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Submitted on July 6, 2005
Division of Biological Sciences, Hokkaido University, Saaporo 001 0021
Corresponding Author: yshinohara{at}glyco.sci.hokudai.ac.jp
Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of the protein glycosylation analysis is still too low to be applied for the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, we employed here a novel approach for a focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high-mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed focused proteomics that carry high-mannose type glycans following direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high-mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover, the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L, -glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase, cathepsin D) and desmosomes (e.g. desmocollin 1, desmocollin 3, desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, respectively, thus indicating N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high-mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high-mannose glycans.
Revised on September 6, 2005
Accepted on September 16, 2005
High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis
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