title: High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.
abstract: Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for 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 high mannose type glycoform-focused proteomics by 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 and gamma-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e.g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that 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.
authors.affiliation: Division of Biological Sciences, Graduate School of Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Uematsu
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Furukawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Kisaburo
authors.initials: K
authors.lastname: Deguchi
authors.forename: Kenji
authors.initials: K
authors.lastname: Monde
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D004817:Epidermis / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D051379:Mice; D008812:Mice, Hairless; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012867:Skin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D020543:Proteome