Cleavage in the GDPH sequence of the C-terminal cysteine-rich part of the human MUC5AC mucin.

• MUC5AC is the main gel-forming mucin expressed by goblet cells of the airways and stomach where it protects the underlying epithelia. We expressed the C-terminal cysteine-rich part of the human MUC5AC mucin in CHO-K1 cells (Chinese-hamster ovary K1 cells) where it formed disulfide-linked dimers in the ER (endoplasmic reticulum). After reducing the disulfide bonds of these dimers, not only the expected monomers were found, but also two smaller fragments, indicating that the protein was partially cleaved. The site of cleavage was located at an Asp-Pro bond situated in a GDPH (Gly-Asp-Pro-His) sequence found in the vWD4 (von Willebrand D4) domain. This sequence is also found in the human MUC2 mucin, previously shown to be cleaved at the same site by a slow, non-enzymatic process triggered by a pH below 6 [Lidell, Johansson and Hansson (2003) J. Biol. Chem. 278, 13944-13951]. In contrast with this, the cleavage of MUC5AC started already in the neutral ER. However, it continued and was slightly accelerated at a pH below 6.5, a pH found in the later parts of the secretory pathway. The cleavage generated a reactive group in the new C-terminus that could link the protein to a primary amine. No cleavage of MUC5AC has so far been reported. By using an antibody reacting with the C-terminal cleavage fragment, we could verify that the cleavage occurs in wild-type MUC5AC produced by HT-29 cells. The cleavage of MUC5AC and the generation of the reactive new C-terminus could contribute to the adherent and viscous mucus found at chronic lung diseases such as asthma and cystic fibrosis, characterized by mucus hypersecretion and lowered pH of the airways.

Nyckelord

Amino Acid Sequence

Animals

CHO Cells

Cell Line

Tumor

Cricetinae

Cysteine

metabolism

Gene Expression Regulation

Humans

Mucins

chemistry

genetics

metabolism

Mutagenesis

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Protease Inhibitors

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