A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae

Owen, R. T.; Boulnois, Graham J.; Andrew, PW; Mitchell, TJ

doi:10.1016/0378-1097(94)90129-5

Cited by 20 publications

(29 citation statements)

References 14 publications

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“…The variant nature of the PLO undecapeptide may be required not only to properly align the undecapeptide sequence but also to maintain the structure of domain 4 in general. Support for the latter comes from minor substitutions at the C terminus of CDCs (Owen et al, 1994 ;Shimada et al, 1999), which disrupt the ability of these toxins to bind to cholesterol and host-cell membranes. Aligned with this final suggestion is the possibility that mutations introduced in this study result in the misfolding of domain 4, or indeed the entire PLO protein structure, leading to the reduction in haemolytic and cholesterolbinding activities.…”

Section: Discussionmentioning

confidence: 99%

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

2002

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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 01 % 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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Section: Discussionmentioning

confidence: 99%

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

2002

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“…12), it is likely that the decrease in ␤-sheet content produced by treatment occurs near these strands. It has been reported that C-terminal truncation of pneumolysin, another cholesterol-binding cytolysin, causes a loss of cell binding activity (17). It would be interesting to know whether this loss in the cell binding activity of pneumolysin is due to the loss of cholesterol binding activity, although conformational studies and molecular mass determination of the truncated species of pneumolysin would be required to draw a conclusion.…”

Section: Discussionmentioning

confidence: 99%

“…Six of the seven total tryptophan residues reside in domain 4, and three are located in the sequence of ECTGLAWEWWR (residues 430 -440), the longest conserved sequence among thiol-activated cytolysins (2, 3). From many efforts to achieve mutagenesis of this toxin family (13)(14)(15)(16)(17), it was shown that all mutations that inhibit cell binding activity reside in domain 4, suggesting that some region in domain 4 binds to membrane cholesterol upon binding to cells. This is consistent with our previous findings that a C-terminal tryptic fragment that contains predominantly domain 4 binds to cholesterol and to cholesterol-containing membrane (18).…”

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confidence: 99%

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Shimada

Nakamura

Naito

et al. 1999

Journal of Biological Chemistry

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Perfringolysin O (-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the ␤-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in -toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native -toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in ␤-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of -toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.

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“…Membrane currents were recorded with an Axopatch 200 amplifier (Axon Instruments, Foster City, CA, USA) connected to a PC for data acquisition and subsequent analysis. Patch-pipettes were made from thick walled, filamented borosilicate glass capillaries, which were coated with Sylgard and fire-polished to give resistances of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] MΩ. The holding potential was set to 0mV and a 1.6 s pulse to +40 followed by a 1.6 s pulse to -40 mV was applied every second throughout the recordings.…”

Section: Recording Conditionsmentioning

confidence: 99%

Pneumolysin generates multiple conductance pores in the membrane of nucleated cells

El-Rachkidy¹,

Davies²,

Andrew³

2008

Biochemical and Biophysical Research Communications

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Patch clamping was used to investigate, for the first time, the electrophysiological properties of pores formed by a cholesterol-dependent cytolysin in the membrane of nucleated cells rather than in artificial membranes as previously. Pneumolysin pores in inside-out patches of CHO cell membranes had a broad range of conductance classified into small (<200 pS), medium (>200 pS and <1 nS) and large (>1 nS). A lytic-deficient mutant of pneumolysin (W433F) induced a similar spectrum of channels but there were more small channels than with wild-type (WT) toxin and the number of events was decreased. The WT toxin also induced channels when given to the inside surface of the plasma membrane.

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A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae

Cited by 20 publications

References 14 publications

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

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

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Pneumolysin generates multiple conductance pores in the membrane of nucleated cells

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