Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells

Molecular chaperones are presumed to associate with large secretory mucin g lycoproteins during their synthesis in the endoplasmic reticulum (ER), but have not been identified to date. We decided to look for possible involveme nt of the chaperones calreticulin (CRT) and calnexin (CLN) during synthesis of two similar gastrointestinal mucins, MUC2 and MUC5AC. Pulse-chase label ling of MUC2 and MUC5AC with [S-35]methionine/cysteine ([S-35]Promix) was p erformed using LS180 and HT29/A1 colonic carcinoma cell lines and was follo wed by immunoprecipitation with anti-mucin and anti-chaperone antibodies. T he precipitated labelled mucin precursors were analysed by SDS/PAGE and aut oradiography. Using antibodies specific for each mucin, newly synthesized m onomeric precursors of both MUC2 and MUC5AC were detected after a Ij,in pul se and then disappeared as oligomers were formed during a 2 h chase period. Only homo-oligomers of MUC2 and MUC5AC were present in the cells. Using an ti-CRT, the MUC2 monomeric precursor and oligomer were co-precipitated from both cell lines after a 15 min pulse and the oligomer less strongly after a 0.5 h chase, but there was little co-precipitation after a 2h chase. At t his time, MUC2 immunoprecipitated by anti- MUC2 was completely oligomerized and was endo-beta-N-acetylglucosaminidase-resistant, indicating that the m ucin had reached the Golgi region. MUC2 co-precipitated with CRT at zero ti me and 0.5 h was endo-beta-N-acetylglucosaminidase-sensitive; therefore CRT must have associated with MUC2 in the ER. Treatment with tunicamycin (TUN) diminished the binding of MUC2 to CRT, suggesting a requirement for initia l N-glycan addition during this process. Using anti-CLN, only a weak co-pre cipitation of MUC2, compared with that seen with anti-CRT, was detected in LS180 cells. In contrast with the findings for MUC2, there was no co-precip itation of MUC5AC with CRT or CLN from either cell line at the various time points. In conclusion, CRT and CLN appear to be involved in MUC2 synthesis at the stage of folding and oligomerization in the ER. Since no interactio n of the chaperones with MUC5AC was detected at a similar stage of synthesi s, these two structurally similar secretory mucins seem to have different c haperone requirements in the ER.