title: Characterisation of mouse monoclonal antibodies for pneumolysin: fine epitope mapping and V gene usage.
abstract: Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) produced by Streptococcus pneumoniae, the main cause of community-acquired pneumonia. We have applied a set of diverse molecular methodologies (PCR-derived PLY peptides, biopanning of a library of phage-displayed random nonapeptides, indirect ELISA and competition tests with soluble peptides) to achieve concordant complementary observations in order to obtain a fine epitope mapping of three mouse monoclonal antibodies (PLY-4, PLY-7 and PLY-8) for PLY. PLY-4 seems to recognise a conformation-dependent epitope with a core reactivity involving R232. The epitopes recognised by PLY-7 and PLY-8 are within the sequences (401)GQDLTAH(407) and (450)KRTISIWGT(458), respectively. PLY-7 also recognises suilysin (SLY), in which the homologous reactive amino acid stretch is (429)GVNLTSH(435). In a homology model of PLY with the crystal structure of perfringolysin O (PFO), R232 is part of a well-exposed contorted loop on the edge of the concave and convex faces of domain 1. The sequences reactive with PLY-7 and PLY-8 would conform one of the loops at the bottom of domain 4 and a beta strand of one of the two beta sheets of this domain, respectively. Western blot analyses carried out with anti-PLY rabbit IgG and polyclonal mouse serum identified stretches comprising residues 40-98, 199-248, 352-414 and 415-471 of PLY as immunogenic and antigenic; altogether with their recognition by the monoclonal antibodies herein considered, these results stress the immunological significance of domains 1 and 4 of the PLY molecule. PLY-4, PLY-7 and PLY-8 share the same Vkappa chain; this chain and that of the PLY-5 monoclonal antibody are essentially in germline configuration, whereas the VH regions of these monoclonals come from diverse gene segments and are mutated.
authors.affiliation: Area de Microbiología, Facultad de Medicina, Universidad de Oviedo, c/Julián Clavería s/n, 33006 Oviedo, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Suárez-Alvarez
authors.forename: María del Mar
authors.initials: Mdel M
authors.lastname: García-Suárez
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Méndez
authors.forename: Juan R
authors.initials: JR
authors.lastname: de los Toyos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001426:Bacterial Proteins; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D009500:Neutralization Tests; D019151:Peptide Library; D011487:Protein Conformation; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000276:immunology; D013301:Streptolysins / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D007106:Immune Sera; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae