Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor.
To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41 kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.
Siffroi-Fernandez
S
S
INSERM U38, Faculté de Médecine, Université de la Méditerranée, 27 Bd J. Moulin, 13385 Marseille Cedex 5, France.
Costagliola
S
S
Paumel
S
S
Giraud
A
A
Banga
J P
JP
Franc
J L
JL
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001216:Asparagine; D001667:Binding, Competitive; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008361:Mannosidases / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D009844:Oligosaccharides / Q000502:physiology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011487:Protein Conformation; D021122:Protein Subunits; D011989:Receptors, Thyrotropin / Q000096:biosynthesis*; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013972:Thyrotropin / Q000378:metabolism
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D021122:Protein Subunits; D011989:Receptors, Thyrotropin; D017485:1-Deoxynojirimycin; D001216:Asparagine; D013972:Thyrotropin; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase