Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.
Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.
Department of Biological and Environmental Sciences, Division of Biochemistry, University of Helsinki, Helsinki, Finland.
Gerard
G
Hernandez Mir
Jari
J
Helin
Kari-Pekka
KP
Skarp
Richard D
RD
Cummings
Antti
A
Mäkitie
Risto
R
Renkonen
Anne
A
Leppänen
D018952:Antigens, CD34 / Q000737:chemistry* / Q000378:metabolism; D042783:Endothelial Cells / Q000737:chemistry*; D018604:Epitope Mapping; D006031:Glycosylation; D006801:Humans; D019041:L-Selectin / Q000378:metabolism*; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D009844:Oligosaccharides / Q000032:analysis*; D014066:Palatine Tonsil; D011134:Polysaccharides / Q000032:analysis*; D011485:Protein Binding; D000080506:Sialyl Lewis X Antigen / Q000031:analogs & derivatives
C086949:6'-sulfated sialyl Lewis x; D018952:Antigens, CD34; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D019041:L-Selectin