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Studies on KDN (=deaminoneuraminic acid) Transferases

KDN（=脱氨基神经氨酸）转移酶的研究

基本信息

批准号：
04453160
负责人：
INOUE Yasuo
金额：
$ 4.35万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

In 1986 we first reported the natural occurrence of deaminated neuraminic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid, KDN) in fish egg glycoprotein. Since then, an increasing number of KDN-containing glycoconjugates have been reported. In this research project, we have carried out some pioneering studies on biosynthesis and developmental expression of polysialic acid- and KDN-containing glycan units.(1) Identification and partial purification of CMP-KDN synthetase. We have identified, partially purified, and characterized CMP-KDN synthetase, a novel enzyme responsible for synthesis of CMP-KDN from KDN and CTP.The enzyme was partially purified from the testis of rainbow trout, and used to synthesize CMP-[^<14>C]KDN.Availability of a ^<14>C-labeled donor molecule, CMP-[^<14>C]KDN, in the synthesis of KDN-glycoconjugates allowed us to use it as a key substrate for biosynthetic studies of KDN-glycoconjugates.(2) Identification and developmental expression of oligo/polysialic acid … More : alpha2->8KDN-transferase. A new glycosyltransferase activity, which is involved in catalysis of the transfer of KDN from the sugar nucleotide CMP-KDN to nonreducing termini of alpha2->8-linked oligo/polysialyl residues of polysialoglycoprotein (PSGP), was found in the ovary of rainbow trout. The KDN-transferase activity was stimulated at neutral pH, in the presence of 2 to 5 mM of Mg^<2+> or Mn^<2+>. Its expression was developmentally regulated in a parallel fashion as found for alpha2->8-plysialyltransferase. Incorporation of the KDN residues into the oligo/polysialyl residues prevented their elongation, resulting in the "capping" of the oligo/polysialyl chains. This is the first example of glycosyltransferase activity that catalyzed the termination of alpha2->8-polysialylation in glycoproteins.(3) Identification, developmental expression, and utilization of Gal : alpha2->3KDN-transferase. Our recent finding of (KDN)GM3 ganglioside on the sperm cell surface of rainbow trout prompted us to search for KDN-transferase responsible for the formation of KDNalpha2->3Galbeta1->sequence. Consequently, we identified the KDN-transferase in trout testis, which catalyzes the reaction of lactosyl ceramide + CMP-KDN->(KDN)GM3 + CMP.We have also identified a few other KDN-transferase activities in trout testis and succeeded in converting sialo-glycoconjugates into the corresponding KDN-containing glycoconjugates by use of these enzyme activities in the reaction of asialo-glycoconjugates with CMP-KDN. Less

1986年，我们首次报道了鱼卵糖蛋白中天然存在的脱氨基神经氨酸(2-酮-3-脱氧-D-甘油-D-半乳糖酮酸，KDN)。从那时起，越来越多的含KdN的糖偶联物被报道。在本研究项目中，我们对含多聚唾液酸和含KDN的糖链单位的生物合成和发育表达进行了一些开创性的研究。(1)CMP-KDN合成酶的鉴定和部分纯化。我们鉴定、部分纯化和鉴定了一种新的负责从KDN和CTP合成CMP-KDN的酶。该酶是从虹鱼的睾丸中部分纯化的，并用于合成CMP-[^&lt；14&gt；C]KDN。(2)寡聚唾液酸…的鉴定与表达更多信息：Alpha2-&gt；8KDN-转移酶。在虹鳟鱼的卵巢中发现了一种新的糖基转移酶活性，它参与催化KDN从糖核苷酸cMP-KDN转移到聚唾液酸糖蛋白(PSGP)的非还原末端。在中性pH条件下，加入2~5 mM的镁离子或锰离子均可刺激KDN-转移酶的活性。它的表达与α2-和gt；8-多唾液酸解转移酶的发育调节方式是平行的。将KDN残基加入到寡聚唾液酸基残基中阻止了它们的伸长，导致了寡聚唾液酸链的“封顶”。这是第一个糖基转移酶活性的例子，它催化糖蛋白中α2-和gt；8-多唾液酸化的终止。(3)半乳糖：α2-和gt；3kdN-转移酶的鉴定、表达和利用。我们最近在虹鱼精子细胞表面发现了(KDN)GM3神经节苷脂，这促使我们寻找与KDNalpha2-&gt；3Galbeta1-&gt；序列形成有关的KdN-转移酶。因此，我们在鲑鱼睾丸中鉴定了催化乳糖基神经酰胺+cMP-kdN-&gt；(KdN)GM3+CMP反应的KdN-转移酶。我们还在鲑鱼睾丸中鉴定了其他几种kdN-转移酶活性，并成功地利用这些酶的活性将唾液酸糖结合物转化为相应的含kdN的糖结合物。较少