Development of Sensitive Probes for Deaminated Neuraminic Acid (KDN) to Search for KDN-Glycoconjugates

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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. We suspected that development of more sensitive assay procedures might demonstrate KDN-glycoconjugates in tissues and cells of many additional animal types. The present investigation has, for its objective, the establishment of chemical and immunochemical probes for the detection of KDN-glycoconjugates.(1) Detection and characterization of oligo/poly(sialic acid) and oligo/poly(KDN) units in glycoconjugates:- We developed new GLC and HPLC procedures for the identification-quantitation of nonulosonates and lower oligomers of NeuAc, NeuGc, and KDN. We examined and compared high-performance anion-exchange chromatographic separation of ^3H-labeled oligo(NeuAc), oligo(NeuGc), and oligo(KDN) alditols by using Mono-Q HR 5/5 resin. We also examined a m … More ethod of selective and quantitative microprecipitation for separation and purification of oligomers and polymers of polysialo- and poly(KDN)-glycoproteins.(2) Generation and use of immunochemical probes for KDN-glycan chains:- The availability of KDN-glycoconjugates prompted us to generate monoclonal antibodies against the KDN-glycan chains found in KDN-ganglioside and KDN-glycoprotein. We first produced a monoclonal antibody IgG3 subclass (mAb.kdn3G) that specifically bound to (KDN)GM3, but not to (NeuAc)GM3. We also generated another monoclonal antibody IgM (mAb.kdn8kdn) by fusion of spleen cells from a BALB/c mouse immunized with KDN-gp and P3U1 mouse myeloma cells. The specificity of these monoclonal antibodies, mAb.kdn3G and mAb.kdn8kdn, was examined and the epitope structures were determined to be KDNalpha2*3Galbeta1* and KDNalpha2*8KDNalpha2* structures, respectively. The potential utility of these monoclonal antibodies is addressed in searching for KDN-glycoconjugates which contain these epitope structures. Less