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AGR2, an endoplasmic reticulum protein, is secreted into the gastrointestinal mucus.

Bergström JH, Berg KA, Rodríguez-Piñeiro AM, Stecher B, Johansson ME, Hansson GC - PLoS ONE (2014)

Bottom Line: We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles.Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal.In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

ABSTRACT
The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2(-/-) mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.

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AGR2 detection in CHO-K1 cells transfected with plasmids encoding the human AGR2 and mutated versions.(A) Cell lysate and (B) spent culture media of the transfected cells separated on a reducing SDS-PAGE and Western blotted. Band intensities of AGR2 normalized to the strongest band (C81S) in the blots of three replicates in (A) and (B) are ahown as a histogram in Fig. S3. All the AGR2 forms were found in the lysate at the expected size. The truncated AGR2 (ΔKTEL) and the C81S were secreted out into the media. C. Loading control for panel A. showing α-actin expression in corresponding lanes. Band intensities normalized to the strongest band in the blot is noted in the corresponding lane. (D) AGR2 transfected CHO-K1 cells immunostained for AGR2 (red) show ER localization, cells co-stained with the ER-marker Calnexin (green).
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pone-0104186-g006: AGR2 detection in CHO-K1 cells transfected with plasmids encoding the human AGR2 and mutated versions.(A) Cell lysate and (B) spent culture media of the transfected cells separated on a reducing SDS-PAGE and Western blotted. Band intensities of AGR2 normalized to the strongest band (C81S) in the blots of three replicates in (A) and (B) are ahown as a histogram in Fig. S3. All the AGR2 forms were found in the lysate at the expected size. The truncated AGR2 (ΔKTEL) and the C81S were secreted out into the media. C. Loading control for panel A. showing α-actin expression in corresponding lanes. Band intensities normalized to the strongest band in the blot is noted in the corresponding lane. (D) AGR2 transfected CHO-K1 cells immunostained for AGR2 (red) show ER localization, cells co-stained with the ER-marker Calnexin (green).

Mentions: The murine Agr2 and the human AGR2 were found in considerable amounts in the intestinal mucus, despite the presence of the non-typical ER retention signal KTEL in their C-termini. AGR2 expressed in CHO-K1 cells was also retained in the ER and not secreted (see Fig. 6D). To clarify these discrepancies, CKO-K1 cells were transiently transfected with plasmids encoding human WT AGR2, AGR2 with its Cys81 replaced by Ser (C81S), AGR2 with its C-terminal KTEL altered to KDEL (T173D), or AGR2 without its C-terminal KTEL (ΔKTEL). Proteins from the cell lysates were separated by reducing PAGE and AGR2 detected by Western blot (Fig. 6A–B). All the AGR2 protein variants were detected in the cell lysates (Fig. 6). However, only the C81S and ΔKTEL AGR2 forms were found in the spent culture media. That the ΔKTEL was not retained has been shown before and was thus expected [23]. However, that its single Cys was involved in controlling ER exit has not been observed previously. The results suggest a specific functional coupling between the ER retaining KTEL signal and the single free Cys for AGR2 retention.


AGR2, an endoplasmic reticulum protein, is secreted into the gastrointestinal mucus.

Bergström JH, Berg KA, Rodríguez-Piñeiro AM, Stecher B, Johansson ME, Hansson GC - PLoS ONE (2014)

AGR2 detection in CHO-K1 cells transfected with plasmids encoding the human AGR2 and mutated versions.(A) Cell lysate and (B) spent culture media of the transfected cells separated on a reducing SDS-PAGE and Western blotted. Band intensities of AGR2 normalized to the strongest band (C81S) in the blots of three replicates in (A) and (B) are ahown as a histogram in Fig. S3. All the AGR2 forms were found in the lysate at the expected size. The truncated AGR2 (ΔKTEL) and the C81S were secreted out into the media. C. Loading control for panel A. showing α-actin expression in corresponding lanes. Band intensities normalized to the strongest band in the blot is noted in the corresponding lane. (D) AGR2 transfected CHO-K1 cells immunostained for AGR2 (red) show ER localization, cells co-stained with the ER-marker Calnexin (green).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4128659&req=5

pone-0104186-g006: AGR2 detection in CHO-K1 cells transfected with plasmids encoding the human AGR2 and mutated versions.(A) Cell lysate and (B) spent culture media of the transfected cells separated on a reducing SDS-PAGE and Western blotted. Band intensities of AGR2 normalized to the strongest band (C81S) in the blots of three replicates in (A) and (B) are ahown as a histogram in Fig. S3. All the AGR2 forms were found in the lysate at the expected size. The truncated AGR2 (ΔKTEL) and the C81S were secreted out into the media. C. Loading control for panel A. showing α-actin expression in corresponding lanes. Band intensities normalized to the strongest band in the blot is noted in the corresponding lane. (D) AGR2 transfected CHO-K1 cells immunostained for AGR2 (red) show ER localization, cells co-stained with the ER-marker Calnexin (green).
Mentions: The murine Agr2 and the human AGR2 were found in considerable amounts in the intestinal mucus, despite the presence of the non-typical ER retention signal KTEL in their C-termini. AGR2 expressed in CHO-K1 cells was also retained in the ER and not secreted (see Fig. 6D). To clarify these discrepancies, CKO-K1 cells were transiently transfected with plasmids encoding human WT AGR2, AGR2 with its Cys81 replaced by Ser (C81S), AGR2 with its C-terminal KTEL altered to KDEL (T173D), or AGR2 without its C-terminal KTEL (ΔKTEL). Proteins from the cell lysates were separated by reducing PAGE and AGR2 detected by Western blot (Fig. 6A–B). All the AGR2 protein variants were detected in the cell lysates (Fig. 6). However, only the C81S and ΔKTEL AGR2 forms were found in the spent culture media. That the ΔKTEL was not retained has been shown before and was thus expected [23]. However, that its single Cys was involved in controlling ER exit has not been observed previously. The results suggest a specific functional coupling between the ER retaining KTEL signal and the single free Cys for AGR2 retention.

Bottom Line: We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles.Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal.In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

ABSTRACT
The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2(-/-) mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.

Show MeSH
Related in: MedlinePlus