Capillary zone electrophoresis and MALDI-mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide.
The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.
Soudan
B
B
Unité INSERM No. 377, Lille, France. soudan@lille.inserm.fr
Hennebicq
S
S
Tetaert
D
D
Boersma
A
A
Richet
C
C
Demeyer
D
D
Briand
G
G
Degand
P
P
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D055271:Mucin 5AC; D009077:Mucins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013912:Threonine / Q000737:chemistry
C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D013912:Threonine; D012694:Serine