On the assembly and O-glycosylation of the MUC2 mucin. Crucial steps in the formation of the intestinal mucus barrier

Abstract

Gel-forming mucins comprise the major barrier between the external and the internal milieu on all mucosal surfaces. In the intestines, where the lumenal milieu is especially demanding, the MUC2 mucin dominates. The aim of this work was to elucidate different aspects of the assembly and O-glycosylation of this mucin, i. e. the processes converting the primary transcript into a native gel. This field is of pathological relevance: Barrier defects are probably important in for instance peptic ulcers and inflammatory bowel disease. Mucin overproduction and hyperviscosity are found in cystic fibrosis and obstructive airway diseases. Although the medical interest, gel-forming mucins are poorly studied, highly due to the difficulties associated with the large molecular size and glycosylation dependent heterogeneity. The MUC2 monomer has a molecular mass of 600 kDa without and presumably 3 MDa with O-glycans.MUC2 mucin assembly was mostly studied by metabolic labeling of LS 174T cells, immunoprecipitation, SDS-agarose gel electrophoresis and autoradiography, occasionally in combination with subcellular fractionation. Size estimation of different MUC2 species was also performed by rate zonal ultracentrifugation. Mucins were purified by isopycnic density gradient ultracentrifugation and assayed by Western blots. O-glycans were studied by gas chromatography, gas chromatography - mass spectrometry and monosaccharide compositional analysis based on HPLC. Glycosyltransferase localization was monitored by fluorescence microscopy and subcellular fractionation of culture cells, where Golgi pH was manipulated with ammonium chloride and bafilomycin A1.It was shown that the MUC2 mucin dimerized within the ER based on disulfide bonds. Both monomers and dimers were transferred to the Golgi, where O-glycosylation started. In the Golgi or later, nonreducible, intermolecular bonds of yet unknown nature were formed between MUC2 molecules. Insolubility of the mucin seemed to be associated with these bonds. A method for deglycosylation of mucins on blotting membranes and in microtiter wells, using gaseous hydrogen fluoride, was developed and found to be valuable in immunoassays. A role of the secretory pathway pH gradient in glycosyltransferase localization was demonstrated, as disruption of this gradient caused relocalization of three different glycosyltransferases. Neutralization of the pH also gave rise to drastic changes in mucin O-glycosylation, suggesting that a proper glycosyltransferase distribution is necessary for mucin O-glycosylation.The MUC2 mucin possesses C-terminal similarities as for cysteines with the von Willebrand factor, making ER-based, disulfide bond stabilized dimerization an expected phenomenon. In contrast, the nonreducible bonds were not predicted, but represent a novel mucosal defense line against reducing agents. They might cause insolubility by crosslinking MUC2 chains, and if so they should be crucial for homeostasis, since mucin insolubility is probably necessary for the barrier function in water filled lumen organs. The pH dependence found for glycosyltransferase localization and mucin O-glycosylation could be pathologically relevant. Golgi pH alterations have been suggested in cystic fibrosis, where O-glycosylation changes could explain the changed mucus quality. In peptic ulcers, ammonia produced by Helicobacter pylori urease could neutralize Golgi pH, thereby changing mucin O-glycosylation, leading to a defective mucus barrier and consequently mucosal injury by hydrochloric acid.