PROCESSING OF N LINKED CARBOHYDRATES & ITS EFFECT ON IGM POLYMERIZATION

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The transport of membrane and secreted glycoproteins is governed by "quality control" mechanisms that mediate the retention and degradation of improperly folded proteins in theendoplasmic reticulum (ER). The molecules involved are calretuculin and calnexin, which specifically bind to the non-reducing terminus of the glucosylated high mannose glycan previous to processing. For oligomeric proteins, (IgM pentamers and hexamers), an additional set of rules appears to govern their transport, such that proteins that have not achieved the proper quaternary structure are retained. Sequences within the C-terminus of the ~t, protein control sIgM sorting, retention, and degradation and when a conserved Cys575 is mutated large amounts of the monomer are secreted. This terminal piece has an N-linked glycan at Gln663,'which is conserved in all species examined; when this residue is mutated polymerization is again abrogated. Thus, we areinterested in the detailed structure of this specific glycan and the general effect of CHO processing on polymerization. Initial work has been undertaken to establish the analytical constraints, sensitivity, and overall feasibility of profiling N-linked glycans released from the immunoglobulins (IgA, IgG, IgD). These materials have been available in larger amounts and the glycans anticipated provide a model for subsequent studies with isolated fractions of Igm. Efficiency of release with endoglycosidases (including studies with several detergents), extraction after methylation, and the overall reproducibility of the method. In this preliminary study, we apply methodology to profile intact glycan distributions using electrospray ionization-mass spectrometry (ESI MS), and further probe these structures by collision-induced dissociation (CID). The absence of chromatographic separations and divergent detecting techniques avoids sample disproportionation. All samples have been profiled, only major ions have been analyzed in detail by CID.