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 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