Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

Harihar Milaganur Mohan,Boning Yang,Nicole A Dean,Malini Raghavan

doi:10.1074/jbc.ra120.014372

Harihar Milaganur Mohan, Boning Yang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra120.014372

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Abstract

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.

Highlights

The endoplasmic reticulum (ER) is responsible for ensuring that protein quality control is maintained throughout various complex steps that allow proteins to reach their native state after being synthesized as an unstructured polypeptide chain [1]
To examine cellular functions of CRT in antitrypsin Z (ATZ) folding and degradation, Calr2/2 mouse embryonic fibroblasts (MEFs, K42 cells [9]) were transduced with retroviruses that encoded either WT human CRT, the Y92A mutant of human CRT that is deficient for binding monoglucosylated glycans [35, 36] or a control virus lacking calreticulin
In the case of eYFPATZ, we observed a small, nonsignificant increase in the media fluorescence of CRT WT over CRT2/2 cells, which was accompanied by a reduction in cellular enhanced YFP (eYFP) mean fluorescence intensity (MFI) (Fig. 1E and Fig. S1B, right panels)

Summary

Introduction

The endoplasmic reticulum (ER) is responsible for ensuring that protein quality control is maintained throughout various complex steps that allow proteins to reach their native state after being synthesized as an unstructured polypeptide chain [1]. In transfections with plasmids encoding eYFP-ATZ, the media fluorescence values were comparable between CRT WT and CRT Y92A, and both were slightly higher than that from CRT2/2 cells (Fig. 1E, right panel).

Results

Conclusion