Joint Research on Polysialo- and PolyKDN-Glycoconjugates

聚唾液酸和聚 KDN 糖复合物的联合研究

• 批准号: 04044055
• 负责人: INOUE Yasuo
• 金额: $ 12.35万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04044055/
• 关键词: Polysialic Acid; Deaminated Neuraminic Acid; KDN; Poly (KDN); KDN-Transferases; Anti-poly (KDN) Monoclonal Antibody; KDN-Cleaving Enzyme (KDNase); New Type Of Polysialic Acid; ポリシアル酸-Ca^<2+>コンプレックス; 単クローン抗体; ポリシアル酸構造の多様性; ウニ卵ポリシアル酸; 抗KDN残基単クロー

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

自从1978年S.Inoue首次证明在真核来源中存在α-2，8-连接的聚唾液酸结构，以及1986年我们首次发现一种新型的唾液酸(KDN)以来，越来越多的聚唾液酸糖结合物被报道。目前的研究目的是：(A)在更广泛的动物物种中寻找这些糖苷，(B)进一步了解它们的功能意义，以及(C)了解这些糖苷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α2，8-聚唾液酸基转移酶和各种类型的KDN-转移酶。(1)聚唾液酸结构的多样性：-我们首次显示存在α-2，8-连接的聚(Neu5Ac)，聚(Neu5Gc)，聚(Neu5Ac，Neu5Gc)，…及其部分乙酰化形式[J.Biol.Chem.268(1993)23675-23684].(2)属KdN进一步利用~(14&gt；C)标记的CMP-KDN鉴定转移酶[J.Biol.Chem.268(1993)2640-2648]--我们在鲑鱼精巢中发现了具有较强活性的CMP-KDN合成酶，并对其进行了部分纯化。使用CMP-[^&lt；14&gt；C]KdN作为供体底物，我们成功地鉴定了一种KdN-转移酶，它特异性地封闭了α-2，8-连接的寡聚(Neu5Ac)和寡聚(Neu5Gc)链[糖偶联物J.11(1994)493-499]。(3)KdN-糖链的免疫化学探针的制备和使用：-我们首次制备了两种不同的单抗，IgG3亚类(mAb.kdn3G)和IgM(mAb.kdn8kdn)，其表位结构分别为KDNalpha2*3Galbeta1*和Alpha2*8-连接的寡聚(KdN)[糖生物学3(1993)31-36；组织化学101(1994)333-340；酶学方法(W.J.Lennarz和G.W.Hart，编辑)230(1994)460-484]。这些抗体被成功地用作灵敏的探针来寻找KdN-糖结合物[GlycoBiology 5(1995)在PRESS；组织化学101(1994)333-340]。(4)聚(Neu5Ac)、聚(Neu5Gc)和聚(KDn)的钙离子结合能力：-我们测定了三个不同的聚(Sia)链的结合常数，并观察到这些聚(Sia)链对Ca^&lt；2+&Gt；必须与聚(SIA)-糖结合物的生理作用有关[生物化学33(1994)1202-1208]。(5)KdN裂解酶的发现：-发现了一种能特异性地降解不同类型的KdN-酮苷键而不是Neu5酰基-酮苷键的酶，并几乎纯化为均一[J.Biol.Chem.269(1994)21415-21419]。(6)发现了一类新的聚唾液酸：-在聚(Neu5Gc)中发现了一种新型的酮苷间键[*5-O_&lt；乙醇基&gt；-Neu5Gcalpha2*]_n，在从海胆卵的凝胶层中分离的聚(Sia)-糖蛋白中发现了[J.Biol.Chem.269(1994)22712-22718]。较少

项目成果

YU SONG: "Expression of novel KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification" Glycobiology. 5(in press). (1995)

宋宇：“精子发生过程中虹鳟睾丸中新型 KDN-神经节苷脂的表达及其结构鉴定”糖生物学。

KITAJIMA KEN: "Analytical methods for identifying and quantitating deamidated sialic acid and α2→8-linked poly-(oligo)nonulosonate residues in glycoconjugates" Anal.Biochem.205. 244-250 (1992)

KITAJIMA KEN：“鉴定和定量糖缀合物中脱酰胺唾液酸和 α2→8-连接的聚（寡）非糖酸残基的分析方法”Anal.Biochem.205（1992）。

KITAZUME SHINOBU: "Developmental expression of trout egg polysialo-glycoproteins and the prerequisite α2,6-,and a2,8-sialyl and a2,8-polysialyltransferase activities required for their synthesis during oogenesis" J.Biol.Chem.269. 10330-10340 (1994)

KITAZUME SHINOBU：“鳟鱼卵聚唾液酸糖蛋白的发育表达以及卵子发生期间合成所需的先决条件 α2,6-、a2,8-唾液酸和 a2,8-聚唾液酸转移酶活性”J.Biol.Chem.269-。 10340 (1994)

KITAZUME SHINOBU: "Detection, isolation, and characterization of oligo/poly(sialic acid) and oligo/poly(deamino-neuraminic acid) units in glycoconjugates" Analytical Biochemistry. 202. 25-34 (1992)

KITAZUME SHINOBU：“糖复合物中寡聚/聚（唾液酸）和寡聚/聚（脱氨基神经氨酸）单元的检测、分离和表征”分析生物化学。

INOUE YASUO: "Glycobiology of fish egg polysialoglycoproteins (PSGP) and deaminated neuraminic acid-rich glycoproteins (KDN-gp)" in Polysialic Acid (J.Roth,U.Rutishauser,and F.A.Troy II.eds.) Birkhauser Verlag Basel,Switzerland. 171-181 (1993)

INOUE YASUO：“鱼卵多唾液酸糖蛋白 (PSGP) 和富含脱氨基神经氨酸的糖蛋白 (KDN-gp) 的糖生物学”，聚唾液酸（J.Roth、U.Rutishauser 和 F.A.Troy II.eds.）Birkhauser Verlag Basel，瑞士