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-selectin using recombinant variants ("LEHis"). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. alpha1-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 SO(4)-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.
Wedepohl
Stefanie
S
Central Institute of Laboratory Medicine and Pathobiochemistry, Charité Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
Kaup
Matthias
M
Riese
Sebastian B
SB
Berger
Markus
M
Dernedde
Jens
J
Tauber
Rudolf
R
Blanchard
Véronique
V
D000116:Acetylgalactosamine / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D019041:L-Selectin / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011134:Polysaccharides; D011994:Recombinant Proteins; D019041:L-Selectin; D000116:Acetylgalactosamine