PSGL-1 recognition of P-selectin is controlled by a tyrosine sulfation consensus at the PSGL-1 amino terminus

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Abstract

P-selectin binding to neutrophils requires a specific protein, P-selectin glycoprotein ligand 1 (PSGL-1), as well as sialyl-Lewis X (sLex) glycan determinants. We have found that a short segment near the amino terminus of PSGL-1 that contains a tyrosine sulfation consensus is essential for P-selectin adhesion and that addition of the amino-terminal segment to some but not all mucin-like molecules confers on those molecules the ability to bind P-selectin. PSGL-1 synthesized in the presence of sulfation inhibitors binds P-selectin weakly, and within the amino-terminal 20 residues, mutation of the tyrosines to phenylalanine abolishes binding. Rolling of HL-60 cells on P-selectin-coated coverslips is strongly attenuated by treatment of cells with an inhibitor of sulfation.

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Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice.

Tanya N. Mayadas, Robert C. Johnson, Helen Rayburn … (1993)

P selectin, expressed on surfaces of activated endothelial cells and platelets, is an adhesion receptor for leukocytes. We report that P selectin-deficient mice, generated by gene targeting in embryonic stem cells, exhibit a number of defects in leukocyte behavior, including elevated numbers of circulating neutrophils, virtually total absence of leukocyte rolling in mesenteric venules, and delayed recruitment of neutrophils to the peritoneal cavity upon experimentally induced inflammation. These results clearly demonstrate a role for P selectin in leukocyte interactions with the vessel wall and in the early steps of leukocyte recruitment at sites of inflammation. These mutant mice should prove useful in deciphering the contributions of P selectin in various inflammatory responses as well as in platelet functions.

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Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

Ryan B MacDonald, M Wigler, Joanne A P Wilson … (1988)

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.

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P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

F. Li, HS Lichenstein, DF Bainton … (1995)

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL- 1 must interact with P-selectin in order for neutrophils to roll on P- selectin at physiological shear stresses.