Studies on KDN (=deaminoneuraminic acid) Transferases

1986年，我们首次报道了鱼卵糖蛋白中自然存在脱胺神经氨酸（2-酮-3-脱氧-d -甘油-d -半乳糖醛酸，KDN）。从那时起，越来越多的含有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的聚糖单元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。我们已经鉴定、部分纯化和表征了CMP-KDN合成酶，这是一种新的酶，负责从KDN和CTP合成CMP-KDN。该酶从虹鳟鱼睾丸中部分纯化，用于合成CMP-[^<14>C]KDN。CMP-[^<14>C]KDN在KDN-糖缀合物合成中的可用性使我们可以将其作为KDN-糖缀合物生物合成研究的关键底物。(2) α 2-> 8kdn转移酶的鉴定和发育表达。在虹鳟鱼卵巢中发现了一种新的糖基转移酶活性，该酶参与催化KDN从糖核苷酸CMP-KDN转移到多唾液糖蛋白（PSGP）的α 2->8-linked oligo/polysialyl残基的非还原端。在中性pH下，在2 ~ 5mm Mg^<2+>或Mn^<2+>的存在下，kdn转移酶活性被刺激。其表达与α 2-> - 8- plysialtransferase的表达方式相似。将KDN残基掺入寡核苷酸/聚硅基残基中阻止了它们的延伸，导致寡核苷酸/聚硅基链的“封顶”。这是第一个糖基转移酶活性催化糖蛋白中α 2-> - 8多唾液化终止的例子。(3) Gal: alpha2-> 3kdn转移酶的鉴定、发育表达和利用。我们最近在虹鳟鱼精子细胞表面发现了（KDN）GM3神经节苷脂，这促使我们寻找KDN转移酶，该转移酶负责形成KDNalpha2- bbb3galbeta1 ->序列。因此，我们在鳟鱼睾丸中发现了KDN-转移酶，它能催化乳糖神经酰胺+ CMP-KDN->(KDN)GM3 + CMP的反应。我们还在鳟鱼睾丸中发现了一些其他的kdn转移酶活性，并利用这些酶活性将唾液糖缀合物与CMP-KDN反应成功地将唾液糖缀合物转化为相应的含有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