title: Large-Scale Identification of N-Glycan Glycoproteins Carrying Lewis x and Site-Specific N-Glycan Alterations in Fut9 Knockout Mice.
abstract: The Lewis x (Le(x)) structure (Galβ1-4(Fucα1-3)GlcNAc-R) is a carbohydrate epitope comprising the stage-specific embryonic antigen-1 (SSEA-1) and CD15, and it is synthesized by α1,3-fucosyltransferase 9 (Fut9). Fut9 is expressed specifically in the stomach, kidney, brain, and in leukocytes, suggesting a specific function in these tissues. In this study, the N-linked glycan mass spectrometry profile of wild-type mouse kidney glycoproteins revealed the presence of abundant terminal fucoses, which were lost following knockout of the Fut9 gene; the terminal fucose was therefore concluded to be Le(x). These results suggested that Le(x) presence is widespread rather than being limited to specific proteins. We endeavored to comprehensively identify the Le(x) carriers in the mouse kidney. Glycopeptides carrying fucosylated glycans were collected by Aleuria aurantia lectin (AAL) affinity chromatography from kidney homogenates of wild-type and Fut9 knockout mice. The site-specific N-glycomes on the glycopeptides were subsequently analyzed by adopting a new glycoproteomic technology composed of dissociation-independent assignment of glycopeptide signals and accurate mass-based prediction of the N-glycome on the glycopeptides. Our analyses demonstrated that 24/32 glycoproteins contained the Le(x) N-glycan structure in wild-type kidney; of these, Le(x) was lost from 21 in the knockout mice. This is the first report of large-scale identification of Le(x)-carrying glycoproteins from a native sample based on the site-specific glycome analysis.
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.|Department of Biomolecular Function, Doctoral Program in Life System Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba , Tsukuba 305-8572, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Noro
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Togayachi
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Tomioka
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Fujita
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Sukegawa
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Nami
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaji
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba 305-8568, Japan.|Department of Biomolecular Function, Doctoral Program in Life System Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba , Tsukuba 305-8572, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
mesh_terms: D000818:Animals; D005647:Fucosyltransferases / Q000235:genetics*; D006023:Glycoproteins / Q000737:chemistry*; D016256:Lewis X Antigen / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D016256:Lewis X Antigen; D011134:Polysaccharides; D005647:Fucosyltransferases; C518194:fucosyltransferase 9, mouse
keywords: IGOT-LC−MS; Lewis x; glycoproteomics; kidney; knockout mouse; site-specific N-glycome analysis; α1,3-fucosyltransferase 9 (Fut9)