyl-core structure is sufficient to form a fully active enzyme.