BIOSYNTHESIS OF THE MUC2 MUCIN - EVIDENCE FOR A SLOW ASSEMBLY OF FULLY GLYCOSYLATED UNITS

The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [C-14]proline and [H-3]glucose over the last 4 8 h in culture. The cell layer was extracted with 6 M guanidinium chlo ride and the mature fully glycosylated mucins were isolated at a densi ty of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M g uanidinium chloride. These mucins were identified as MUC2 with an anti -peptide antibody. The macromolecules were fragmented by reduction int o two distinct populations of MUC2 subunits as assessed by agarose ele ctrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed b y rate-zonal centrifugation, ranged from 5 x 10(6) to 40 x 10(6) Da. H owever, the metabolically labelled MUC2 mucins, though found throughou t the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the for mation of the largest species must be preceded by glycosylation and oc cur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axels son, Carlstedt, and Hansson (1995) Biochem. J. 308, 873-880].