title: Gentamicin binds to the lectin site of calreticulin and inhibits its chaperone activity.
abstract: Recently, it became clear that aminoglycoside antibiotics affect protein-protein interactions involving protein disulfide isomerase as well as protein synthesis in the endoplasmic reticulum. In this study, we used affinity column chromatography to screen gentamicin-binding proteins in microsomes derived from bovine kidney in order to learn about the possible mechanisms of gentamicin-associated nephrotoxicity. One of the gentamicin-binding proteins was identified as calreticulin (CRT) by N-terminal amino acid sequence analysis. Interestingly, gentamicin inhibited the chaperone and oxidative refolding activities of CRT when N-glycosylated substrates such as alpha1-antitrypsin and alpha-mannosidase were used as substrates, but it did not inhibit the chaperone activity of CRT when unglycosylated citrate synthase was used. Moreover, CRT suppressed the aggregation of deglycosylated and denatured alpha-mannosidase, but gentamicin did not inhibit its chaperone activity. Experiments with domain mutants suggest that the lectin site of CRT is the main target for gentamicin binding and that binding of gentamicin to this site inhibits the chaperone activity of CRT.
authors.affiliation: Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Horibe
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Matsui
authors.forename: Mami
authors.initials: M
authors.lastname: Tanaka
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nagai
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kikuchi
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D037282:Calreticulin / Q000737:chemistry*; D002417:Cattle; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D037102:Lectins / Q000737:chemistry*; D008861:Microsomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry*; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000494:pharmacology; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000737:chemistry; D043323:alpha-Mannosidase / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D037282:Calreticulin; D005839:Gentamicins; D037102:Lectins; D018832:Molecular Chaperones; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D012685:Sepharose; D043323:alpha-Mannosidase; D019704:Protein Disulfide-Isomerases; D010100:Oxygen