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.

Branched Sialylated <i>N</i>-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H

“…The N-glycan structure of neurotrimin and LAMP in the adult rat brain has previously been reported (Itoh et al, 2008), in which the structure of the identified Nglycans was consistent with sialylated A2G´2F on neurotrimin, but not on LAMP.…”

IgLON Cell Adhesion Molecules Are Shed from the Cell Surface of Cortical Neurons to Promote Neuronal Growth

“…IgLONs are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules and are the most abundant glycosylphosphatidylinositolanchored proteins expressed in neurons (16). The IgLON proteins contain three immunoglobulin domains followed by a glycosylphosphatidylinositol anchor protein and possess 6 -7 potential glycosylation sites (17). IgLON family members include neurotrimin (NTM), opioid-binding cell adhesion molecule (OBCAM), limbic system-associated membrane protein (LSAMP), and neuronal growth regulator 1 (NEGR1) (16,18,19).…”

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol‐anchored proteins through selective glycan enrichment

“…In addition to its GPI anchor, most characterized GPI-APs also possess additional carbohydrate modifications such as N-and/or O-linked glycans (Fig. 1A) [1][2][3][4].…”