| Budget Amount *help |
¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Research Abstract |
Thyroxine-Binding Globulin (TBG) is a principal thyroxine-binding protein in serum and is synthesized in the liver. The protein belongs to alpha1-antitrypsin family because of its structural similarity. In this investigation, physiological significance of carbohydrate moiety in TBG was studied. At first, we employed hepatome cell line HepG2 which synthesizes TBG and corticosteroid-binding globulin (CBG, also a member of alpha1-antitrypsin family). When the cell was treated with tunicamycin (inhibitor of core glycosylation), TBG was never secreted into the medium. On the other hand CBG was secreted into the medium without glycosylation. However, intracellular degradation was increased in both TBG and CBG when treated with tunicamycin. It was also noted that TBG synthesized in the presence of tunicamycin cross-reacted with anti-denatured TBG which had no thyroxine binding. Thus, it is concluded that carbohydrate moiety in TBG play important roles in T4-binding, secretion and intracellula
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r degradation.
To further investigate the role of carbohydrate residues in TBG, TBG-Gary found in a patient by Murata et al was studied. TBG-Gary was characterized by decreased T4-binding, annodal shift on isoelectric focusing and heat instability. The analysis of structure of TBG genome revealed that single point mutation in exon I causes amino acid substitution of 96th amino acid "isoleucine" to "asparagine". This amino acid substitution resulted in a creation of a potential glycosylation site (asparagine-cystine-serine). Complete cDNAs coding for normal TBG as well as TBG-Gary was constructed and inserted in expression vector pEUK-C1. Transfection of the respective plasmids into Cos 1 cells and subsequent analysis by pulse-chase experiment revealed that TBG-Gary was indeed has an additional carbohydrate residues, reduced T4-binding, and cross-reacted with anti-denatured TBG. Moreover, it was demons trated that TBG-Gary was much more rapidly degraded in the cells. When the mutant asparagine residue in TBG-Gary was substituted to aspartic acid by site-specific mutagenesis, the mutated TBG behaved like normal TBG.
These studies demonstrate that carbohydrate residues in TBG play important role in hormone binding, cellular transport, and metabolism. Less
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