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

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. 35 S pulse-chase kinetic experiments revealed that a truncated mutant lacking the C-terminal 14 amino acids (pRMCDCT) 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.Keywords: mucin; furin; goblet cell; dimer; Muc2.The present study deals with a novel cleavage reaction in rat intestinal Muc2, one which was not anticipated from previous mucin research, and has no known parallel in other mucins. Because of sequence homology however, it is also expected to occur in human MUC2. Our experiments document a posttranslational proteolytic cleavage at the extreme C-terminus of the intestinal secretory mucin rat Muc2.The C-terminus of rat Muc2 (earlier called MLP, mucin-like peptide) [1], consists of over 800 residues containing cysteinerich domains that are structurally homologous with many other secretory mucins [2±6], and with the D4, B, C and cystine knot (CK) domains of human von Willebrand factor (vWF) [7,8]. The CK domain, which includes 85±90 residues near the C-terminal end, has been shown to be important in mediating dimerization of vWF and porcine submandibular mucin [9]. Immediately C-terminal to the CK domain in human intestinal MUC2 and rat Muc2 there is a unique 14-amino-acid cationic domain, which completes the polypeptide chain. In this sequence there are 5 arginines and 1 lysine in rat Muc2, and 5 arginines in the corresponding human MUC2 sequence.In a previous report [10] we showed that rat Muc2 in crude tissue extracts and homogenates, or purified from rat intestine, did not interact with an antibody specific for the synthetic peptide E20±14, which corresponds to the C-terminal 14 amino acids. The antibody localized to the ER, golgi, and condensing vacuole region of goblet cells in intestinal sections, and signals also appeared along the lower margins of the theca, a filamentrich structure which surrounds the apical granule mass [11,12].Immunofluorescence was not seen over the mucin granule mass, however. By contrast, an antibody raised against a second synthetic peptide, G21±10, i...