N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.
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.
Otto
Vivianne I
VI
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland. vivianne.otto@pharma.ethz.ch
Damoc
Eugen
E
Cueni
Leah N
LN
Schürpf
Thomas
T
Frei
Renate
R
Ali
Sarah
S
Callewaert
Nico
N
Moise
Adrian
A
Leary
Julie A
JA
Folkers
Gerd
G
Przybylski
Michael
M
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
D011134:Polysaccharides; D020033:Protein Isoforms; D018799:Intercellular Adhesion Molecule-1; D019158:N-Acetylneuraminic Acid