title: Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.
abstract: Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders.
authors.affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.|Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Tsumagari
authors.affiliation: Department of Organ Network and Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan.
authors.forename: Kyoko
authors.identifier: http://orcid.org/0000-0002-7080-582X
authors.initials: K
authors.lastname: Shirakabe
authors.affiliation: Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Ogura
authors.affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
authors.forename: Fuminori
authors.initials: F
authors.lastname: Sato
authors.affiliation: Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Sehara-Fujisawa
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D000818:Animals; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D055252:Cell-Derived Microparticles / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D045726:Metalloproteases / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D040901:Proteomics; D015398:Signal Transduction
chemical_list: C403514:CADM1 protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D007136:Immunoglobulins; D008565:Membrane Proteins; D045726:Metalloproteases; D051722:ADAM Proteins