In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113

• Published in: The Journal of biological chemistry 1992-07, Vol.267 (20), p.13998-14004
• Main Authors: JANSSEN, O. E, REFETOFF, S
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Cloning, Molecular; DNA - genetics; DNA - isolation & purification; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic Variation; Genetic Vectors; Humans; Liver - physiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Oocytes - metabolism; Polymerase Chain Reaction - methods; Protein Biosynthesis; Proteins; Recombinant Proteins - biosynthesis; Restriction Mapping; RNA - genetics; RNA - isolation & purification; Thyroxine-Binding Proteins - biosynthesis; Thyroxine-Binding Proteins - genetics; Transcription, Genetic; Xenopus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)49669-5

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.