ANTIBODY REAGENTS FOR DETECTING PROTEINS WITH O-MANNOSE MODIFICATIONS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 存在大量哺乳动物O-甘露糖基化糖蛋白的强有力证据，特别是从释放聚糖的质谱分析推断的脑中。 然而，除了肌营养不良聚糖外，这些糖蛋白仍然是神秘的。 我们建议通过开发可以检测这种修饰的抗体来鉴定该家族的其他成员以及寻找这些和<$-肌营养不良蛋白聚糖的加工中间体。 这样的抗体然后将用于检测来自POMGnT 1敲除小鼠的脑的O-Man糖蛋白，因为该酶的不存在将阻止延伸超过初始Man残基，呈现用于免疫沉淀或相关抗体检测的感兴趣的表位。 这些抗体将与M.G.芬兰人在斯克里普斯研究所使用那里开发的技术。 这利用了豇豆花叶病毒的衣壳，其中表面赖氨酸已经用炔官能团修饰作为呈递抗原的支架。 用叠氮化物官能团制备抗原，因此可以使用“点击化学”方法将其缀合至衣壳。 O-Man糖肽抗原将在CCRC制备，用于连接到衣壳，然后注射到鸡中用于产生IgY抗体，以评估该方法的有效性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There is strong evidence for the presence of a significant number of mammalian O-mannosylated glycoproteins, particularly in the brain as deduced from mass spectral analysis of released glycans. However, with the exception of ¿-dystroglycan these glycoproteins have remained cryptic. We propose to identify additional members of this family as well as search for processing intermediates of these and of ¿-dystroglycan by developing antibodies that can detect this modification. Such antibodies would then be used to detect O-Man glycoproteins from the brains of POMGnT1 knock-out mice since the absence of this enzyme would prevent extension beyond the initial Man residue, presenting the epitope of interest for immune precipitation or related antibody detection. The antibodies will be developed in collaboration with the laboratory of M.G. Finn at The Scripps Research Institute using a technology developed there. This exploits the capsid of the cow pea mosaic virus where surface lysines have been modified with alkyne functional groups as a scaffold for presentation of an antigen. The antigen is prepared with an azide functional group so it can be conjugated to the capsid using the "click chemistry" methodology. O-Man glycopeptide antigens will be prepared at the CCRC for linking to the capsid and then injected into chickens for generating IgY antibodies to evaluate the effectiveness of the approach.