Specificity of thyroid hormone synthesis The role of thyroid peroxidase

73

Biochimica et Biophysica Acta, 540 (1978) 73--82

© Elsevier/North-Holland Biomedical Press

BBA 28511

SPECIFICITY OF THYROID HORMONE SYNTHESIS THE ROLE OF THYROID PEROXIDASE

D. DEME, J. POMMIER and J. NUNEZ Unitd de Recherche sur la Glande Thyroide et la Rdgulation Hormonale-I.N.S.E.R.M. and Equipe de Recherche Associde No. 449-C.N.R.S. 78, avenue du Gdndral Leclerc, 94270 Bicdtre (France)

(Received July 28th, 1977)

Summary Thyroglobulin has been iodinated in vitro using thyroid peroxidase, lactoperoxidase and horseradish peroxidases plus I2 at different pH values. The iodine distribution between thyroxine, triiodothyronine monoiodotyrosine and diiodotyrosine has been determined in several samples of thyroglobulin. 1. It has been shown that the highest hormone contents are always obtained with thyroid peroxidase. At pH 7.4 lactoperoxidase catalyzes the coupling reaction which produces thyroid hormones as efficiently as thyroid peroxidase. However at lower pH values (6.6 and 6.3) the yield of both thyroxine and triiodothyronine is greatly reduced with this enzyme whereas it is only slightly decreased with the thyroid enzyme. These data show that enzyme specificity plays a role in the selection of the tyrosine residues of thyroglobulin which undergo iodination and which are then able to couple to thyroid hormones. 2. This conclusion is supported by the results obtained with horseradish peroxidase. With this enzyme far fewer diiodotyrosine residues able to form thyroxine are synthesized and therefore a much lower thyroxine content is found. On the other hand the amount of triiodothyronine produced with horseradish and lactoperoxidase at pH 6.3 is the same. 3. With chemical iodination thyroxine and triiodothyronine begin to b e formed only when 30--40 iodine atoms have been introduced whereas with thyroid peroxidase significant amounts of hormones are already synthesized at much lower levels of iodination (less than 10 atoms). The iodine distribution between mono- and diiodotyrosine is also different when chemical iodination is compared to enzymatic iodination (thyroid or lactoperoxidase) at pH 7.4, 4. These results demonstrate that the regulation of iodination and coupling processes depends not only on the native structure of thyroglobulin but also on the properties of thyroid peroxidase.

74 Introduction in the past few years it has been established that the same enzyme, thyroid peroxidase, catalyzes both the iodination of the tyrosine residues of thyroglobulin, producing mono- and diiodotyrosine residues, and the coupling of some of these residues to form the hormones thyroxine and triiodothyronine [1--2]. Non-iodinated thyroglobulin contains approx. 140 ~yrosine residues; a maximum of 20 25 of these residues can be iodinated in vivo and approx. 40 in vitro. Only approx, g of these iodotyrosine residnes can be coupled to thyroid hormones. Previous results have shown: (I) that iodination of thyroglobulin takes place in a rigid sequential order predetermined by the microenvironment of each tyrosine residue and hence by the native structure of the protein [3]; (2) that the highest efficiency of hormone synthesis is obtained in a very narrow range of iodination [4]. These results sugges5 that the tyrosine which are coupled are located in a very precise sequence of thyroglobulin. Thus the native structure of thyroglobulin seems to be very important in regulating the iodination and coupling processes. Krinsky and Fruton [5] studying the iodination of a series of synthetic tyrosine-eontaining di- and tripeptides with thyroid peroxidase, lactoperoxidase and horseradish peroxidase observed differences in specificity of these enzymes. In contrast Taurog [6--7] studying the iodination of thyroglobulin with lacto and thyroid peroxidases showed that thyroid peroxidase posseses no marked specificity in its ability to catalyze iodination and thyroxine formation at pH 7. The experiments reported in this work demonstrate that thyroid peroxidase itself also plays an important role both in the choice of the tyrosine residues which are iodinated and in maximal coupling of the hormonogenic iodotyrosine residues. This conclusion has been obtained by comparing the ability of different peroxidases, thyroid peroxidase, lactoperoxidase, horseradish peroxidase and chemical iodination to perform the iodination and coupling reactions° Materials and Methods

(1) Materials Iodide and glucose were purchased from Prolabo, glucose oxidase~ horseradish peroxidase (Rz = 3) and lactoperoxidase (Rz = 0.6) from Boehringer Mannheim G m b H (Mannheim0 G.F.R.), pronase from Calbiochem, Na12Sl from Commissariat ~ l'Energie Atomiqueo The following were prepared as previously described; hog thyroid peroxidase [6], human goitre thyrogtobulin [9 10]. Goitre thyroglobulin was poorly iodinated (0.02--0.04% iodine i.e° 1.3 2.6 iodine atoms per mol of thyroglobulin).

(2) Thyroglobulin iodination This reaction was performed at 20°C in 0.05 M phosphate buffer pH 7.4, 6.g or 6.3 with an H202-generating system (glucose 6 mM), glucose oxidase 1 pg/ ml). The concentration of the H~O2-generating system was carefully determined as previously described [11] in order to maintain a steady-state production of hydrogen peroxidase neither limiting nor inhibitory: it has been shown by us

75 that when the glucose oxidase concentration was t o o low no linearity in the kinetics of iodination was obtained. In contrast when glucose oxidase concentration was t o o high an inhibition was observed; the proportionality between peroxidase concentration and initial rate of the reaction was considered the best p r o o f that the concentration of the H202-generating system was neither inhibitory nor limiting. Control experiments were performed which showed that the same amount of H202-generating system could be used for a large range of iodide concentrations and for given amounts of the different enzymes used in this work. It was also controlled that sufficient amounts of H20~ were still available after long periods of incubation and thus that no inactivation of the H202-generating system occurred in these conditions. The incubation medium contained human goitre thyroglobulin and increasing concentrations of iodide (0.1--200 pM) labeled with 12sI, and either thyroid peroxidase (1 U/ml, specific activity 1000 U/mg, lactoperoxidase (2 pg/ml) or horseradish peroxidase (10 gg/ml). The unit of peroxidase activity (iodide peroxidation to iodine) is defined [1] as the quantity of enzyme required to increase the absorbance at 353 nm by 1 min -1 under the following standard conditions: 10 mM iodide, 5 pg/ml glucose oxidase and 6 mM glucose in a 0.05 M phosphate buffer, pH 7.4, at 20°C. After 1.5 h of incubation the coupling reaction was complete and the reaction was stopped by NaHSO3 addition. Chemical iodination was performed at 4°C with vigourous stirring of human goitre thyroglobulin in 0.05 M phosphate buffer pH 7.4 and varying volumes of an Is (10 -3 M) solution in the same phosphate buffer containing KI (1.5 • 10 -3 M). Both the chemical iodination and coupling being much slower processes than the enzymatic ones the incubation was pursued for 3 h.

(3) Estimation of iodoamino acids The iodinated amino acids in iodinated thyroglobulin were measured after removal of excess iodide by dialysis. The samples were then hydrolysed (15 h) by pronase dissolved in a 0.05 M phosphate buffer pH 7.6. The iodinated amino acids released were analysed using paper chromatographic methods with two solvents, ethanol/ammonium carbonate 0.2 M (1 : 0.5, v/v) and amyl-3-ol saturated with 2 M NH4OH. Under these conditions of hydrolysis, deiodination was less than 2% and the chromatographic studies revealed only minute amounts of labeled material remaining at the origin. Results and Discussion Chemical iodination of tyrosine residues by I2 is a substitution reaction which is catalyzed at an alkaline pH. Mayberry et al. [12] have shown that the a m o u n t of iodination of free tyrosine is proportional to the a m o u n t of phenoxide formed and that a second alkaline catalysis is required for the removal of the proton present in the ortho position of the phenolic ring. Thus chemical iodination and coupling by I2 can be studied only at pH ~> 7.5 where the phenoxide derivative of tyrosine is formed. On the contrary the enzymatic reactions are catalyzed at neutral or acidic pH values depending on the enzyme used; with thyroid peroxidase the optimum pH for iodination is 7.5, with lactoperoxidase approx. 6.5 (Fig. 1) and with horseradish peroxidase approx. 5.5

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[13]. For these reasons iodination and coupling were compared at different pH values. Fig. 2 shows the relationship between the leve! of iodination and the a m o u n t of thyroxine formed at pH 7.4 with thyroid peroxidase and lactoperoxidase. Clearly at this pH the two enzymes catalyze identically the coupling reaction which produces thyroxine from two hormonogenic diiodotyrosine residues. At pH 7.4 maximal thyroxine contents (approx. 2.5 mol thyroxine/tool of thyroglobulin) were obtained when the iodine c o n t e n t reached approx. 70 iodine atoms. This means that, with both enzymes, approx. 5 diiodotyrosine residues can be coupled to thyroxine (diiodotyrosine hormonogenic residues) and that a m a x i m u m of 10 iodine atoms out of approx. 70 are in the form of thyroxine: the other approx. 60 iodine atoms remain in the form of monoiodo- and diiodotyrosme. Similar conclusions can be made a~ pH 7.4 for 3,5,3'-triiodothyronine ~Fig. 2b). However Figs. i and 2 show that with lactoperoxidase the a m o u n t of thy o roxine or triiodothyronine which can be formed at every !evel of iodination decreases when the pH is lowered. For instance from pH 7.4 to pH 6.6 the thyroxine c o n t e n t only decreases from 2.4 to 2 mol/mol of thyroglobulin with thyroid peroxidase (Fig. la) whereas it is reduced 100% with laetoperoxidase (Fig. lb). Two hypotheses can be made to explain these results: (1) the Tyr residues which are iodinated with thyroid peroxidase and lactoperoxidase are the same at all the pH values (i.e. they occupy the same position in the primary sequence of thyroglobulin) but lactoperoxidase is unable to interact at lower pH v~Jues with all the residues which are potentially able to eoupte, and (2) the distribu° tion of iodine between t h e hormonogenic and nonhormonogenic residues differs with the pH when lactoperoxidase is used. Figs. 3a and b represent the relationship between the number of iodine atoms

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incorporated into thyroglobulin and the number of residues of m o n o i o d o t y r o sine and diiodotyrosine formed; whatever the enzyme, lactoperoxidase or thyroid peroxidase, and the pH the number of monoiodotyrosine residues is identical (Fig. 3a). At pH 7.4 the number of diiodotyrosine residues is identical with lactoperoxidase and thyroid peroxidase (Fig. 3b), whereas it seems to be higher with lactoperoxidase at pH 6.6 or 6.3 (Fig. 3b). However this apparent excess is due to the fact that at these pH values part of the diiodotyrosine residues has n o t been coupled to thyroxine; if the number of diiodotyrosine

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residues which have been transformed to thyroxine is added to the number of residues actually present in the form of diiodotyrosine the results depicted in Fig. 3b are obtained: no difference is apparent whatever the nH or the enzyme. This result suggests that lactoperoxidase forms at pH 6.6 or 6.3 as many iodotyrosine residues potentially able to couple to hormones as at pH 7.4 (and as thyroid peroxidase) but is unable to interact with a large proportion of them to form thyroxine. A better answer to this problem would be provided by a direct determination of the position in the primary and tertiary structure of thyroglobulin of each tyrosine residue involved in the iodination and coupling processes. Unfortunately the knowledge of thyroglobulin structure remains very incomplete because of the high complexity of the molecule [14--17] ; thus fingerprinting of iodine-containing peptides is difficult and has so far been unsuccessful.

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A minimal conclusion is therefore that the enzyme plays a role either in the choice of the tyrosine residues which undergo iodination or in the ability to couple the hormonogenic residues to thyroid hormones. This conclusion is strengthened when one examines the results obtained with lactoperoxidase or chemical iodination. Fig. 4a represents the relationship between the number of iodine atoms incorporated into thyroglobulin and the number of mol of thyroxine formed in the presence either of lactoperoxidase or horseradish peroxidase at pH 6.3. It is again clear that depending on the enzyme, different amounts of thyroxine are formed; the maximal quantity of this hormone which can be obtained is approx. 0.35 mol with horseradish peroxidase and 0.8 mol with lactoperoxidase. Surprisingly the same amount of triiodothyronine was found with both enzymes (Fig. 4b). These results suggest that the marked differences found for thyroxine depend on the fact that lactoperoxidase synthesizes many fewer hormonogenic

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diiodotyrosine residues than lactoperoxidase: Fig. 5a and b show actually that the distribution of iodine between mono- and diiodotyrosine residues differs depending on the enzyme. More monoiodotyrosine and less diiodotyrosme, as measured directly (Fig. 5b) or measured directly plus that converted co thyroxine (Fig. 5c), is formed with horseradish peroxidase. It is clear therefore that fewer diiodotyrosine residues potentially able to couple to thyroxine are formed in the presence of horseradish peroxidase. On the contrary the number of iodotyrosine residues which become coupled to triiodothyronine is obviously the same with both enzymes. Once again it is evident that the amount of hormone formed depends no~ oniy on the structure of thyroglobulin but also on the ability of the enzyme to interact with a number of residues precisely located in the primary structure of protein. The results obtained by chemical iodination strongly support this conclusion. Chemical iodination of thyroglobulin was performed at pH 7.4 and the results could therefore be compared with those obtained with thyroid peroxi-

81 dase at the same pH (Fig. 2). Fig. 6 represents the formation of thyroxine as a function of the iodine content. When one compares Fig. 6 and Fig. 2 it is clear that the formation of thyroxine or triiodothyronine begins only when much higher levels of iodination are reached: with thyroid peroxidase hormones are already f o u n d when the iodine c o n t e n t is lower than 10 atoms whereas with chemical iodination sig~nificant amounts of hormones are detected only above 30--40 iodine atoms. Fig. 7 also shows that the iodine distribution between mono- and diiodotyrosine residues is quite different. It is clear from these data that during chemical iodination most of the tyrosine residues which are iodinated first are n o t those which are able to couple. From the data reported in Figs. 2, 4 and 6 the efficiency of thyroid hormone formation may be calculated for the iodine atoms successively incorporated into thyroglobulin in the presence of thyroid peroxidase, lactoperoxidase, horseradish peroxidase and in chemical iodination. Fig. 8 represents the change in thyroxine formation for 5 iodine atoms successively incorporated as a function of the a m o u n t of iodine added between 0 and 85 atoms. Maximal efficiency of conversion was f o u n d for thyroid peroxidase at pH 7.4 between 25 and 35 iodine atoms. With lactoperoxidase at pH 7.4 the efficiency was the same whereas it was greatly reduced with lactoperoxidase at pH 6.6 and 6.3 and further still with horseradish peroxidase at pH 6.3. However, in all the enzymatic conditions the highest proportion of iodine incorporated into hormones was always f o u n d in approximatively the same interval of iodination. The situation was quite different for chemical iodination, where maximal efficiency was obtained above 60 iodine atoms. These data therefore show that a c o m m o n minimal number of tyrosine always interacts with the enzyme whatever the pH and the enzyme. In other words thyroglobulin contains a limited number of tyrosine residues which are able, after iodination, to interact with thyroid peroxidase, lactoperoxidase or horseradish peroxidase to form hormones. This group is probably located in the same sequence of the native protein of the different enzymes and undergoes coupling as soon as the level of iodination has reached 25--35 iodine atoms. Another group of hormonogenic iodotyrosine residues is also formed in the same interval of iodination and will couple only with thyroid peroxidase in a broad range of pH and with lactoperoxidase at pH 7.4. As expected t h y r o i d peroxidase is the enzyme which appears the best adapted to perform coupling in a broad range of pH. It is also unlikely that the variations in h o r m o n e synthesis observed with lactoperoxidase at different pH depend directly on changes concerning the structure of thyroglobulin or the ionization of the tyrosine or iodotyrosine residues of this protein: if such as the case one would expect to obtain the same types of modifications in thyroid h o r m o n e synthesis with thyroid peroxidase as well as with lactoperoxidase. Thus this work provides clear evidence that the enzyme thyroid peroxidase plays an important role in the selection of the residues which are able to be iodinated and coupled to t h y r o i d hormones. Hence it would appear that the iodination of thyroglobulin, although it is a post-translational process [18--21] may be very selective, selectivity being provided by the structure of both the enzyme, thyroid peroxidase and the hormone-synthesizing protein matrix, thyroglobulin. It is interesting to note that some specificity has been acquired

82 during evolution for this particular peroxidase which belongs to a class of enzymes reputed to be very poorly specific.

Acknowledgements We are grateful to Madame De Prailaune for her excellent technical assistance. The radioisotopes used in this work were purchased with a grant from the Commissariat fi l'Energie Atomique.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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