Endocrine, vol. 6, no. 3, 309-315, June 1997 0969-711X/97/6:309-315/$9.75 9 1997 by Humana PressInc. All rights of any nature whatsoever reserved. Thyroxine Binding to Transthyretin Met 119 Comparative Studies Of Different Heterozygotic Carriers And Structural Analysis Maria Rosario Almeida, 1,2Ana Margarida Damas/Martine C. Lans, 3 Abraham Brouwer, 3 and Maria Joao Saraiva 1,~ lCentro de Estudos de Paramiloidose, Porto, Portugal; 21nstituto de Ciencias Biomedicas Abel Salazar, Universidade do Porto, Porto, Portugal; and 3Department of Toxicology, Agricultural University Wageningen, Wageningen, Netherlands. The majority of the known transthyretin (TTR) variants are associated with amyloidosis, but there are also variants associated with euthyroid hyperthyroxin- emia and others are apparently nonpathogenic. TTR Met 119 is a nonpathogenic variant found to be fre- quent in the Portuguese population. Previous studies on thyroxine (T4) binding to semi-purified TTR from heterozygotic carriers of TTR Met 119, reported by us and other groups, revealed different results. There- fore, to further characterize T 4 binding to TTR Met 119 we performed T4-TTR binding studies in homotetra- meric recombinant TTR Met 119 variant and normal TTR. We also studied T 4 binding to TTR purified from serum of different heterozygotic carriers of TTR Met 119 including compound heterozygotic individuals carriers of a TTR mutation in the other allele. We observed an increased T 4 binding affinity to TTR Met 119 from heterozygotic individuals and compound heterozygotes and this effect of increasing T4 binding affinity was consistent and independent from the mutation present in the other allele. Recombinant homotetrameric TTR Met 119 and heterotetrameric protein from heterozygotic carriers of TTR Met 119 presented similar T 4 binding affinity demonstrating the increased T 4 binding affinity of TTR Met 119. X-ray crystallography studies performed on the recom- binant TTR Met 119 variant revealed structural alter- ations mainly at the level of residue Leu 110 allowing a closer contact between the hormone and the mutant protein. These results are consistent with the observed T 4 binding results. Key Words: Transthyretin; TTR Met 119; thyroxine binding; structural analysis. Received September 24, 1996; Revised December 5, 1996 and February 18, 1997; Accepted March 20, 1997. Author to whom all correspondence and reprint requests should be addressed: Maria Jo~o Saraiva, Centro de Estudos de Parmiloidose, Hospital de Santo Ant6nio, 4050 Porto, Portugal. E-mail:mjsaraiv@ncc.mp.pt Introduction Transthyretin (TTR) is a thyroxine (T4) binding protein, carrying about 20% ofT 4 in serum. TTR is also a carrier of retinol binding protein (RBP). The TTR molecule is a tet- ramer of identical subunits with 127 amino acid residues each, coded by a single copy gene. About 50 different point mutations have been described in TTR (1). Most of them are associated with familial amyloidotic polyneuropathy (FAP), a disease characterized by the deposition of TTR as amyloid; others are responsible for euthyroid hyper- thyroxinemia and there are still some apparently non-pathogenic mutations. In addition to the high fre- quency of occurrence of the FAP associated TTR Met 30 variant in the Portuguese population, screening studies revealed also a high frequency of occurrence of some apparently nonpathogenic variants, in particular of TTR Ser 6 (2) and TTR Met 119 (3) in heterozygotic carriers and in compound heterozygotic carriers. Some studies have already been performed with TTR from heterozygotic individuals carriers of each of these variants. TTR Ser 6 was first reported by Fitch et al. (4) and described in association with hyperthyroxinemia; how- ever, recent studies on recombinant TTR Ser 6 have demonstrated that this variant has a normal T4 binding affinity (5). TTR Met 30, the most studied variant, has low T 4 binding affinity in the heterozygotic form (6), and the homozygotic TTR Met 30 has almost no affinity for T 4 (7). Concerning T 4 binding to TTR Met 119, its carriers were first described as presenting no differences in thyroid hormone levels as compared to normals (8). Scrimshaw et al. (9) found also normal levels of free T 4 in carriers of TTR Met 119, but increased T 4 binding to TTR. A study by Alves et al. (10) reported normal T 4 levels in serum of TTR Met 119 carriers but an increased T 4 binding poten- tial as compared to control, attributed to the relatively high levels of TTR in their serum. However, recently, Curtis et al. (11) described an increased T 4 binding affinity for TTR semi-purified from sera of heterozygotic TTR Met 119 carriers. To further characterize T 4 binding to 309