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Proteoglycans support proper granule formation in pancreatic acinar cells

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Abstract

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.

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Abbreviations

CEL:

Carboxyl ester lipase

TGN:

Trans-Golgi network

ZG:

Zymogen granule

ZGC:

Zymogen granule content

ZGM:

Zymogen granule membrane

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Acknowledgments

We would like to thank Waltraud Ackermann (Marburg) and Tina Schrader (Exeter) for excellent technical assistance, Katja Schmidt for helpful comments, Ines Castro for help with the Figures, Hans and Beate Schrader for data mining and all those colleagues who provided antibodies (see “Materials and methods” section). This work was supported by the German Research Foundation (DFG; SCHR 518/5-1, 2) and the Portuguese Foundation for Science and Technology (FCT) [SFRH/BD/48722/2008 to M. A.].

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Authors and Affiliations

  1. Centre for Cell Biology and Department of Biology, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal

    Miguel Aroso & Michael Schrader

  2. Department of Cell Biology and Cell Pathology, University of Marburg, 35037, Marburg, Germany

    Brigitte Agricola

  3. College of Life and Environmental Sciences, Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD, UK

    Christian Hacker & Michael Schrader

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  1. Miguel Aroso
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  2. Brigitte Agricola
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  4. Michael Schrader
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Corresponding author

Correspondence to Michael Schrader.

Electronic supplementary material

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418_2015_1339_MOESM1_ESM.jpg

Supplementary Figure S1. Inhibition of proteoglycan synthesis in AR42J cells. Dexamethasone-stimulated AR42J cells were treated with different concentrations of β-d-xyloside (b-D-xyl) (0–2.5 mM) for 48 h, and the relative amounts of precipitable proteoglycans in cell homogenates were analyzed using the Blyscan assay. The data are from 4 independent experiments and are expressed as mean ± S.D. (JPEG 179 kb)

418_2015_1339_MOESM2_ESM.jpg

Supplementary Figure S2. Co-localisation of carboxypeptidase A and the Golgi marker p115 in unstimulated AR42J cells. Unstimulated AR42J cells (- dexa) were processed for immunofluorescence and stained with antibodies directed against the Golgi marker protein p115 (A) and the granule enzyme carboxypeptidase A (CBP A) (B). Note the co-localisation of both proteins at the Golgi complex (arrows) and the presence of fine punctate structures positive for carboxypeptidase A, which may represent constitutive cargo containers (B). Bar, 10 µm. (JPEG 500 kb)

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Aroso, M., Agricola, B., Hacker, C. et al. Proteoglycans support proper granule formation in pancreatic acinar cells. Histochem Cell Biol 144, 331–346 (2015). https://doi.org/10.1007/s00418-015-1339-x

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