Abstract
E-selectin and P-selectin are two closely related vascular cell adhesion proteins. Each selectin has an amino-terminal C-type lectin domain that is thought to possess the carbohydrate binding site that binds the sialylated Lewisx antigen (sLex or CD15s) (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc). In addition to the sLex carbohydrate, P-selectin binds sulfated proteoglycan, 3-sulfated galactosyl ceramide (sulfatide), and heparin. Both E- and P-selectin have an EGF-like (EGF) domain that is immediately adjacent to and COOH-terminal to the lectin domain. We report that mutagenic substitution of single amino acid residues in either the P- or E-selectin EGF domain can dramatically alter selectin binding to sLex, heparin, or sulfatide. Substitution of E- and P-selectin EGF domain residue Ser128 with an arginine results in E- and P-selectin proteins that have lost the requirement for alpha1-3-linked fucose and are thus able to bind to sialyllactosamine. A similar phenotype is reported for an E-selectin mutation within the lectin domain. Additionally, we have determined that conservative substitution of EGF domain residues 124 and 128 can alter E-selectin binding such that it is able to adhere to heparin or sulfatide and can reduce P-selectin adherence to these ligands. The distance between the substituted EGF domain amino acid residues and the primary carbohydrate binding site within the lectin domain and their relative positioning as determined by the three-dimensional crystal structure of the E-selectin lectin and EGF domains (Graves, B. J., Crowther, R. L., Chandran, C., Rumberger, J. B., Li, S., Huang, D.-S., Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K. (1994) Nature 367, 532-538) suggest that there is little direct contact between the two domains. However, we report mutant binding characteristics which indicate that selectin oligosaccharide binding may be modulated by both domains and that wild-type E- and P-selectin/sLex binding interactions may be significantly different from those previously hypothesized.
Highlights
The selectins (E, P, and L-selectin) make up a family of three vascular cell adhesion proteins that appear to modulate the migration of leukocytes from blood into extravascular tissue
In an attempt to elucidate the molecular mechanism(s) by which the S128R mutation alters selectin binding, we have completed a more extensive mutagenesis of several amino acid residues within the E- and P-selectin EGF domains and report that P-selectin sulfatide binding is dependent upon amino acids that are located within the EGF domain of that protein
To determine how the E-selectin S128R mutation might cause an increase in the incidence, early onset, and severity of atherosclerotic disease in individuals that possess this allele, we used site-directed mutagenesis to make the same adenine to cytosine mutation [16, 17] in both E- and P-selectin cDNAs and assessed binding of the altered proteins to several common selectin ligands
Summary
Materials—Tissue culture media, dialyzed fetal calf serum, phosphate-buffered saline (PBS), and antibiotics were obtained from Life Technologies, Inc., and fetal calf serum was from Hyclone. Transfected COS-1 supernatants were either directly coated onto ELISA plates or the recombinant IgG fusion proteins in these supernatants were captured by incubation in ELISA wells that were precoated with rabbit anti-mouse IgGA2 antibody (Cappel catalog number 50228). ELISA plates were subsequently washed repeatedly with PBS and incubated with a 1:5000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (Calbiochem). ELISAs were performed as follows, 8 ϫ 104 beads were added to duplicate wells of 96-well flexible assay plates that had been previously blocked with PBS supplemented with 3% BSA, equivalent amounts of wild-type E-selectin or P-selectin coated beads together with beads prepared from mock transfected cell lysates were tested at the same time on the same plate as positive and negative controls. The absorbance was monitored with a 405 nm filter in a Bio-Rad model 450 microplate reader
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