The sequence-specific peptide-binding activity of the protein sulfide isomerase AGR2 directs its stable binding to the oncogenic receptor EpCAM

M. Aiman Mohtar , Lenka Hernychova , J. Robert O'Neill , Melanie L. Lawrence , Euan Murray , Borek Vojtesek , Theodore Hupp

Abstract

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131–135 (VDPSL). A peptide binding site consensus of Tx[IL][YF] [YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro. Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM’s detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.
Author M. Aiman Mohtar
M. Aiman Mohtar,,
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, Lenka Hernychova
Lenka Hernychova,,
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, J. Robert O'Neill
J. Robert O'Neill,,
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, Melanie L. Lawrence
Melanie L. Lawrence,,
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, Euan Murray
Euan Murray,,
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, Borek Vojtesek
Borek Vojtesek,,
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, Theodore Hupp (ICCVS)
Theodore Hupp,,
- International Centre for Cancer Vaccine Science
Journal seriesMolecular & Cellular Proteomics, ISSN 1535-9476, (A 40 pkt)
Issue year2018
Vol17
No4
Pages737-763
Publication size in sheets1.3
DOIDOI:10.1074/mcp.RA118.000573
URL https://doi.org/10.1074/mcp.RA118.000573
Languageen angielski
LicenseOther; published final; Uznanie Autorstwa (CC-BY); with publication
Score (nominal)45
ScoreMinisterial score = 40.0, ArticleFromJournal
Ministerial score (2013-2016) = 45.0, ArticleFromJournal
Publication indicators WoS Impact Factor: 2017 = 5.232 (2) - 2017=6.323 (5)
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