Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 15, Pages 5645-5650Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1120269109
Keywords
mucus; bicarbonate; cystic fibrosis; unpacking
Categories
Funding
- Swedish Research Council [7461, 21027]
- Swedish Cancer Foundation
- Knut and Alice Wallenberg Foundation
- IngaBritt and Arne Lundberg Foundation, Sahlgren's University Hospital (LUA-ALF)
- Wilhelm and Martina Lundgren's Foundation, Torsten och Ragnar Soderbergs Stiftelser
- Sahlgrenska Academy, National Institute of Allergy and Infectious Diseases [U01AI095473]
- Swedish Foundation for Strategic Research - The Mucus-Bacteria-Colitis Center
- Center for Biosciences at Karolinska Institutet
- Swedish Cystic Fibrosis Foundation
- Erica Lederhausen's Foundation
- Lederhausen's Center for Cystic Fibrosis Research
- University of Gothenburg
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MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. TheMUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.
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