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A MUC1 tandem repeat reporter protein produced in CHO-K1 cells has sialylated core 1 O-glycans and becomes more densely glycosylated if coexpressed with polypeptide-GalNAc-T4 transferase

Artikel i vetenskaplig tidskrift
Författare Fredrik J. Olson
Malin Bäckström
Hasse Karlsson
J. Burchell
Gunnar C. Hansson
Publicerad i Glycobiology
Volym 15
Nummer/häfte 2
Sidor 177-91
ISSN 0959-6658 (Print)
Publiceringsår 2005
Publicerad vid Institutionen för medicinsk och fysiologisk kemi
Sidor 177-91
Språk en
Länkar www.ncbi.nlm.nih.gov/entrez/query.f...
Ämnesord Animals, CA-15-3 Antigen/genetics/*metabolism, COS Cells, Cricetinae, Gene Expression/*genetics, Genes, Reporter/genetics/physiology, *Glycosylation, N-Acetylgalactosaminyltransferases/genetics/*metabolism, N-Acetylneuraminic Acid/genetics/*metabolism
Ämneskategorier Medicin och Hälsovetenskap

Sammanfattning

A recombinant mucin O-glycosylation reporter protein, containing 1.7 tandem repeats (TRs) from the transmembrane mucin MUC1, was constructed. The reporter protein, MUC1(1.7TR)-IgG2a, was produced in CHO-K1 cells to study the glycosylation of the MUC1 TR and the in vivo role of polypeptide-GalNAc-T4 glycosyltransferase. N-terminal sequencing of MUC1(1.7TR)-IgG2a showed that all five potential O-glycosylation sites within the TR were used, with an average density of 4.5 glycans per repeat. The least occupied site was Thr in the PDTR motif, where 75% of the molecules were glycosylated, compared to 88-97% at the other sites. This glycan density was confirmed by an alternative liquid chromatography-mass spectrometry (LC-MS) based approach. The O-linked oligosaccharides were released from MUC1(1.7TR)-IgG2a and analyzed by nano-LC-MS and LC-MS/MS. Four oligosaccharides were present, NeuAcalpha2-3Galbeta1-3GalNAcol, NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAcol, Galbeta1-3(NeuAcalpha2-6)GalNAcol, and Galbeta1-3GalNAcol, the two first being most abundant. Coexpression of the human polypeptide-GalNAc-T4 transferase with MUC1(1.7TR)-IgG2a increased the glycan occupancy at Thr in PDTR, Ser in VTSA, and Ser in GSTA, supporting the function of GalNAc-T4 proposed from previous in vitro studies. The expression of GalNAc-T4 with a mutation in the first lectin domain (alpha) had no glycosylation effect on PDTR and GSTA but surprisingly gave a dominant negative effect with a decreased glycosylation to around 50% at the Ser in VTSA. The results show that introduction of glycosyltransferases can specifically alter the sites for O-glycosylation in vivo.

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