THE SOLUBLE FORM OF E-SELECTIN IS AN ASYMMETRIC MONOMER - EXPRESSION,PURIFICATION, AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN

The gene coding for a soluble form of human E-selectin (sE-selectin) h as been expressed in Chinese hamster ovary (CHO) cells. Cells seeded i nto a hollow fiber reactor secreted protein at a level of 160 mg/liter . The protein was purified to >95% pure and low endotoxin (<2 ng/mg), using physiological pH and buffers. The amino acid composition and N-t erminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose dependent manner, and th is binding could be blocked up to 100% by pretreatment of HL60 cells w ith sE-selectin. The concentration of sE-selectin required for 50% inh ibition was 1 mu M. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited infla mmatory migration of neutrophils in a selective fashion. Purified sE-s electin exhibited a broad band of M(r) similar to 75,000 on nonreducin g SDS-PAGE. sE-selectin eluted with M(r) similar to 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting a n oligomeric state. Matrix-assisted laser-desorption MS gave a molecul ar weight of 80,000, while the minimum monomer molecular weight from t he gene sequence should be 58,571, demonstrating that the monomeric mo lecule thus expressed had 27% carbohydrate. Equilibrium analytical ult racentrifugation gave an average solution molecular weight of 81,600 ( +/- 4,500). Velocity ultracentrifugation gave a sedimentation coeffici ent of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assumi ng a prolate ellipsoid of revolution. An analysis of the NMR NOESY spe ctra of sE-selectin, sialyl-lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X prod uctively. Hence, in solution, sE-selectin is a functional elongated mo nomer.