Identification of a Glycoprotein Ligand for E-selectin on Mouse Myeloid Cells

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. T HE selectins form a class of three cell adhesion molecules which mediate the binding of leukocytes to endothelial cells (McEver, 1991; Vestweber, 1992). One of them, L-selectin (LAMA, MEL-14-antigen), is expressed on the surface of most leukocyte types (Gallatin et al., 1986; Siegelman et al., 1989; Lasky et al., 1989; Tedder et al., 1990; Camerini et al., 1989) and is involved in the homing of lymphocytes into peripheral lymph nodes (Gallatin et al., 1983) as well as in the migration of neutrophils into sites of inflammation (Lewinsohn et al., 1987; Watson et al., 1991). The other two selectins, E-selectin (ELAM-1) and P-selectin (GMP-140, PADGEM, CD62), are expressed on endothelial cells (Bevilacqua et al., 1987; McEver et al., 1989) and bind to neutrophils, monocytic cells, and certain subsets of lymphocytes (Bevilacqua et al., 1989; Picker et al., 1991a; Shimizu et al., 1991; Geng et al., 1990; Damle et al., 1992). In contrast to the vast majority of all known cell adhesion molecules, the selectins function as carbohydrate lectins. They share a highly homologous (60-65%) NH2-terminal lectin domain which resembles the Ca~+-dependent lectin domains of animal lectins (Drickamer, 1988). Binding of the same tetrasaccharide structure, sialyl Lewis x, to E-, P-, and L-selectin has been demonstrated (Phillips et al., 1990; Polley et al., 1991; Foxall et al., 1992). However, the stereoisomerle form, sialyl Lewis a, can also bind to all three selectins (Berg et al., 1991a; Handa et al., 1991; Tyrrell et al., 1991; Berg et al., 1992), and lately also sulphated Lewis x and sulphated Lewis a carded by glycolipids, and lacking sialic acid have been reported to bind Land E-selectin (Yuen et al., 1992; Green et al., 1992). To what extent the physiological ligands resemble these structures is still unknown. Also, whether different selectins share the same physiological ligands or have different ones, is an important question to answer. This is especially interesting for the two endothelial selectins, Eand P-selectin, since they can interact with the same types of leukocytes. Differences in the regulation of the cell surface expression of the two selectins suggest that both can support leukocyte adhesion to endothelium in different situations. Only P-selectin is stored in intracellular granula, from which it can rapidly be mobilized to the cell surface within minutes upon induction with preinflammatory agents such as thrombin or histamine (Geng et al., 1990). However, both selectins can also be induced by the same stimuli and expressed simultaneously on the cell surface. The TNF-ot induced synthesis of E-selectin, which leads to a transient increase of the protein on the cell surface with maximal expression after 3-4 h of induction, could also be demonstrated for P-selectin on mouse endothelial cells (Weller et al., 1992; Hahne et al., 1993). Since both selectins can be expressed on the cell surface simultaneously and since both interact with neutrophils, the physiological ligands for both selectins on neutrophils need to be identified to understand how Eand P-selectin cooperate in the process of neutrophil extravasation. Some glycoprotein ligands for the selectins have been identified in the past. Best characterized is the L-selectin ligand GlyCAM-1. This is a 50-kD mucin-like molecule which was affinity-isolated from mesenteric lymph nodes using an L-selectin-IgG chimeric protein (Imai et al., 1991; Lasky et al., 1992). GlyCAM-1 is also recognized by the mAb MECA 79 which reacts with several glycoproteins on endothelial cells of peripheral lymph nodes (Berg et al., © The Rockefeller University Press, 0021-9525/93/04/449/11 $2.00 The Journal of Cell Biology, Volume 121, Number 2, April 1993 449--459 449 on Jne 4, 2017 D ow nladed fom Published April 15, 1993