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Glycobiology Advance Access originally published online on July 28, 2006
Glycobiology 2006 16(11):1033-1044; doi:10.1093/glycob/cwl032
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

N-Glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Vivianne I. Otto1,2, Eugen Damoc4,5, Leah N. Cueni2, Thomas Schürpf2, Renate Frei2, Sarah Ali2, Nico Callewaert3, Adrian Moise4, Julie A. Leary5, Gerd Folkers2 and Michael Przybylski4

2 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences; 3 GlycoINIT and Institute of Microbiology, Department of Biology, ETH Zurich, Switzerland; 4 Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany; and 5 Genome Center, UC Davis, Davis, CA 95616

1 To whom correspondence should be addressed; e-mail: vivianne.otto{at}pharma.ethz.ch

Received on January 2, 2006; revised on July 26, 2006; accepted on July 26, 2006

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 ~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.

Key words: Fourier transform ion cyclotron resonance mass spectrometry / glycosylation–function relationships / N-glycosylation / oligosialic acid / mouse soluble intercellular adhesion molecule-1


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