Glycosulfopeptides with O-glycans containing sialylated and polyfucosylated polylactosamine bind with low affinity to P-selectin.
P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO3-) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO3- residues and a short, monofucosylated C2-O-sLe(x) bound to P-selectin with high affinity (K(d) approximately 650 nm). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6' and GSP-6", with three TyrSO3- residues and either difucosylated polylactosamine (C2-O-Le(x)-sLe(x)) or trifucosylated polylactosamine (C2-O-Le(x)-Le(x)-sLe(x)). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6' and GSP-6" bound to P-selectin with low affinity (K(d) approximately 37 microm for GSP-6' and K(d) approximately 50 microm for GSP-6"). Binding of GSP-6' and GSP-6" to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6' and GSP-6". These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.
Leppanen
Anne
A
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Penttila
Leena
L
Renkonen
Ossi
O
McEver
Rodger P
RP
Cummings
Richard D
RD
D000606:Amino Sugars / Q000737:chemistry*; D019921:Biotinylation; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins; D018922:HL-60 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D016256:Lewis X Antigen / Q000378:metabolism; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D019007:P-Selectin / Q000737:chemistry* / Q000378:metabolism; D010452:Peptide Biosynthesis; D010455:Peptides; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000606:Amino Sugars; D002352:Carrier Proteins; C121374:GSP-6 glycosulfopeptide; D006023:Glycoproteins; D007074:Immunoglobulin G; D037102:Lectins; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D019007:P-Selectin; D010455:Peptides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D012492:Salts; C066929:polylactosamine; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid