Studies on KDN (=deaminoneuraminic acid) Transferases

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-半乳糖醛酸，KDN）。从那时起，越来越多的含KDN的糖缀合物被报道。在本研究项目中，我们对含聚唾液酸和含KDN的聚糖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。我们从虹鳟鱼精巢中分离纯化了一种新的合成CMP-KDN的酶-CMP-KDN合成酶，并将其用于合成CMP-[^C] KDN。由于CMP-[^C] KDN可用于合成KDN-糖缀合物，因此可以将其作为研究KDN-糖缀合物生物合成的关键底物。<14><14><14>(2)寡聚唾液酸的鉴定及发育性表达 ...更多信息 ：α 2-&gt; 8KDN-转移酶。在虹鳟鱼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转移到多聚唾液酸糖蛋白（PSGP）的α 2-&gt; 8-连接的寡聚/多聚唾液酸残基的非还原末端。在中性pH下，在2至5 mM Mg ^2 +&gt;或Mn ^2 +&gt;的存在下，可刺激KDN-转移酶的活性。其表达以与α 2-&gt; 8-β-唾液酸转移酶相同的方式发育调节。将KDN残基掺入寡聚/聚唾液酸残基中防止了它们的延伸，导致寡聚/聚唾液酸链的"加帽"。这是催化糖蛋白中α 2-&gt; 8-聚唾液酸化终止的糖基转移酶活性的第一个实例。(3)Gal：α 2-&gt; 3KDN-转移酶的鉴定、发育表达和利用。我们最近在虹鳟鱼精细胞表面发现了（KDN）GM3神经节苷脂，这促使我们寻找负责形成KDNalpha2-&gt; 3Galbeta1-&gt;序列的KDN-转移酶。因此，我们在鳟鱼睾丸中鉴定了催化乳糖基神经酰胺+CMP-KDN-&gt;（KDN）GM 3 + CMP反应的KDN-转移酶。我们还在鳟鱼睾丸中鉴定了一些其他的KDN-转移酶活性，并且通过利用这些酶活性在去唾液酸糖缀合物与CMP-KDN的反应中成功地将唾液酸糖缀合物转化为相应的含KDN的糖缀合物。少

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