Abstract
The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into 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-terminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose-dependent manner, and this binding could be blocked up to 100% by pretreatment of HL60 cells with sE-selectin. The concentration of sE-selectin required for 50% inhibition was 1 microM. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited inflammatory migration of neutrophils in a selective fashion. Purified sE-selectin exhibited a broad band of M(r) approximately 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with M(r) approximately 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting an oligomeric state. Matrix-assisted laser-desorption MS gave a molecular weight of 80,000, while the minimum monomer molecular weight from the gene sequence should be 58,571, demonstrating that the monomeric molecule thus expressed had 27% carbohydrate. Equilibrium analytical ultracentrifugation gave an average solution molecular weight of 81,600 (+/- 4,500). Velocity ultracentrifugation gave a sedimentation coefficient of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assuming a prolate ellipsoid of revolution. An analysis of the NMR NOESY spectra of sE-selectin, sialyl-Lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X productively. Hence, in solution, sE-selectin is a functional elongated monomer.
Highlights
From the Departments of $2MacromolecularSciences, IProtein Biochemistry, llGene Expression Sciences, **Cellular Biochemistry, $$Inflammation and Respiratory Pharmacology, and @Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939
This weak attachment is stimulated by tinhas been expressed in Chinese hamster cytokines, such as tumor necrosis factor-aand interleukin-1,as ovary (CHO) cells
Selectins are a class of cell adhesion molecules which mediate thisrolling behavior via low-avidity transient interactions betweenleukocytes andendothelial cells (Lawrence and Springer, 1991;von Andrian et al, 1991)
Summary
Prior to assay, coated plates were washed once with 0.2 ml of HBSS', 0.1 ml of dye-labeled cell suspension was added to coated wells. SE-selectin-coated wells were pretreated with 0.1 ml of monoclonal antibody diluted in HBSS'for 30 min at room temperature. To demonstrate inhibition of adhesion with sE-selectin, dye-labeled HL60 cells were pretreated with increasingconcentrations of purified protein for 30 min at room temperature prior to plating onto sE-selectin-coated plates. The partial specific volume, 6, was calculated from the weight percent average of the partial specific volumes of the component amino acids (Zamyatnin, 1972) and the weight percent average of the partialspecific volumes of the sugars, assuming a ratioof hexose:hexosamine:sialicacid of 5:4:2 (biantennary, typical for CHO cells), with 21,474Da due to carbohydrate (fromabove) This yields a value of U = 0.688 ml g". Flf, is the component of the frictional ratio contributed by hydration, which is defined as, flL= (1+ w/Up)'"
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