title: Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.
abstract: Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes.
authors.affiliation: Department of Life Science, Graduate School of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Nakagawa
authors.affiliation: Master Course, Graduate School of Medical Science, Akita University, Akita 010-8543, Japan.
authors.forename: Kotomi
authors.initials: K
authors.lastname: Sugawara
authors.affiliation: Department of Life Science, Graduate School of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Tatsufumi
authors.initials: T
authors.lastname: Goto
authors.affiliation: Department of Life Science, Graduate School of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Wakui
authors.affiliation: Department of Life Science, Graduate School of Engineering Science, Akita University, Akita 010-8502, Japan; Research Center for Engineering Science, Graduate School of Engineering Science, Akita University, Akita 010-8502, Japan. Electronic address: nunomura@gipc.akita-u.ac.jp.
authors.forename: Wataru
authors.initials: W
authors.lastname: Nunomura
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D006886:Hydroxychloroquine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors; D008565:Membrane Proteins / Q000378:metabolism; D000072417:Protein Domains
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D008565:Membrane Proteins; C454201:SLC4A1 protein, human; D006886:Hydroxychloroquine; D007770:L-Lactate Dehydrogenase
keywords: Band 3; Human erythrocytes; Hydroxychloroquine; Lactate dehydrogenase