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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)
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