Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display

• Published in: International journal of medical microbiology Vol. 295; no. 2; pp. 87 - 98
• Main Authors: Benedek, Orsolya, Salam Khan, A., Schneider, György, Nagy, Gábor, Autar, Reshma, Pieters, Roland J., Emődy, Levente
• Format: Journal Article
• Language: English
• Published: Germany Elsevier GmbH 01.06.2005; Elsevier Science Ltd
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Cloning, Molecular; Epitope mapping; Escherichia coli; Laminin; Laminin - metabolism; Models, Molecular; Molecular Sequence Data; Peptide Library; Phage display; Plasminogen activator; Plasminogen Activators - chemistry; Plasminogen Activators - genetics; Plasminogen Activators - metabolism; Protein Binding; Sequence Alignment; Yersinia pestis; Yersinia pestis - genetics; Yersinia pestis - metabolism; Epitope mapping; Plasminogen activator; Phage display; Laminin; Yersinia pestis
• ISSN: 1438-4221; 1618-0607
• DOI: 10.1016/j.ijmm.2005.02.002

Yersinia pestis plasminogen activator (Pla), a surface virulence factor contributes to the highly invasive nature of the pathogen by binding various tissue matrix components. In this study we characterised the laminin-binding site(s) of Pla via phage display and alanine-scanning mutagenesis. Previously we isolated 18 different heptamer peptide sequences from a phage display library with biopanning on laminin, and have shown that two of them with sequences of WSLLTPA or YPYIPTL completely inhibited laminin binding of a Pla-expressing recombinant Escherichia coli strain. These phages themselves strongly bound laminin in an ELISA assay utilising horseradish peroxidase-labelled anti-M13 antibody. In the present study, with the application of synthetic peptides, a 55% and a 33% inhibition was demonstrated using WSLLTPA and YPYIPTL, respectively. Peptide pattern alignment showed two homologous regions for WSLLTPA and one for YPYIPTL inside the Pla sequence. Amino acids were changed for alanine in one of the WSLLTPA regions and in the YPYIPTL region in order to prove the role of the LTP/PTL motifs in laminin binding. Of the four constructed mutants, the triple mutant L65A-T66A-L67A in the WSLLTPA region and the double mutant G178A-L179A in the YPYIPTL region decreased the laminin binding capacity of the Pla-expressing recombinant E. coli by about 50%.