Joint Research on Polysialo- and PolyKDN-Glycoconjugates

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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