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.
Godl
Klaus
K
Department of Medical Biochemistry, Göteborg University, Gothenburg, Sweden.
Johansson
Malin E V
ME
Lidell
Martin E
ME
Mörgelin
Matthias
M
Karlsson
Hasse
H
Olson
Fredrik J
FJ
Gum
James R
JR
Kim
Young S
YS
Hansson
Gunnar C
GC
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D006858:Hydrofluoric Acid / Q000494:pharmacology; D015151:Immunoblotting; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology
D004220:Disulfides; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D010455:Peptides; D014357:Trypsin; D006858:Hydrofluoric Acid