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Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagesesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2

Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagesesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2
Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagesesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2
An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC- rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365]
0006-2960
7326-7338
Beers, S.A.
a02548be-3ffd-41ab-9db8-d6e8c3b499a2
Buckland, A.G.
378d0e1a-8bde-4754-9d1a-dd3cf1eb8207
Giles, N.
ef6f7af2-b4cb-4ae6-9f2b-100380d8f39f
Gelb, M.H.
be15b679-9672-486f-8d02-3eb65da4dad2
Wilton, D.C.
4b995f66-ad6c-4d96-9179-c64f3b54466a
Beers, S.A.
a02548be-3ffd-41ab-9db8-d6e8c3b499a2
Buckland, A.G.
378d0e1a-8bde-4754-9d1a-dd3cf1eb8207
Giles, N.
ef6f7af2-b4cb-4ae6-9f2b-100380d8f39f
Gelb, M.H.
be15b679-9672-486f-8d02-3eb65da4dad2
Wilton, D.C.
4b995f66-ad6c-4d96-9179-c64f3b54466a

Beers, S.A., Buckland, A.G., Giles, N., Gelb, M.H. and Wilton, D.C. (2003) Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagesesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2. Biochemistry, 42 (24), 7326-7338. (doi:10.1021/bi0343222).

Record type: Article

Abstract

An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC- rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365]

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Published date: 2003

Identifiers

Local EPrints ID: 43102
URI: http://eprints.soton.ac.uk/id/eprint/43102
ISSN: 0006-2960
PURE UUID: ee373efc-db5d-4751-80ac-ed25b854d70e
ORCID for S.A. Beers: ORCID iD orcid.org/0000-0002-3765-3342

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Date deposited: 11 Jan 2007
Last modified: 16 Mar 2024 03:18

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Contributors

Author: S.A. Beers ORCID iD
Author: A.G. Buckland
Author: N. Giles
Author: M.H. Gelb
Author: D.C. Wilton

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