Mutations in a Specific Human Serum Albumin Thyroxine Binding Site Define the Structural Basis of Familial Dysalbuminemic Hyperthyroxinemia

Abstract

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants. Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA. Sitedirected mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA. These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine. Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA. Familial dysalbuminemic hyperthyroxinemia (FDH),1 an autosomal dominant condition in which the total thyroxine level in serum is elevated while the free thyroxine level is normal, results from the presence of an abnormal human serum albumin (HSA) with an enhanced affinity for thyroxine (1). Although this condition had been widely reported in the medical literature (1-8), the molecular basis of FDH was not known until the identification of a single point mutation in the HSA gene of several FDH individuals resulting in the substitution of histidine for arginine at amino acid position 218 (9). This result was confirmed by another study in which the same mutation was identified in FDH individuals from eight unrelated families (10). Recently, it was shown that recombinantly produced FDH HSA has an enhanced affinity for thyroxine similar to that seen for natural FDH HSA (11), a result that confirmed that all of the information necessary to generate the FDH phenotype is contained in the FDH mutation.The binding of thyroxine to HSA has been extensively studied (12-23), yet the molecular basis of this interaction remains obscure. Early studies used equilibrium dialysis to measure the binding of radiolabeled thyroxine and radiolabeled thyroxine analogs to HSA. Interpretation of these results was complicated by the observation of several binding components, which were difficult to resolve. For example, some of these studies assigned four equal binding sites for thyroxine with dissociation constants (K d ) of 6.6 M (12-14), whereas other studies resolved the binding data into two sites with The aforementioned results indicated that HSA has multiple thyroxine binding sites, whereas the existence of a specific thyroxine binding site in the 2A subdomain of HSA was suggested by other observations. ...

A point mutation in the albumin gene in a Chinese patient with familial dysalbuminemic hyperthyroxinemia

“…The diagnosis was eventually confirmed by demonstrating the point mutation in exon 7 of the albumin gene as described in western white populations (14,15), although this differs from the mutation reported in a Japanese family (16). The guanine to adenosine transition in the second nucleotide of codon 218 of the albumin gene, resulting in the replacement of the normal arginine by histidine in one of the two alleles has been confirmed to be responsible for the phenotype of FDH in western white families (18,19). The guanine to cytosine transition in the same position, causing the replacement of the arginine by proline in the Japanese kindred, produced a distinct phenotype of FDH that displays greater serum tT 4 , tT 3 and rT 3 concentrations (16).…”

“…Recently, the molecular basis of FDH has been found to be a substitution of histidine (CAC) and proline (CCC) for arginine (CGC) in codon 218 of the albumin gene in unrelated western families and in a Japanese kindred, respectively (14)(15)(16). Recombinant human serum albumin with a substitution of His 218 for Arg 218 has been shown to have an increased affinity for T 4 , similar to the human serum albumin purified from the FDH patients, confirming that the substitution of His 218 for Arg 218 generates the phenotype of FDH (17)(18)(19). The K a of FDH albuminPro 218 for T 4 in the Japanese kindred was approximately 80-fold that of albumin in normal individuals and the replacement of arginine by proline is believed to be responsible for this condition (16).…”

“…In human serum albumin, the guanidino group of arginine at codon 218 is involved in an unfavorable binding interaction with the amino group of T 4 (19). As neither histidine nor proline has the guanidino group, the FDH His 218 -albumin or Pro 218 -albumin produces a more favorable binding interaction with T 4 .…”

The extraordinary ligand binding properties of human serum albumin

“…His and Arg218 ! Pro mutations within subdomain IIA greatly enhance the affinity for thyroxine and causes the elevated serum total thyroxine levels associated with familial dysalbuminemic hyperthyroxinemia (52).…”

A Dynamic Model for Bilirubin Binding to Human Serum Albumin

“…The techniques used in this study to mutate the HSA coding region and to synthesize and purify the mutated protein have been previously described (32)(33)(34)(35).…”