A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.
P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.
Leppänen
A
A
Department of Biochemistry and Molecular Biology, Oklahoma City, Oklahoma 73104, USA.
Mehta
P
P
Ouyang
Y B
YB
Ju
T
T
Helin
J
J
Moore
K L
KL
van Die
I
I
Canfield
W M
WM
McEver
R P
RP
Cummings
R D
RD
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D006023:Glycoproteins; D006801:Humans; D016256:Lewis X Antigen / Q000737:chemistry; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D010455:Peptides; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding
D002352:Carrier Proteins; C121374:GSP-6 glycosulfopeptide; D006023:Glycoproteins; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D010455:Peptides; D011134:Polysaccharides