In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113.
Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood. Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency. In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes. Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed. TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding. TBG-CD5 had altered electrophoretic mobility and did not bind T4. TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly. Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M. The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.
Janssen
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Department of Medicine, University of Chicago, Illinois 60637.
Refetoff
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D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013975:Thyroxine-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014981:Xenopus
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013975:Thyroxine-Binding Proteins; D012313:RNA; D004247:DNA