An autocatalytic cleavage in the C terminus of the human MUC2 mucin occurs at the low pH of the late secretory pathway.
During purification of a recombinant MUC2 C terminus expressed in CHO-K1 cells, the protein was partly cleaved when buffers with a pH of 6.0 were used. When buffers with higher pH values were used, less cleavage was found. Disulfide bonds held the two fragments generated together as these were only observed after reduction. Edman sequencing of the C-terminal 110-kDa fragment revealed that the cleavage had occurred at an Asp-Pro bond, a site described previously to generate the so-called "link peptide" after disulfide bond reduction. In vitro studies on the conditions for cleavage showed that it occurred in a time-dependent manner at a pH below 6.0. Furthermore, the reaction was not enzyme-mediated as it occurred in pure preparations of the MUC2 C terminus and was not inhibited by protease inhibitors. When expressed in the mucin producing cell line LS 174T, the C terminus was cleaved to a higher extent compared with the CHO-K1 cells. Neutralizing the secretory pathway with either NH(4)Cl or bafilomycin A1 inhibited this cleavage. Altogether, our results suggest that the cleavage is an autocatalytic reaction that occurs in the acidic environment of the late secretory pathway. Furthermore, the cleavage produced a new, reactive C terminus that has the potential to attach the mucin to itself or other molecules. Because a pH below 6 can be reached in the late secretory pathway and on mucosal surfaces, the cleavage and possible cross-linking are likely to be of biological importance.
Lidell
Martin E
ME
Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, Sweden.
Johansson
Malin E V
ME
Hansson
Gunnar C
GC
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D001710:Biotin / Q000494:pharmacology; D019921:Biotinylation; D015153:Blotting, Western; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018942:Macrolides; D008969:Molecular Sequence Data; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000900:Anti-Bacterial Agents; D003432:Cross-Linking Reagents; D004220:Disulfides; C524625:MUC2 protein, human; D018942:Macrolides; D055262:Mucin-2; D009077:Mucins; D010455:Peptides; D011994:Recombinant Proteins; D001710:Biotin; C040929:bafilomycin A1