title: Identification of binding sites of Lactobacillus plantarum enolase involved in the interaction with human plasminogen.
abstract: The enolase EnoA1 of Lactobacillus plantarum is here shown to interact with human plasminogen (Plg). By sequence alignment of EnoA1 with Streptococcus pneumoniae and Bifidobacterium lactis enolases, we identified BS1 and BS2 Plg-binding sites. A structure prediction of EnoA1 showed lysine residues in position 255 (BS2), and 422 (BS1) exposed on protein surface. A lysine residue in position 259 was as well identified as surface-exposed amino acid. The enoA1 gene was site directed-mutagenized to generate four mutated proteins, carrying K255A, K259A, K422A and K259A/K422A substitutions. The functional role of these lysine residues was assessed evaluating specific Plg-binding activity of the mutated proteins. While the binding activity of the mutated proteins was drastically reduced, the residual enzymatic activity was more than 50% of EnoA1. Our results show that L. plantarum EnoA1 exhibits the Plg-BS1, and the Plg-BS2 extending up to the lysine residue in position 259, therefore consisting of 12-aa residues instead of 9-aa residues described in S. pneumoniae. A test performed on whole cells of L. plantarum, demonstrated that after inducing conversion of the cell-bound plasminogen to plasmin, this was released into the medium, unlike the mechanism reported for most pathogens, that retained plasmin bound to the cell surface.
authors.affiliation: Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Caserta, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Vastano
authors.forename: Ugo
authors.initials: U
authors.lastname: Capri
authors.forename: Marco
authors.initials: M
authors.lastname: Candela
authors.forename: Rosa Anna
authors.initials: RA
authors.lastname: Siciliano
authors.forename: Luigi
authors.initials: L
authors.lastname: Russo
authors.forename: Mario
authors.initials: M
authors.lastname: Renda
authors.forename: Margherita
authors.initials: M
authors.lastname: Sacco
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D019936:Probiotics; D011485:Protein Binding; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D010958:Plasminogen; D005341:Fibrinolysin; D010751:Phosphopyruvate Hydratase