title: Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase.
abstract: Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice α-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI-1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 μm, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50,0.22 μm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti-inflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved anti-endotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan.|Center for Transdisciplinary Research, Niigata University, Niigata, 950-2181, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Taniguchi
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Ochiai
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan.
authors.forename: Ryu
authors.initials: R
authors.lastname: Toyoda
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Sato
authors.affiliation: Graduate School of Technology, Niigata Institute of Technology, Niigata, 945-1195, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Saitoh
authors.affiliation: Department of Chemistry, Tokyo Dental College, Tokyo, 101-0062, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D001120:Arginine / Q000737:chemistry*; D002460:Cell Line; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000737:chemistry*; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D012275:Oryza / Q000737:chemistry; D010940:Plant Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D000516:alpha-Amylases / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D023181:Antimicrobial Cationic Peptides; D008070:Lipopolysaccharides; D010940:Plant Proteins; D009569:Nitric Oxide; D001120:Arginine; D000516:alpha-Amylases; D007930:Leucine
keywords: amino acid substitution; anti-endotoxic activity; anti-inflammatory activity; endotoxin-binding activity; endotoxin-neutralizing peptide; multifunctional peptide