The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity - PubMed (original) (raw)

. 2002 Dec;148(Pt 12):3947-3954.

doi: 10.1099/00221287-148-12-3947.

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The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity

Stephen J Billington et al. Microbiology (Reading). 2002 Dec.

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Abstract

The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P(499)), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P(499) is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 0.1% activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C(492)) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C(492), which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

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