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.
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