Joint Research on Polysialo- and PolyKDN-Glycoconjugates

自1978年S.Inoue首次证明在真核生物中存在α 2,8-链聚唾液酸结构和1986年我们首次发现一种新型唾液酸（KDN）以来，越来越多的聚唾液酸-和聚KDN-糖缀合物被报道。本研究的目的是：(a)在更广泛的动物物种中寻找这些糖基，(b)进一步了解它们的功能意义，以及(c)了解这些糖基的生物合成和降解的基本机制，从而开发参与这些过程的酶的可能用途，如alpha2,8-多涎酰基转移酶和各种类型的kdn转移酶。(1)证明了聚唾液酸结构的多样性：-我们首次发现了α 2,8-linked poly (Neu5Ac), poly (Neu5Gc), poly (Neu5Ac, Neu5Gc), poly （KDN）及其部分乙酰化形式的存在[j](1993) 23675 - 23684]。(2) ^<14>C标记的CMP-KDN在KDN转移酶鉴定中的应用：-我们发现CMP-KDN合成酶在鲑鱼睾丸中具有较强的活性，并对其进行部分纯化以制备CMP- [^<14>C] KDN [j] .中国生物医学工程，2006(1993) 2640 - 2648]。通过使用CMP- [^<14>C] KDN作为供体底物，我们成功地鉴定了一种KDN转移酶，该酶特异性地覆盖了α 2,8链寡聚/聚（Neu5Ac）和寡聚/聚（Neu5Gc）链[Glycoconjugate J.11(1994) 493-499]。(3) kdn -聚糖链免疫化学探针的制备和使用：-我们首先制备了两种不同的单克隆抗体，IgG3亚类（mAb. kdn3g）和IgM （mAb. kdn3g）。kdn8kdn)，其中表位结构分别为KDNalpha2*3Galbeta1*和alpha2*8-linked oligo/poly (KDN)[糖生物学3 (1993)31-36；组织化学101 (1994)333-340；酶学方法（W.J.Lennarz & G.W.Hart主编）230(1994) 460-484]。这些抗体被成功地用作寻找kdn -糖缀合物的敏感探针[糖生物学5 (1995)]；组织化学101(1994)333-340]。(4)聚（Neu5Ac）、聚（Neu5Gc）和聚（KDN）的钙离子结合能力：-我们确定了三种不同聚（Sia）链的Ca^<2+>结合常数，并且观察到这些聚（Sia）链对Ca^<2+>具有较高的亲和力，这一定与聚(Sia) -糖缀合物的生理作用有关[生化33(1994)1202-1208]。(5) kdn - cleaved酶（KDNase）的发现：发现了一种能水解不同类型kdn -酮苷键但不能水解neu5acyl -酮苷键的酶，并对其进行了纯化[j] .中国生物医学工程学报，2009(1994) 21415 - 21419]。这种酶在聚(KDN) -糖缀合物的结构和功能研究中特别有用[Science（1995）提交]。(6)从海胆卵中分离的聚(Sia) -糖蛋白聚（Neu5Gc）中发现了一种新的酮苷间键[*5-O_<glycolyl>- neu5gcalpha2 *]_n [j](1994) 22712 - 22718]。少

Since the first demonstration of the occurrence of alpha2,8-linked polysialic acid structure in eukaryotic source in 1978 by S.Inoue and our first finding of a new type of sialic acid (KDN) in 1986, an increasing number of both polysialo- and polyKDN-glycoconjugates have been reported. The present investigation is, for its objective, (a) to search for these glycotopes in a wider variety of animal species, (b) to obtain further insight into their functional significance, and (c) to understand the basic mechanism for biosynthesis and degradation of such glycotopes, thereby developing the possible use of the enzymes involved in these processes such as alpha2,8-polysialyltransferases and varous types of KDN-transferases.(1) Demonstration of diversity in polysialic acid structures : - We showed for the first time the presence of alpha2,8-linked poly (Neu5Ac), poly (Neu5Gc), poly (Neu5Ac, Neu5Gc), poly (KDN) and their partially acetylated forms [J.Biol.Chem.268 (1993) 23675-23684].(2) Genera … More tion and use of ^<14>C-labeled CMP-KDN in identification of KDN-transferases : - We found the strong activity of CMP-KDN synthetase in trout testis and partially purified it to prepare CMP- [^<14>C] KDN [J.Biol.Chem.268 (1993) 2640-2648]. By use of CMP- [^<14>C] KDN as a donor substrate we were successful of identification of a KDN-transferase which specifically capped the alpha2,8-linked oligo/poly (Neu5Ac) and oligo/poly (Neu5Gc) chains [Glycoconjugate J.11 (1994) 493-499].(3) Generation and use of immunochemical probes for KDN-glycan chains : - We first produced two different monoclonal antibodies, IgG3 subclass (mAb.kdn3G) and IgM (mAb.kdn8kdn), of which epitope structures were KDNalpha2*3Galbeta1* and alpha2*8-linked oligo/poly (KDN), respectively [Glycobiology 3 (1993) 31-36 ; Histochemistry 101 (1994) 333-340 ; Methods in Enzymology (W.J.Lennarz & G.W.Hart, Eds.) 230 (1994) 460-484]. These antibodies were successfully used as the sensitive probes in searching for KDN-glycoconjugates [Glycobiology 5 (1995) in press ; Histochemistry 101 (1994) 333-340].(4) Calcium ion binding capacity of poly (Neu5Ac), poly (Neu5Gc), and poly (KDN) : - We determined Ca^<2+> binding constant for three different poly (Sia) chains and the observed relatively high affinity of these poly (Sia) chains toward Ca^<2+> must be relevant to the physiological role of poly (Sia) -glycoconjugates [Biochemistry 33 (1994) 1202-1208].(5) Discovery of KDN-cleaving enzyme (KDNase) : - An enzyme hydrolyzing specifically different types of KDN-ketosidic linkages but not Neu5Acyl-ketosidic bonds was found and purified almost to homogeneity [J.Biol.Chem.269 (1994) 21415-21419]. This enzyme was particularly useful in both structural and functional studies of poly (KDN) -glycoconjugates [Science (1995) submitted].(6) Discovery of a new class of polysialic acids : - A novel type of interketosidic linkages, [*5-O_<glycolyl>-Neu5Gcalpha2*]_n, in poly (Neu5Gc) was found in poly (Sia) -glycoprotein isolated from the jelly coat of sea urchin eggs [J.Biol.Chem.269 (1994) 22712-22718]. Less