The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment.

• The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine (hsv//eng)

Nyckelord

Amino Acid Sequence

Animals

CHO Cells

Chromatography

Gel

Cricetinae

Dimerization

Disulfides

Electrophoresis

Polyacrylamide Gel

Genetic Vectors

Glycosylation

Hydrofluoric Acid

pharmacology

Immunoblotting

Microscopy

Electron

Molecular Sequence Data

Mucin-2

Mucins

chemistry

metabolism

Mutagenesis

Site-Directed

Peptides

chemistry

Precipitin Tests

Protein Binding

Protein Structure

Tertiary

Sequence Homology

Amino Acid

Spectrometry

Mass

Electrospray Ionization

Spectrometry

Mass

Matrix-Assisted Laser Desorption-Ionization

Trypsin

pharmacology

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