Abstract
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.
Highlights
The last stages of thyroglobulin maturation occur in the thyroid follicular lumen and include thyroid hormone formation and glycan completion
Isolation of a Human Thyroglobulin Fragment-Fusion Protein Recognized by mAb240 —pEX human thyroglobulin expression libraries were prepared from PstI fragments of thyroglobulin cDNA inserted into the three forms of the pEX plasmid, corresponding to the three reading frames
Nitrocellulose culture disk replicas were tested for the presence of peptides encoding the receptor thyroglobulin binding domain by using mAb78 and mAb240 antibodies
Summary
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. 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 for the intrafollicular retention or lysosomal homing of the prohormone. The glycoprotein is synthesized and partially matured within thyrocytes (intramonomer disulfide bonding, glycosylation, sulfatation, phosphorylation) and secreted into the follicular lumen In this compartment, thyroglobulin is further processed by iodination, hormonogenesis, and completion of some complex type oligosaccharide units [2]
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