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

开发脱氨基神经氨酸 (KDN) 敏感探针来寻找 KDN-糖缀合物

基本信息

批准号：
02558014
负责人：
INOUE Yasuo
金额：
$ 9.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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

1986年，我们首次报道了鱼卵糖蛋白中天然存在的脱氨基神经氨酸(2-酮-3-脱氧-d-甘油-d-半乳糖酮酸，KDN)。从那时起，越来越多的含KdN的糖偶联物被报道。我们怀疑，开发更灵敏的检测程序可能会在许多其他动物类型的组织和细胞中显示KdN-糖结合物。本研究的目的是建立检测KdN-糖结合物的化学和免疫化学探针。(1)糖结合物中寡聚(唾液酸)和寡聚(KdN)单元的检测和表征：-我们建立了新的GLC和HPLC法，用于鉴定和定量NeuAc、NeuGc和KdN的非磺酸盐和较低的寡聚体。用Mono-Q HR 5/5树脂对~3H标记的寡糖醇(NeuAc)、寡糖醇(NeuGc)和寡糖醇(KdN)进行了高效阴离子交换层析分离。我们还检查了一个m…更多的选择性和定量的微沉淀方法用于分离和纯化聚唾液酸糖蛋白和聚(KDN)-糖蛋白的低聚物和聚合物。(2)KDN-糖链免疫化学探针的产生和使用：-KDN-糖偶联物的可用性促使我们产生针对KDN-神经节苷脂和KDN-糖蛋白中的KDN-糖链的单抗。我们首先制备了一种与(KdN)GM3特异结合而不与(NeuAc)GM3结合的单抗IgG3亚类(mAb.kdn3G)。我们还将KDN-gp免疫的BALB/c小鼠的脾细胞与P3U1小鼠骨髓瘤细胞融合，获得了另一种单抗IgM(mAb.kdn8kdn)。检测了mAb.kdn3G和mAb.kdn8kdn的特异性，确定其表位结构分别为KDNalpha2*3Galbeta1*和KDNalpha2*8KDNalpha2*结构。这些单抗在寻找包含这些表位结构的KdN-糖偶联物方面的潜在用途被提出。较少