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

Earlier immunolocalization experiments showed that the extreme cationic C-t erminus of the rat intestinal mucin Muc2 (RMC) was present at the base of i ntestinal goblet cells in the vicinity of ER and golgi compartments, but wa s not found with the rest of the mucin in apical storage granules. This pro mpted us to investigate the possibility that an early proteolytic cleavage reaction occurs post-translationally. A plasmid pRMC, encoding the C-termin al 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 1 4 amino acids. Cleavage did not occur with the construct RMCfH, a furin sit e-mutated (A-T-A-A) counterpart of pRMCH (poly His6 tagged RMC). Addition o f a furin inhibitor to COS-7 cell incubations also prevented cleavage of RM C and RMCH products. S-35 pulse-chase kinetic experiments revealed that a t runcated mutant lacking the C-terminal 14 amino acids (pRMC Delta CT) 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-ter minus of rMuc2 apppears to facilitate the correct formation of normal Muc2 domain dimers.