Abstract
E-selectin (ELAM-1) is a member of the selectin family of cellular adhesion molecules. This family of proteins possesses an amino-terminal Ca(2+)-dependent lectin or carbohydrate recognition domain that is essential for ligand binding. A known E-selectin ligand is the carbohydrate antigen, sialyl Lewis(x) (sLe(x) (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc). We have developed a model of E-selectin binding to the sLe(x) tetrasaccharide, (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc), using the NMR-determined, E-selectin-bound solution conformation of sLe(x) (Cooke, R.M., Hale, R.S., Lister, S. G., Shah, G., and Weir, M. P. (1994) Biochemistry 33, 10591-10596) together with the x-ray crystallographic structures of E-selectin (Graves, B. J., Crowther, R. L., Chandran, C., Rumberger, J. B., Li, s., Huang, K.-S., Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K. (1994) Nature 367, 532-538) (ligand unbound) and a related C-type animal lectin, the mannose-binding protein (Weis, W. I., Drickamer, K., and Hendrickson, A. (1992) Nature 360, 127-134) (ligand bound). Analysis of this model indicated that the alteration of one E-selectin amino acid, alanine 77, to a lysine residue might shift binding specificity from sLe(x) to mannose. The results presented here show that an E-selectin mutant protein possessing this change displays preferential binding to mannose containing oligosaccharides and that further mutagenesis of this mannose-binding selectin confers galactose recognition in a predictable manner. These mutagenesis data support the presented model of the detailed interactions between E-selectin and the sLe(x) oligosaccharide.
Highlights
The selectins are a family of three carbohydrate-binding proteins.These proteins are expressed on the surface of vascular endothelial cells (E· and Pvselectin), platelets (P-selectin), and leukocytes (L-selectin) and function in binding ligands present on the surface of heterologous cells to promote intercellular adhesion
The results presented here demonstrate that a mutant E-selectin is able to bind oligomannose in a manner similar to the rat mannose-binding protein which has a Klnhibition of 7.2 mM for free mannose (3, 17)
To elucidate the interactions that occur between selectin and ligand, a model of sl.e" bound by the E-selectin carbohydrate recognition domain (CRD) was developed
Summary
(Received for publication, December 28, 1994, and in revised form, April 5, 1995). Timothy P. E-selectin (ELAM-l) is a member of the selectin family of cellular adhesion molecules This family of proteins possesses an amino-terminal Ca2 + -dependent lectin or carbohydrate recognition domain that is essential for ligand binding. Analysis of this model indicated that the alteration of one E-selectin amino acid, alanine 77, to a lysine residue might shift binding specificity from sLex to mannose. The selectins, the asialoglycoprotein receptor, the low-affinity IgE receptor (CD23), the pulmonary surfactant apoprotein SP-A, together with the serum and liver mannose-binding proteins are included in this class of proteins because multiple sequence alignment indicates that they are evolutionarily related (16) Each of these proteins possesses a relatively high degree of sequence similarity that includes 14 invariant and 17 highly conserved amino acid residues within their CRDs (15, 16). The mutagenesis data we present support our proposed fucose coordination (2, 3, 20) as well as the modeled binding conformation
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