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Studies of the roles of high charge and a lack of tryptophan residues on the properties of human group IIA secreted phospholipase A2

Studies of the roles of high charge and a lack of tryptophan residues on the properties of human group IIA secreted phospholipase A2
Studies of the roles of high charge and a lack of tryptophan residues on the properties of human group IIA secreted phospholipase A2

The structure of the human group IIA sPLA2 is dominated by two molecular characteristics. Firstly, the enzyme has a very high surface positive charge with an isoelectric point of approximately 10. Secondly, the enzyme does not have tryptophan residues on its membrane-binding surface. The enzyme is an acute phase antibacterial protein with little or no activity on 'self' membranes under normal conditions. The overall aim of this thesis was to establish that the molecular characteristics of this enzyme are responsible for its unique physiological role in the body.

A strategy of charge reversal mutagenesis has been used to show that it is the high global positive charge on the surface of the protein that is required for it to exhibit its potent Gram-positive antibacterial properties. This high charge is responsible for the enzymes' ability to penetrate the bacterial cell wall and gain access to the underlying cell membrane where hydrolysis can take place. A reduction in the overall charge on the enzymes' surface is associated with reduced cell membrane hydrolysis and consequently antibacterial potency.

A strategy involving tryptophan replacement of the non-polar residues within the hydrophobic collar that surrounds the active site entrance has demonstrated that it is the lack of membrane-binding tryptophan residues that enable the protein to exhibit little or no activity on phosphatidylcholine interfaces. The introduction of two tryptophan residues (V3,31W) into the putative membrane-binding surface of the enzyme produced a protein with similar characteristics to the human group V enzyme. These characteristics included the ability of the mutant protein to hydrolyse 'self' membranes and to be internalised into cells. The introduced tryptophan residues have also been used as fluorescence reporter groups to propose a binding model for the protein on the membrane surface.

University of Southampton
Beers, Stephen A
Beers, Stephen A

Beers, Stephen A (2002) Studies of the roles of high charge and a lack of tryptophan residues on the properties of human group IIA secreted phospholipase A2. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The structure of the human group IIA sPLA2 is dominated by two molecular characteristics. Firstly, the enzyme has a very high surface positive charge with an isoelectric point of approximately 10. Secondly, the enzyme does not have tryptophan residues on its membrane-binding surface. The enzyme is an acute phase antibacterial protein with little or no activity on 'self' membranes under normal conditions. The overall aim of this thesis was to establish that the molecular characteristics of this enzyme are responsible for its unique physiological role in the body.

A strategy of charge reversal mutagenesis has been used to show that it is the high global positive charge on the surface of the protein that is required for it to exhibit its potent Gram-positive antibacterial properties. This high charge is responsible for the enzymes' ability to penetrate the bacterial cell wall and gain access to the underlying cell membrane where hydrolysis can take place. A reduction in the overall charge on the enzymes' surface is associated with reduced cell membrane hydrolysis and consequently antibacterial potency.

A strategy involving tryptophan replacement of the non-polar residues within the hydrophobic collar that surrounds the active site entrance has demonstrated that it is the lack of membrane-binding tryptophan residues that enable the protein to exhibit little or no activity on phosphatidylcholine interfaces. The introduction of two tryptophan residues (V3,31W) into the putative membrane-binding surface of the enzyme produced a protein with similar characteristics to the human group V enzyme. These characteristics included the ability of the mutant protein to hydrolyse 'self' membranes and to be internalised into cells. The introduced tryptophan residues have also been used as fluorescence reporter groups to propose a binding model for the protein on the membrane surface.

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Published date: 2002

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Local EPrints ID: 464906
URI: http://eprints.soton.ac.uk/id/eprint/464906
PURE UUID: ffa59f9a-9bcc-4f7e-bd75-018c0fa2e09b

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Date deposited: 05 Jul 2022 00:09
Last modified: 05 Jul 2022 03:31

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Author: Stephen A Beers

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