The antigenicity of thyroxine residues in human thyroglobulin

Thyroglobulin (Tg) is a large glycoprotein of mol.wt. 660000 synthesized in the thyroid. It is the vehicle for the unique route of biological synthesis of thyroxine and contains up to six thyroxyl residues in peptide linkage within its peptide chain. Despite the fact that small quantities are present in the human circulation from birth onwards auto-antibodies to thyroglobulin are commonly found. Some of these auto-antibodies are directed against antigenic sites in thyroglobulin that contain thyroxyl residues and also cross-react with thyroxine free in solution. We have compared the interactions between such a human auto-antibody and three different preparations of human thyroglobulin, their tryptic digests and thyroxine. Thyroglobulin was purified from three post-mortem samples of thyroid tissue by extraction with 50m~-sodium phosphate (pH 7.4) containing 50m~-6-aminohexanoic acid, followed by gel filtration on SepharosedB. The three preparations TgR, TgE and TgC contained 4.2, 4.0 and 1.1 residues of thyroxine respectively per mol of thyroglobulin. They, their tryptic digests and thyroxine were tested for their ability to inhibit the binding of 12SI-labelled thyroxine tracer to the antibody. Inhibition curves of percentage tracer bound against increasing quantity for TgR, TgE and thyroxine were parallel whereas that for TgC diverged slightly (results for TgE and TgC shown in Fig. 1). In molar terms the ratios of the quantities required to achieve the same level of inhibition of tracer binding (half the maximum) were: TgR : T4, 1.1 ; TgE : T4, 1 .O; TgC : T4, 1.5. These relative potencies of the thyroglobulins bear no relation to their thyroxyl contents. We infer that some thyroxyl-containing sites are unreactive towards this antiserum and, since TgC with only 1.1 thyroxyl residues reacts as well as the other two preparations it may be that only one thyroxyl-containing site reacts with this antibody. A corollary is that the immediate surroundings of the thyroxyls vary and may largely determine the antigen-antibody interaction. This inference is supported by binding studies with the tryptic digests of the thyroglobulin preparations. The inhibition curves for the tryptic digests of TgR and TgE were the same but quite different from those of the parent proteins in shape as well as showing an increase in binding energy (results for TgE are shown in Fig. 1). Thus the different members of the population of antibodies in the antiserum have responded differently to the structural and conformational changes caused by the breakdown of the thyroglobulin structure. Hence the binding is influenced by conformational determinants in the antigenic sites. The inhibition curve of the tryptic digest of TgC shows little change in shape and a minor decrease in binding energy compared with that of the original protein (see Fig. 1). This is consistent with the low content of thyroxyl residues ( I . 1) in this preparation; opening up the structure cannot expose more to antibody binding. The possibility that the lack of reactivity of some thyroxyls was solely due to steric hindrance was examined in a two-site binding experiment with a sheep anti-thyroxine antiserum shown previously to cross-react with thyroglobulin (Pearce et a/., 1981). An immunoglobulin fraction from this antiserum was immobilized by reaction with cyanogen bromide-activated Sepharose-4B (Pharmacia). Thyroglobulin was then introduced into a column of the solid-phase antibody and was adsorbed, presumably by reaction with thyroxyl residues. Portions of the same anti-thyroxine antiserum were then passed through the column of the now immobilized thyroglobulin. Analysis of the effluent showed an adsorption of antibodies that was related to 60 -