N-Glycan Analysis of Recombinant L-Selectin Reveals Sulfated GalNAc and GalNAc−GalNAc Motifs

The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-select...

Full description

Saved in:
Bibliographic Details
Published in:Journal of proteome research 2010-07, Vol.9 (7), p.3403-3411
Main Authors: Wedepohl, Stefanie, Kaup, Matthias, Riese, Sebastian B, Berger, Markus, Dernedde, Jens, Tauber, Rudolf, Blanchard, Véronique
Format: Article
Language:English
Subjects:
Acetylgalactosamine - chemistry
Amino Acid Motifs
Amino Acid Sequence
Cell Line
Humans
L-Selectin - chemistry
L-Selectin - genetics
Molecular Sequence Data
Polysaccharides - chemistry
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants (“LEHis”). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. α1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc−GalNAc or SO4−GalNAc−GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr100170c