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Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification

Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification
Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification
Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ERp57 and the ERp57-tapasin conjugate by rapid acidification of the intracellular environment with trichloroacetic acid (TCA), followed by thiol modification with the alkylating agent 4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). By using TCA/AMS treatment, non-tapasin-associated ERp57 is shown to exist almost exclusively in a reduced state, suggesting that both thioredoxin-like CXXC motifs are exposed and reduced. A 110-kDa product is readily detected with this TCA/AMS protocol and is confirmed as an ERp57-tapasin conjugate by its absence from the tapasin-deficient .220 cell line and by immunoblotting with both ERp57- and tapasin-specific antisera. The ERp57-tapasin conjugate can also be modified with the oxidizing agent diamide, indicating that within the pool of ERp57-tapasin complexes the free, non-tapasin-linked CXXC motif exists in both oxidized and reduced states, suggesting availability to undergo redox reactions.
1523-0864
375-379
Antoniou, Antony N.
3d711a3b-f688-46f9-823e-5bb925d10ea8
Powis, Simon J.
1bdcdc68-9097-459d-9675-7bb408316bf9
Antoniou, Antony N.
3d711a3b-f688-46f9-823e-5bb925d10ea8
Powis, Simon J.
1bdcdc68-9097-459d-9675-7bb408316bf9

Antoniou, Antony N. and Powis, Simon J. (2003) Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification. Antioxidants & Redox Signaling, 5 (4), 375-379. (doi:10.1089/152308603768295104).

Record type: Article

Abstract

Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ERp57 and the ERp57-tapasin conjugate by rapid acidification of the intracellular environment with trichloroacetic acid (TCA), followed by thiol modification with the alkylating agent 4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). By using TCA/AMS treatment, non-tapasin-associated ERp57 is shown to exist almost exclusively in a reduced state, suggesting that both thioredoxin-like CXXC motifs are exposed and reduced. A 110-kDa product is readily detected with this TCA/AMS protocol and is confirmed as an ERp57-tapasin conjugate by its absence from the tapasin-deficient .220 cell line and by immunoblotting with both ERp57- and tapasin-specific antisera. The ERp57-tapasin conjugate can also be modified with the oxidizing agent diamide, indicating that within the pool of ERp57-tapasin complexes the free, non-tapasin-linked CXXC motif exists in both oxidized and reduced states, suggesting availability to undergo redox reactions.

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Published date: 2003

Identifiers

Local EPrints ID: 26199
URI: http://eprints.soton.ac.uk/id/eprint/26199
ISSN: 1523-0864
PURE UUID: 91772956-00b2-464f-bbc7-d5a604e1ffe5

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Date deposited: 21 Apr 2006
Last modified: 08 Jan 2022 09:54

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Author: Antony N. Antoniou
Author: Simon J. Powis

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