title: Distribution of surface-exposed and non-accessible amino acid sequences among the two major structural domains of the S-layer protein of Aeromonas salmonicida.
abstract: The tetragonally arranged crystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor. Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate showed that the secondary structure of A-protein, and its 39,439 molecular weight amino-terminal trypsin-resistant peptide, were altered. In both cases alpha-helix was increased significantly at the expense of beta-structure when SDS was added. Western and dot immunoblotting, immuno-microscopy and enzyme-linked immunosorbent assay with monospecific polyclonal antiserum and eight monoclonal antibodies specific for epitopes exposed on the surface of native A-layer showed that the 481 residue A-protein subunit and the surface of A-layer were conserved antigenically. Mimeotope analysis of nonapeptides representing the sequence of A-protein allowed identification of 146 residues in presumed linear epitopes accessible on the surface of A-layer. Inaccessible or non-epitopic residues accounted for 70% of the protein. The majority of inaccessible residues were in the N-terminal 301 residues of A-protein. Dispersed among these were 65 surface-accessible residues in five linear epitope clusters illustrating the complex folding of this major structural domain of A-protein. The C-terminal 180 residues carried fewer linear epitopes but contained the major region of A-layers surface-accessible sequence, including four linear epitopes in predominantly hydrophobic sequence. Four A-layer surface-binding monoclonal antibodies also bound to this minor structural domain, although the epitopes of only two were identified by mimeotope analysis. The epitopes of six A-layer surface-binding monoclonals could not be identified, suggesting that A-layer may also contain conformation dependent surface epitopes.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, BC, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Doig
authors.forename: W D
authors.initials: WD
authors.lastname: McCubbin
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: T J
authors.initials: TJ
authors.lastname: Trust
mesh_terms: D000333:Aeromonas / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins; D015153:Blotting, Western; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D014357:Trypsin; D014461:Ultracentrifugation
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D014357:Trypsin