title: N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.
abstract: Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland. vivianne.otto@pharma.ethz.ch
authors.forename: Vivianne I
authors.initials: VI
authors.lastname: Otto
authors.forename: Eugen
authors.initials: E
authors.lastname: Damoc
authors.forename: Leah N
authors.initials: LN
authors.lastname: Cueni
authors.forename: Thomas
authors.initials: T
authors.lastname: Schürpf
authors.forename: Renate
authors.initials: R
authors.lastname: Frei
authors.forename: Sarah
authors.initials: S
authors.lastname: Ali
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.forename: Adrian
authors.initials: A
authors.lastname: Moise
authors.forename: Julie A
authors.initials: JA
authors.lastname: Leary
authors.forename: Gerd
authors.initials: G
authors.lastname: Folkers
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000818:Animals; D016466:CHO Cells; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011134:Polysaccharides; D020033:Protein Isoforms; D018799:Intercellular Adhesion Molecule-1; D019158:N-Acetylneuraminic Acid