Abstract
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%.
| Original language | English |
|---|---|
| Pages (from-to) | 87-98 |
| Number of pages | 12 |
| Journal | International Journal of Medical Microbiology |
| Volume | 295 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jun 1 2005 |
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Keywords
- Epitope mapping
- Laminin
- Phage display
- Plasminogen activator
- Yersinia pestis
ASJC Scopus subject areas
- Microbiology
- Microbiology (medical)
Cite this
Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display. / Benedek, Orsolya; Salam Khan, A.; Schneider, G.; Nagy, Gábor; Autar, Reshma; Pieters, Roland J.; Emődy, L.
In: International Journal of Medical Microbiology, Vol. 295, No. 2, 01.06.2005, p. 87-98.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display
AU - Benedek, Orsolya
AU - Salam Khan, A.
AU - Schneider, G.
AU - Nagy, Gábor
AU - Autar, Reshma
AU - Pieters, Roland J.
AU - Emődy, L.
PY - 2005/6/1
Y1 - 2005/6/1
N2 - 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%.
AB - 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%.
KW - Epitope mapping
KW - Laminin
KW - Phage display
KW - Plasminogen activator
KW - Yersinia pestis
UR - http://www.scopus.com/inward/record.url?scp=18844444264&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18844444264&partnerID=8YFLogxK
U2 - 10.1016/j.ijmm.2005.02.002
DO - 10.1016/j.ijmm.2005.02.002
M3 - Article
C2 - 15969469
AN - SCOPUS:18844444264
VL - 295
SP - 87
EP - 98
JO - International Journal of Medical Microbiology
JF - International Journal of Medical Microbiology
SN - 1438-4221
IS - 2
ER -