The contribution of tandem repeat number to the O-glycosylation of mucins.
The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.
Silverman
Howard Scott
HS
Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK.
Sutton-Smith
Mark
M
McDermott
Kimberley
K
Heal
Paul
P
Leir
Shih-Hsing
SH
Morris
Howard R
HR
Hollingsworth
Michael A
MA
Dell
Anne
A
Harris
Ann
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D018938:Caco-2 Cells / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D018598:Minisatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009077:Mucins / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment / Q000379:methods
D000939:Epitopes; C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D009077:Mucins; D009844:Oligosaccharides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins