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Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells
M Lenter1, A Levinovitz, S Isenmann
1Hans Spemann Laboratory, Max Planck Institute for Immunology, Freiburg, Germany.
Abstract:
E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-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-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific 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.
Insights
E-selectin and P-selectin bind to distinct glycoprotein ligands on myeloid cells. One type requires N-linked carbohydrates and is selectin-specific, while another binds independently of these carbohydrates and is common to both selectins.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- E-selectin and P-selectin are inducible endothelial cell adhesion molecules.
- They function as Ca(2+)-dependent lectins mediating neutrophil and monocyte binding.
- Previous work identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells.
Purpose of the Study:
- To characterize the glycoprotein ligands recognized by P-selectin.
- To compare the binding specificities of E-selectin and P-selectin ligands.
- To elucidate the roles of N-linked carbohydrates and sialic acid in selectin-ligand interactions.
Main Methods:
- Utilized recombinant chimeric selectin-IgG fusion proteins (E-selectin and P-selectin).
- Investigated binding to mouse myeloid cells, neutrophils, and human HL60 cells.
- Enzymatically removed N-linked carbohydrates and sialic acid to assess their impact on binding.
Main Results:
- P-selectin recognized a 160-kD glycoprotein ligand distinct from the E-selectin ligand.
- Both E-selectin and P-selectin monospecific ligands required N-linked carbohydrates for binding.
- A second category of ligands (230- and 130-kD) bound both E- and P-selectin, required sialic acid, and were independent of N-linked carbohydrates.
Conclusions:
- E- and P-selectin recognize two distinct categories of glycoprotein ligands.
- Monospecific ligands depend on N-linked carbohydrates, while common ligands depend on sialic acid.
- These findings reveal differential recognition mechanisms for selectin ligands in cell adhesion.