title: Glycosylation analysis of IgLON family proteins in rat brain by liquid chromatography and multiple-stage mass spectrometry.
abstract: IgLON family proteins, including limbic-associated membrane protein (LAMP), opioid-binding cell adhesion molecule (OBCAM), neurotrimin, and Kilon, are immunoglobulin (Ig) superfamily cell adhesion molecules. These molecules are composed of three Ig domains and a glycosylphosphatidylinositol (GPI) anchor and contain six or seven potential N-glycosylation sites. Although their glycosylations are supposed to be associated with the development of the central nervous system like other Ig superfamily proteins, they are still unknown because of difficulty in isolating individual proteins with a high degree of homology in performing carbohydrate analysis. In this study, we conducted simultaneous site-specific glycosylation analysis of rat brain IgLON proteins by liquid chromatography and multiple-stage mass spectrometry (LC-MS ( n )). The rat brain GPI-linked proteins were enriched and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The four proteins were extracted from the gel, and subjected to LC-MS ( n ) after proteinase digestions. A set of glycopeptide MS data, including the mass spectrum, the mass spectrum in the selected ion monitoring mode, and the product ion spectra, was selected from all data based on carbohydrate-related ions in the MS/MS spectrum. The peptide portion and the carbohydrate structure were identified on the basis of peptide-related ion and carbohydrate-related ions, and the accurate mass. The site-specific glycosylations of four proteins were elucidated as follows. N-Glycans near the N-terminal were disialic acid-conjugated complex- and hybrid-type oligosaccharides. The first Ig domains were occupied by Man-5-9. Diverse oligosaccharides, including Lewis a/x-modified glycans, a brain-specific glycan known as BA-2, and Man-5, were found to be attached to the third Ig domain. Three common structures of glycans were found in the GPI moiety of LAMP, OBCAM, and neurotrimin.
authors: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Satsuki
authors: S
authors: Itoh
authors: Akiko
authors: A
authors: Hachisuka
authors: Nana
authors: N
authors: Kawasaki
authors: Noritaka
authors: N
authors: Hashii
authors: Reiko
authors: R
authors: Teshima
authors: Takao
authors: T
authors: Hayakawa
authors: Toru
authors: T
authors: Kawanishi
authors: Teruhide
authors: T
authors: Yamaguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D058851:GPI-Linked Proteins; D006031:Glycosylation; D050440:Immunoglobulin Subunits / Q000032:analysis / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000032:analysis / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; D058851:GPI-Linked Proteins; D050440:Immunoglobulin Subunits; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; C118293:Negr1 protein, rat; D019006:Neural Cell Adhesion Molecules; C489215:Opcml protein, rat; C092530:neurotrimin