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Glycobiology Advance Access published online on July 28, 2006
Glycobiology, 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
Received January 2, 2006
Revised July 26, 2006
Accepted July 26, 2006

Article

N-Glycan Structures and N-Glycosylation Sites of Mouse Soluble Intercellular Adhesion Molecule-1 Revealed by MALDI-TOF and FTICR Mass Spectrometry

Vivianne I. Otto 1 *, Eugen Damoc 2, Leah N. Cueni 1, Thomas Schürpf 1, Renate Frei 1, Sarah Ali 1, Nico Callewaert 3, Adrian Moise 4, Julie A. Leary 5, Gerd Folkers 1, and Michael Przybylski 4

1 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences
2 Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany; Genome Center, UC Davis, Davis, CA 95616, U.S.A
3 GlycoINIT and Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology (ETH), 8093 Zurich, Switzerland
4 Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany
5 Genome Center, UC Davis, Davis, CA 95616, U.S.A

* To whom correspondence should be addressed.
Vivianne I. Otto, E-mail: vivianne.otto{at}pharma.ethz.ch


  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 MALDI-TOF mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and tri-sialylated 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 ca. 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.

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