CD62/P-selectin binding sites for myeloid cells and sulfatides are overlapping.
P-Selectin (CD62/GMP140/PADGEM) is an inducible cell-surface glycoprotein expressed by endothelial cells and platelets following stimulation by inflammatory mediators such as thrombin, histamine, or peroxides. P-Selectin mediates the binding of leukocytes to activated vascular endothelium at sites of inflammation and plays a role in mediating the binding of activated platelets to leukocytes and the vascular cell wall. The adhesive function of P-selectin is mediated by its calcium-dependent (or C-type) lectin domain, which is known to bind to carbohydrate ligands including fucosyl-N-acetyllactosamine (Lex, CD15), sialyl-Lex, and 3-sulfated galactosylceramides (sulfatides). Sulfatides can efficiently block P-selectin/myeloid cell binding in vitro and are excreted at high levels by activated granulocytes. These observations led to the hypothesis that sulfatide may play a role in facilitating the disengagement of CD62, allowing the efficient exit of granulocytes from the blood stream at sites of inflammation. In this report, we extend our previous mutagenesis analysis of the P-selectin binding site [Hollenbaugh, D., Bajorath, J., Stenkamp, R., & Aruffo, A. (1993) Biochemistry 32, 2960] and show that replacement of Tyr48 with Ser or Lys113 with Arg results in P-selectin mutants that, although correctly folded, do not bind to HL60 cells. These results suggest that the conservation of charged and hydrogen-bonding site chains is not sufficient to maintain the P-selectin function and that the exact stereochemistry provided by the side chains of residues lining the P-selectin binding pocket is critical for P-selectin binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Bajorath
J
J
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Hollenbaugh
D
D
King
G
G
Harte
W
W
Eustice
D C
DC
Darveau
R P
RP
Aruffo
A
A
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D002448:Cell Adhesion; D003198:Computer Simulation; D004563:Electrochemistry; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013433:Sulfoglycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D013433:Sulfoglycosphingolipids