Large-Scale Identification of N-Glycan Glycoproteins Carrying Lewis x and Site-Specific N-Glycan Alterations in Fut9 Knockout Mice

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• 作者：Erika Noro ; Akira Togayachi ; Takashi Sato ; Azusa Tomioka ; Mika Fujita ; Masako Sukegawa ; Nami Suzuki ; Hiroyuki Kaji ; Hisashi Narimatsu
• 刊名：Journal of Proteome Research
• 出版年：2015
• 出版时间：September 4, 2015
• 年：2015
• 卷：14
• 期：9
• 页码：3823-3834
• 全文大小：770K
• ISSN：1535-3907

文摘

The Lewis x (Le^x) structure (Gal尾1鈥?(Fuc伪1鈥?)GlcNAc-R) is a carbohydrate epitope comprising the stage-specific embryonic antigen-1 (SSEA-1) and CD15, and it is synthesized by 伪1,3-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.