TY - JOUR
T1 - Evaluation of MUC6 mucin tandem repeats
AU - Parry, Simon
AU - Sutton-Smith, Mark
AU - Heal, Paul
AU - Leir, Shih Hsing
AU - Palmai-Pallag, Timea
AU - Morris, Howard R.
AU - Hollingsworth, Michael A.
AU - Dell, Anne
AU - Harris, Ann
N1 - Funding Information:
We thank Dr Colm Reid and Deborah Harrison for assistance. This work was supported by the Cystic Fibrosis Trust (UK), the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust including a Biomedical Research Collaboration Grant. AD is a BBSRC Professorial Fellow. M.S-S. was a recipient of a Medical Research Council studentship.
PY - 2005/2/11
Y1 - 2005/2/11
N2 - The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.
AB - The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.
KW - Caco2 cell
KW - Fast atom bombardment mass spectrometry
KW - MUC6 mucin
KW - Panc1 cell
KW - Pancreas
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U2 - 10.1016/j.bbagen.2004.11.010
DO - 10.1016/j.bbagen.2004.11.010
M3 - Article
C2 - 15716126
AN - SCOPUS:13544251475
SN - 0304-4165
VL - 1722
SP - 77
EP - 83
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 1
ER -