Abstract Number: LPB0065
Meeting: ISTH 2021 Congress
Background: Plasminogen-binding group A streptococcal M-like protein (PAM) is the primary protein responsible for binding human plasminogen (hPg) in several Group A streptococcus (GAS) strains. PAM is bound to the cell wall through sortase A (SrtA) interaction with the LPXTG motif. The inactivation of SrtA has shown a reduction in the binding activity of PAM to hPg. Mutating the amino acid in the fourth position of the LPXTG motif has shown reduced affinity of SrtA to cleave the peptides in vitro.
Aims: Investigating the impact of inhibiting SrtA cleavage of PAM through mutation of the LPXTG motif in GAS.
Methods: LPSXG peptides were tested using FRET pairs incubated with SrtA protein and LPSYG was shown to have the lowest activity. AP53 GAS strain containing a mutated PAM protein was generated. Cell wall fractions were separated, and relative abundance was determined by western blot. Binding of hPg by PAM was determined by whole cell ELISA. Cell membrane fractions were collected and tested with fluorescent peptides to determine cleavage rates.
Results: The LPSYG peptide showed reduced activity with SrtA protein in vitro. When tested in GAS cells, the PAM mutants were attached to the cell wall and bound to hPg at the same level as wild type (Figure 1).
Cell membrane fractions were tested with fluorescent peptides and the LPSYG peptide was cleaved at significantly higher rates than LPSTG (Figure 2).
Conclusions: In vitro SrtA demonstrated no interaction with the LPSYG peptide. When mutated into GAS, PAM showed similar binding affinity to hPg. Cell membrane fractions were added to the peptides and LPSYG was cleaved at higher rates than LPSTG. This result suggests another protein can recognize the LPSYG motif and cleave in place of SrtA.
To cite this abstract in AMA style:Readnour B, Ayinuola Y, Ploplis V, Castellino F. Mutation of Sortase A Cleavage Site and Potential Impact on Human Plasminogen Binding by PAM [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/mutation-of-sortase-a-cleavage-site-and-potential-impact-on-human-plasminogen-binding-by-pam/. Accessed November 29, 2023.
« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/mutation-of-sortase-a-cleavage-site-and-potential-impact-on-human-plasminogen-binding-by-pam/