|Budget Amount *help
¥6,800,000 (Direct Cost : ¥6,800,000)
Fiscal Year 1993 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1992 : ¥5,100,000 (Direct Cost : ¥5,100,000)
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
: 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