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The cationic C-terminus of rat Muc2 facilitates dimer formation post translationally and is subsequently removed by furin.

European journal of biochemistry / FEBS

Transfection, Animals, Blotting, Western, COS Cells, Dimerization, Electrophoresis, Polyacrylamide Gel, Epitopes, metabolism, Furin, In Situ Hybridization, Intestines, Kinetics, Mucin-2, Mucins, Mutagenesis, Site-Directed, Plasmids, Precipitin Tests, Protein Processing, Post-Translational, Rats, Subtilisins

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      Abstract

      Earlier immunolocalization experiments showed that the extreme cationic C-terminus of the rat intestinal mucin Muc2 (RMC) was present at the base of intestinal goblet cells in the vicinity of ER and golgi compartments, but was not found with the rest of the mucin in apical storage granules. This prompted us to investigate the possibility that an early proteolytic cleavage reaction occurs post-translationally. A plasmid pRMC, encoding the C-terminal 534 amino acids of the mucin, was expressed in COS-7 cells and was shown to undergo cleavage at an R-T-R-R sequence located within the C-terminal 14 amino acids. Cleavage did not occur with the construct RMCfH, a furin site-mutated (A-T-A-A) counterpart of pRMCH (poly His6 tagged RMC). Addition of a furin inhibitor to COS-7 cell incubations also prevented cleavage of RMC and RMCH products. 35S pulse-chase kinetic experiments revealed that a truncated mutant lacking the C-terminal 14 amino acids (pRMCDeltaCT) forms faulty (doublet) dimers in the ER. These were not secreted as efficiently as the normal dimer of wild-type (pRMC) constructs. Thus the cationic C-terminus of rMuc2 apppears to facilitate the correct formation of normal Muc2 domain dimers.

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      10806399

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