title: Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.
abstract: Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.
authors.affiliation: BioResearch Technologies and Proteins, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Darling
authors.forename: Uma
authors.initials: U
authors.lastname: Kuchibhotla
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Glaesner
authors.forename: Radmila
authors.initials: R
authors.lastname: Micanovic
authors.forename: Derrick R
authors.initials: DR
authors.lastname: Witcher
authors.forename: John M
authors.initials: JM
authors.lastname: Beals
mesh_terms: D002236:Carbohydrate Conformation; D002460:Cell Line; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000592:standards; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000378:metabolism*; D012015:Reference Standards; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D014162:Transfection
chemical_list: D007141:Immunoglobulin Fc Fragments; D009844:Oligosaccharides; D020033:Protein Isoforms; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid