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The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells.

Journal article
Authors Martin Lidell
Malin E V Johansson
Matthias Mörgelin
Noomi Asker
James R Gum
Young S Kim
Gunnar C. Hansson
Published in The Biochemical journal
Volume 372
Issue Pt 2
Pages 335-45
ISSN 0264-6021
Publication year 2003
Published at Institute of Medical Biochemistry
Pages 335-45
Language en
Links dx.doi.org/10.1042/BJ20030003
Keywords Animals, Blotting, Western, CHO Cells, Colonic Neoplasms, chemistry, metabolism, Cricetinae, DNA Primers, chemistry, Dimerization, Disulfides, metabolism, Electrophoresis, Polyacrylamide Gel, Gastric Mucins, chemistry, metabolism, Glycoside Hydrolases, metabolism, Glycosylation, Green Fluorescent Proteins, Humans, Hydrofluoric Acid, pharmacology, Immunoglobulin Isotypes, metabolism, Luminescent Proteins, metabolism, Microscopy, Electron, Mucin 5AC, Mucin-2, Mucins, genetics, immunology, metabolism, Mutagenesis, Site-Directed, Plasmids, Polymerase Chain Reaction, Precipitin Tests, Proto-Oncogene Proteins c-myc, metabolism, Recombinant Fusion Proteins, Transfection
Subject categories Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Abstract

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

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