E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca(2+)-dependent lectin. We have recently identified a 150K glycoprotein as the major ligand for E-selectin on myeloid cells, using a recombinant antibody-like form of mouse E-selectin as an affinity probe. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor, except for a unique 70-amino-acid aminoterminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin.

Abstract. E-and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca2+-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca 2+-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide:N-glycosidase F inhibited cell binding to P-and E-select.in.In addition, glyeoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E-and P-selcctin in a Cae+-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospeciflc ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E-and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.

Abstract. E-selectin is an inducible endothelial cell adhesion molecule for neutrophils which functions as a Ca2+-dependent lectin. Using a recombinant, antibody-like form of mouse E-selectin, we have searched for glycoprotein ligands on mouse neutrophils and the neutrophil progenitor cell line 32D cl 3. We have identified a 150-kD glycoprotein as the only protein which could be affinity-isolated with soluble E-selectin from [3sS]methionine/[35S]cysteine-labeled 32D cl 3 cells. Binding of this protein was strictly Ca2÷-dependent, was blocked by a cell adhesionblocking mAb against mouse E-selectin, and required the presence of sialic acid on the 150-kD ligand. This glycoprotein was also afffinity-isolated from mature neutrophils, in addition to a minor component at 250 kD, but could not be isolated from several other nonmyeloid cell lines. The 150-kD glycoprotein was the only protein from 32D cl 3 cells, which was detectable by silverstaining after a one-step affinity-isolation.

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