Localization of O-glycans in MUC1 glycoproteins using electron-capture dissociation fragmentation mass spectrometry.
MUC1 is a mucin glycoprotein containing multiple tandem repeats of 20 amino acids, with five serines and threonines that can be O-glycosylated. Here, we investigated the O-glycosylation site occupancy in MUC1 glycoproteins produced in two mutant CHO cell lines, Lec3.2.8.1 and ldlD. We found that the average site occupancy was higher in MUC1 from Lec3.2.8.1 than from ldlD and that the occupancy increased with the number of tandem repeats in the protein and also depended on the culture conditions used for production. Moreover, we describe the successful use of electron-capture dissociation (ECD) fragmentation, coupled to online liquid chromatography mass spectrometry, to determine the glycosylation of individual sites in recombinant MUC1 proteins with 16 tandem repeats. We analyzed MUC1 tandem repeat peptides with 1-5 GalNAc residues by ECD fragmentation and found that the first site to be glycosylated was either Ser-5 or Thr-6, with the addition of a second GalNAc at Thr-14. For peptides with three GalNAc residues, several different variants of glycopeptides were found, indicating a heterogeneous order of glycosylation at this stage. In contrast, only one variant was found for peptides with four GalNAc residues, where Thr-19 in the PDTR motif was left unglycosylated, indicating that this site is glycosylated last. The results gave novel insight into the order of GalNAc substitution in MUC1 in vivo.
Sihlbom
Carina
C
Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
van Dijk Härd
Iris
I
Lidell
Martin E
ME
Noll
Thomas
T
Hansson
Gunnar C
GC
Bäckström
Malin
M
D000116:Acetylgalactosamine / Q000737:chemistry*; D000818:Animals; D016466:CHO Cells; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D013058:Mass Spectrometry; D018396:Mucin-1 / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010455:Peptides
D018396:Mucin-1; D010455:Peptides; D000116:Acetylgalactosamine