Identification of the membrane receptor binding domain of thyroglobulin. Insights into quality control of thyroglobulin biosynthesis.
The last stages of thyroglobulin maturation occur in the thyroid follicular lumen and include thyroid hormone formation and glycan completion. In this compartment, newly secreted thyroglobulins interact with a thyrocyte membrane receptor that prevents their premature lysosomal transfer and degradation. Both GlcNAc moieties and thyroglobulin peptide determinants are involved in receptor interaction. Here we used monoclonal antibodies (mAbs) directed against human thyroglobulin either to inhibit (mAb78) or to enhance (mAb240) the thyroglobulin binding and to identify the region of the thyroglobulin involved in the receptor recognition. Peptides containing the mAb epitopes were obtained by immunoscreening cyanogen bromide-derived native human thyroglobulin peptides and a cDNA thyroglobulin expression library. Three peptides, localized in the thyroglobulin N-terminal domain, were obtained. Peptides N1 (Ala1148-Gln1295) and N2 (Ser789-Met1008) were recognized by mAb240 and mAb78, respectively. None of them bound the receptor. The third peptide, N3 (Ser789-Met1172), (i) overlapped all or part of the N1 and N2 peptide sequences and was recognized by both mAbs, (ii) carried two complex glycans at Asn797 and Asn928, of which a subset presented accessible GlcNAc residues, and (iii) inhibited the thyroglobulin binding to FRTL5 cell membrane preparations. The N3 peptide includes tyrosine residues that have been reported to be involved in hormone formation. These results suggest that structural modifications closely associated with hormone formation within this domain act as sensors for the receptor interaction and thus for the intrafollicular retention or lysosomal homing of the prohormone.
Mezghrani
A
A
Laboratoire de Biochimie, Ingénierie des Protéines, UMR 6560, Institut Fédératif Jean Roche, Faculté de Médecine-Nord, Boulevard P. Dramard, 13916 Marseille Cedex 20, France.
Mezgrhani
H
H
Mziaut
H
H
Courageot
J
J
Oughideni
R
R
Bastiani
P
P
Miquelis
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D018604:Epitope Mapping; D000939:Epitopes; D006020:Glycopeptides / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D013954:Thyroglobulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006020:Glycopeptides; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C032198:thyroglobulin receptor; D013954:Thyroglobulin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D003488:Cyanogen Bromide