title: Isoleucine 61 is important for the hemolytic activity of pyolysin of Trueperella pyogenes.
abstract: Pyolysin (PLO) is a hemolysin secreted by Trueperella pyogenes (T. pyogenes) and is important for the pathogenicity of T. pyogenes. Oligomerization of PLO monomers is a critical step in the process of hemolysis. However, the mechanisms of intermolecular interaction of PLO monomers are still not clearly illuminated. In this study, two monoclonal antibodies (mAbs) against PLO, named AP-1A3 and AP-4F12, respectively, were generated firstly, of which AP-1A3 showed no or undetectable hemolysis inhibition activity against recombinant PLO (rPLO), whereas AP-4F12 could markedly inhibit the hemolytic activity of rPLO. Epitope mapping revealed that AP-1A3 recognized amino acid residues ranging from 64 to 79 of mature PLO (91-106 including the signal peptide), whereas AP-4F12 recognized amino acid residues ranging from 58 to 75 (85-102 including the signal peptide). Comparison of the amino acid sequence of two epitopes revealed that six amino acid residues ranging from 58 to 63 of PLO were associated with the hemolytic activity of PLO. Alanine scan showed that substitution of each amino acid ranging from 58 to 62 with alanine had apparent impact on the hemolytic activity of rPLO, especially for the substitution of isoleucine 61 which caused almost complete loss of hemolytic activity of rPLO. Our findings identified a region in PLO and an amino acid in that region might play important role in the process of oligomerization of PLO monomers.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China.
authors.forename: Minghui
authors.initials: M
authors.lastname: Yan
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China.
authors.forename: Yunhao
authors.initials: Y
authors.lastname: Hu
authors.affiliation: College of Animal Science and Technology, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China; National Food Safety and Nutrition Collaborative Innovation Center, Wuxi, Jiangsu 214122, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Bao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China.
authors.forename: Lingxiao
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China; National Food Safety and Nutrition Collaborative Innovation Center, Wuxi, Jiangsu 214122, PR China. Electronic address: jwwang@neau.edu.cn.
authors.forename: Junwei
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China. Electronic address: zhangwenlong@neau.edu.cn.
authors.forename: Wenlong
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D055667:Arcanobacterium / Q000276:immunology* / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006460:Hemolysin Proteins / Q000276:immunology*; D006461:Hemolysis / Q000276:immunology*; D007532:Isoleucine / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D000939:Epitopes; D006460:Hemolysin Proteins; C108448:Pyolysin, Arcanobacterium pyogenes; D007532:Isoleucine
keywords: Hemolytic activity; Isoleucine 61; PLO; mAbs