Calcium-induced changes in thyroglobulin conformation

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 227, No. 2, December, pp. 351-357, 1983

Calcium-Induced

Changes in Thyroglobulin

Conformation’

SILVESTRO FORMISAN0,2 BRUNO DI JESO, RENATO ACQUAVIVA, EDUARDO CONSIGLIO, AND GIUSEPPE PALUMBO Centro di Endwrinologia ed Oncologia Speriwzntale, C.N.R. 2-” Facoltci di Medicina e chimrgia, Via S. Pansini, 5 80131 Naples, Italy Received February

14, 1983, and in revised form June 30, 1983

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes’ radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.

The organized structure of the thyroglobulin molecule is responsible for the efficient conversion of iodine into the two hormones, thyroxine and triiodothyronine. The concentration of thyroglobulin in the colloid is remarkably high (possibly of the order of 20%) (1) so that extensive molecular interactions, including gel formation, may be possible (2, 3). It is of interest that the thyroid gland contains a relatively high concentration of calcium for a soft tissue (more than 5 mM) (3,4), amounting to as much as three and a half times the level of calcium in the serum. Recent studies (5) have been directed towards an understanding of the specific interaction of thyroglobulin with specific membrane receptors. Consiglio et al. (6) reported that bovine thyroglobulin binds to a specific receptor on thyroid plasma membranes. The presence of Ca2+ has been shown to enhance thyroglobulin binding to thyroid membranes (7). It is widely reported (8) the solubility of proteins decreases in presence of polyeth‘This work was partly supported by NIH Grant lRO1 AM 21689. ’ To whom correspondence should be addressed.

ylene glycol, a water-soluble synthetic polymer. In spite of its relatively inert chemical properties, PEG3 has been found to have some interesting biological effects. For example, it induces the growth of crystals in a large variety of proteins which have not been crystallized before (9), promotes the fusion and hybridization of cells (lo), and causes an enhancement of microtubule assembly of tubulin (11). These observations prompted us to investigate the use of polyethylene glycol solutions as a tool to study the properties of thyroglobulin. In this solvent, the solubility of thyroglobulin is drastically reduced and the effects of binding can be observed from the changes in solubility. MATERIAL

AND

METHODS

Bovine thyroids were collected at the local abattoir. Thyroids were cleaned of the surrounding tissues, minced with scissors into small pieces, and then soaked for 15 min in 0.01 M Tris-HCl, 0.1 M KCl, pH 7.5. These operations were carried out at 4°C. After fractional precipitation (1.4-1.8 M) with saturated ammonium sulfate in 0.05 M sodium phosphate buffer ‘Abbreviation used: PEG, Polyethylene (Nominal average molecular weight 6000).

351

glycol

0003-9861/83 $3.00 Copyright All rights

SE 1083 by Academic Press, Inc. of reproduction in any form reserved.

352

FORMISANO

at pH 7.2, the extracted proteins were quickly dialyzed against the extraction buffer and layered on the top of a linear sucrose gradient (lo-40% w/v) and centrifuged in a SW 25.2 rotor at 20°C for 23 h at 23,000 rpm. The 19 S thyroglobulin was collected and dialyzed against several changes of 0.01 M EDTA, pH 7.5, to remove divalent cations. Finally, the sample was dialyzed against 0.01 M Tris-HCl buffer, pH 7.5. Also, the latter operations were carried out at 4°C. Thyroglobulin concentration was routinely measured spectrophotometrically using an A?% (1 cm) of 10 at 280 nm. Thyroglobulin samples having different iodine contents were obtained by isopycnic centrifugation in RbCl gradients as previously described by Schneider and Edelhoch (12). Iodine determinations, carried out according to the method of Palumbo et al (13), of all significant fractions from the isopycnic gradient showed a linear increase of the halogen content from 0.35 to 1.00% w/w. Unfractionated thyroglobulin, which was used for most of the experiments, had an average iodine content of 0.71% w/w. Precipitation of th~ogbbulin with PEG. Precipitation curves were obtained by mixing 0.5 ml of a stock solution containing thyroglobulin, buffer, and salt with an equal volume of buffered PEG solution. Polyethylene glycol having a nominal average molecular weight of 6,000 was purchased from Fischer Scientific Company under the trade name of Carbowax, which was used without further purification. Solutions were mixed on a Vortex mixer, incubated at room temperature (24°C) for 66 min, and then centrifuged for 5 min at approximately 3OOOg.Longer incubation or centrifugation times did not cause any additional precipitation. The supernatant was carefully removed by suction and analyzed for thyroglobulin content by measuring the optical density at 230 nm (after appropriate dilution with buffer). Although the intrinsic absorbance of PEG at 230 nm was quite small, appropriate blanks were run for each experiment. Glass-redistilled water was used in the preparation of all solutions. All chemicals used throughout were reagent grade. Gel chromatography. Gel permeation was performed with Sepharose 6B (Pharmacia Fine Chemicals) in 10% w/v PEG 6000,O.Ol M Tris-HCl, 0.001 M sodium azide, pH 7.5, at room temperature (24OC). The column used (Pharmacia K/90) was packed with preswollen gel to the height of 56 cm. A differential pressure of 60 cm was kept constant during all the runs by using a 250-ml Mariotte flask. The flow rate was 6 ml/h. Elution profiles were obtained by reading the absorbance of each fraction (0.5 ml) at 280 nm. The void and salt volumes of the column were determined with Dextran blue and iv’-acetyltryptophanamide. The standards used for column calibration were Dextran blue 2000 and egg albumin (A& = 45,000).

ET AL. Sucrose density gradient centtifugatim Centrifugation were performed with SW 41 rotor at a temperature setting of 15°C. Linear gradients were performed by mixing equal volumes of 5 and 20% (w/v) sucrose, each containing 10% (w/v) PEG 6000 in the same buffer 0.01 M, Tris-HCl, pH 7.5. Gradients contained either 0.1 M KC1 or 0.033 M CaCl,. Solutions (0.4 ml) of thyroglobulin (1 mg/ml) were layered on the top of gradients and spun for 20 h at 33,000 rpm. After centrifugation the gradients were collected (0.3 ml/fraction) by suction from the bottom of the tubes with the aid of a needle lowered through the gradient. An LKB peristaltic pump set a 2 ml/min was used for this purpose. The protein-concentration profile was monitored by measuring the protein-intrinsic tryptophan fluorescence. The fluorescence emission was monitored at 340 nm using an excitation wavelength of 280 nm on a Perkin-Elmer MPF3 Spectrofluorimeter. RESULTS

S&&i& The solubility profile of thyroglobulin as a function of PEG concentration at two protein concentrations (i.e., 1 and 4 mg/ ml) at pH 7.5 in 0.01 M Tris-HCl is depicted in Fig. 1. Precipitation does not occur at a concentration of thyroglobulin of 1 mg/ ml until the PEG concentration reaches about 14% w/v.

FIG. 1. Effect of thyroglobulin concentration on its precipitation curve by PEG 6000. The solutions contained 0.01 M Tris-HCl, Oil M KC1 at pH 7.5 and room temperature. The thyroglobulin concentration was 1 (0) and 4 mg/ml (A).

CALCIUM-THYROGLOBULIN

19 S Thyroglobulin, although homogeneous with respect to protein, actually consists of a wide spectrum of molecules differing in their iodine content. We have analyzed the precipitates produced by increasing the concentration of PEG in order to determine if the different degrees of iodination have any effect on the solubility properties of unfractionated thyroglobulin. It was found that the iodine content at different degrees of precipitation remained constant, indicating that the precipitation of thyroglobulin is independent of its iodine content. This fact is also more evident from the observation that the precipitation curves of two thyroglobulin samples from the same isopycnic gradient, having 0.40 and 0.85%w/w iodine content, respectively, are totally superimposable (Fig. 2). All our experiments have been performed in 10% (w/v) PEG and 1 mg/ml thyroglobulin. In this solvent thyroglobulin remains soluble when the concentration of several monovalent anions (Cl-, Br-, CH&OO-) or cations (Rb+, K+ Na+, Lit, NH:) are varied. However, an important loss in solubility of thyroglobulin was observed with certain divalent cations, i.e., calcium and magnesium. Figure 3 indicates that the solubility of thyroglobulin first

FIG. 2. Effect of iodine content on the precipitation curve of thyroglobulin by PEG 6000. The solutions contained 0.01 M Tris-HCI, 0.1 M KC1 at pH 7.5 and room temperature. The thyroglobulin concentration was 1 mg/ml; iodine content was 0.40% w/w (0) and 0.85% w/w (O), respectively.

INTERACTION

353

decreases as the concentration of CaClz or MgClz increases until about 0.05 M and then increases at higher concentrations of these two salts, approaching the initial value. The loss in solubility indicates that the bivalent salts are bound to thyroglobulin. The recovery in solubility suggest that the binding of Ca2+(and Mgz+) is ionic-strength dependent. We tested this assumption either by simply increasing the concentration of the buffer or by adding LiCl, KCl, NH&l, and CH&OOK to a solution containing 0.05 M CaC12in 10% w/v PEG, in which thyroglobulin is almost completely precipitated. In all four solutions, the solubility of thyroglobulin returned to 100% and no difference was observed among the different salts (Fig. 4). Gel Permeation Chromatography The elution profiles of 19 S thyroglobulin from Sepharose 6B columns at 25°C are shown in Fig. 5. The column was equilibrated with potassium or calcium chlorides at the same ionic strength, i.e., 0.10 or 0.033 M, respectively; in addition, the solvent contained 10% PEG, 0.001 M NaN3, 0.01 M Tris-HCl buffer, pH 7.5. As seen in Fig. 5, the presence of 0.033 M CaC12retards the position of the thyroglobulin peak in comparison to that obtained in 0.1 M KCl. Also MgC12 (0.033 M, data not reported) shows a similar effect, although much smaller. The change in the elution volume of a protein in a column is related to a change in the Stokes’ radius of the protein (14,15). Consequently, the greater elution volume of thyroglobulin in the presence of calcium rules out the possibility that this ion causes a dimerization or polymerization of thyroglobulin. The observed change may result from a calcium-induced modification in either the hydrodynamic shape of the protein or the degree of dissociation of the protein into its subunits. Actually, 19 S bovine thyroglobulin consists of two families of molecules in which the same two chains (Mr = 330,000) are bound together either by covalent or noncovalent bonds. If dissociation is produced by Ca2+, only the noncovalently bonded dimer could be affected. Consequently the elution profile

354

FORMISANO

ET AL.

01 0.1

0.2 SALT

0.3

I 0.5

0.4

CONCENTRATION

1 0.6

M

FIG. 3. Solubility of thyroglobulin as a function of salt concentration in the presence of 10% w/v PEG 6000: CaCIZ (X), MgC& (O), and NH&l, LiCl, RbCI, KCI, CHaCOOK, (NH&SO,, and sodium phosphate (A). Thyroglobulin concentration was 1 mg/ml in 0.01 M Tris-HCI buffer, pH 7.5, at 25°C.

should be resolved into two separate peaks, one for the undissociated (M, = 660,000) and the other for the dissociated form (Mr = 330,000). Since we have observed a symmetrical elution pattern consisting of a single peak displaced towards larger elution volumes, the most likely explanation

of this shift is that Ca2+produces a change in protein dimensions of thyroglobulin leading to a more compact shape, i.e., a decrease in Stokes’ radius. The possibility that calcium-induced changes in thyroglobulin conformation lead to the absorption of the protein to the gel matrix is an

I

01 0.1

0.2

0.3

0.4

0.5

0.6

SALT CONCENTRATION M FIG. 4. Solubility of thyroglobulin as a function of the salt concentration in the presence of 10% w/v PEG 6000 and 0.05 M CaCl,: NaCl (X), KC1 (A), NH&l (0). and LiCl (0). Thyroglobulin concentration was 1 mg/ml in 0.01 M Tris-HCl, pH 7.5, at 25’C.

CALCIUM-THYROGLOBULIN

0

41

’

15

b 25 Volume

35 lmll

FIG. 5. Elution profiles of thyroglobulin on a Sepharose 6B column (60 X 0.9 cm) equilibrated with 0.01 M Tris-HCl, PEG 6000 (10% w/v), pH 7.5, in: (0) 0.1 M KC1 or (0) 0.033 M CaCla. The sample consisted of 0.8 ml of a solution of thyroglobulin (0.6 mg/ml) in 10% PEG 6000. Following chromatography fluorescence intensities were determined as described under Materials and Methods.

alternative explanation but it seems rather unlikely since it is not in agreement with the sucrose gradient experiments reported in the next paragraph.

INTERACTION

355

The question of whether the sizing properties of Sepharose 6B remain unchanged in the presence of PEG needs further elaboration. Since our main purpose was to study the effect of calcium on the properties of thyroglobulin in the presence of PEG, we have checked whether the resolving power of the gel survives the addition of 10% (w/v) PEG. In Fig. 6 the effect of 10% PEG on the elution profiles of the following mixtures is reported: thyroglobulin, 1.0 mg/ml; egg albumin, 2.0 mg/ml; and Nacetyltryptophanamide. It is known that the presence of PEG changes the elution behavior of proteins on exclusion columns due to osmotic effects of the polymer. This has been described by Hellsing (16) and more recently confirmed by Ingham (1’7). Nevertheless, the data clearly indicate that in the presence of 10% PEG, Sepharose 6B retains its sieving properties. An additional conclusion implicit in this result is that alterations in the solvent (10% w/v PEG) do not significantly alter the pore-size distribution and elution characteristics of Sepharose 6B. It should also be noted that the retardation caused by 0.033 M CaCl, of the thyroglobulin peak (Fig. 5) is more than half that

0.200 -

0.1602 E 0.120z d O aoml-

Volume fmlJ FIG. 6. Comparison of the sieving properties of Sepharose 6B in the presence (0) or absence of 10% PEG 6000 (A). The sample used in this experiment was constituted by thyroglobulin (1 mg/ ml), egg albumin (2 mg/ml), and N-acetyltryptophanamide (about 0.02 mg/ml) in 0.8 ml of 0.01 M, Tris-HCl, 0.1 M KCl. The other conditions are the same as in Fig. 5.

356

FORMISANO

caused by 10% PEG (Fig. 6). It seems unlikely that this small concentration of CaCl, could produce an effect this large on the elution properties of the 10% PEG column. Sedimentation The conclusion reached from the chromatographic experiment concerning the effect of Cazf on the conformation of thyroglobulin was confirmed by sedimentation experiments on sucrose gradients. Figure 7 presents the sedimentation profile of thyroglobulin obtained on a density gradient with and without calcium (in presence of 10% PEG (w/v)). It is evident from this figure that the sedimentation rate is greater in Ca2+ than in K+ (at the same ionic strength) and that, in both cases, the curves retain the same degree of symmetry. In the absence of association, conformational changes resulting in greater compactness of the molecule are required for an increase in sedimentation rate, since the effects of bound calcium on the density of thyroglobulin and of free calcium on the density of sucrose are incapable of explaining the magnitude of the observed change.

Bottom

ET AL. DISCUSSION

There are several reports indicating that the thyroid gland of man and rat contains a concentration of Ca2+amounting to three to four times that of serum (3,4). Haeberli et al. (3) have shown that an excess of Ca” is associated with the epithelial cells and primarily located in the luminal region. Furthermore, they have studied the kinetics of transport of Ca2+between serum and the thyroid gland in an attempt to follow the accumulation of this ion in the lumen. However, although the mechanism by which Ca2+is concentrated in the lumen remains unclear, it seems possible that some of the calcium that is present may be due to its association with thyroglobulin. The solubility curve (in PEG) obtained with increasing concentrations of Ca2+ (Fig. 3) is quite unusual in that it is biphasic, an initial loss in solubility being followed by a recovery. We suggest that the loss in solubility is a direct consequence of the binding of Ca2+ since membranedialysis experiments using 45Cahave shown that Ca2+is bound to thyroglobulin under the same experimental conditions (see below). The loss in solubility could result either from the reduction of the net negative

TOP

FIG. ‘7. Sucrose density gradient centrifugation of thyroglobulin in 0.01 M Tris-HCl and 10% w/v PEG 6000, pH 7.5, in: (0) 0.1 M KC1 and (0) 0.033 M CaCl,. The sample applied to the gradient consisted of 0.4 ml of thyroglobulin (0.8 mg/ml) dissolved in Tris-HCI buffer. Both samples were sedimented in the same rotor to maintain experimental conditions as close as possible.

CALCIUM-THYROGLOBULIN

charge of thyroglobulin or to a Ca’+-induced structural change. The binding of Ca2+ has been found to decrease from 15 mol/mol in 0.01 M KC1 to 7 mol/mol in 0.05 M KC1 when Ca2+ was present at 0.033 M in 10% PEG. Thus it appears that the increase in solubility on adding neutral salt results from the loss of bound Ca2+. The increase in KC1 (or ionic strength) either competes and displaces Ca2+ or reverses the structural change and thereby releases Ca2+. The binding of Ca2+ and the effects of the other ions could explain the solubility changes but can not account for the increase in elution volume and increase in sedimentation rate of thyroglobulin. Since both of these changes depend primarily on modifications in the size and shape of thyroglobulin, these parameters appear to be modified. The only type of molecular change that can fit both the gel chromatography and sedimentation data is a decrease in Stokes’ radius. This reduction in effective hydrodynamic volume can arise in several ways depending on the structure of the molecule in the absence of Ca2+. It is known from hydrodynamic measurements that thyroglobulin is an asymmetric molecule in solution, having an axial ratio of about 10, when considered as a prolate ellipsoid of revolution (18). For a molecule as large as thyroglobulin (44, = 660,000), this asymmetry is likely to be the result of the presence of several independent domains rather than that of a rigid ellipsoid of revolution with an axial ratio of 10. Moreover, there are relaxation data (19) which indicate the existence of two rotational units smaller than that of the native structure or even of its two subunits. If this is the case, the binding of Ca2+could bring two domains together and lead to a more compact structure. This reaction would not necessarily involve any change in secondary or tertiary structure of the domains. The loss in polar surfaces with domain association could really lead to a reduction in solubility. Alternate mechanisms, such as a change in the axial ratio of a compact ellipsoid of revolution

357

INTERACTION

or a refolding of random polypeptide chains involve major changes in binding of a larger number of residues and therefore appear much less likely. The latter event occurs when calmodulin binds Ca2+ (20). However, in this case the binding is much stronger, more specific, and presumably not sensitive to ionic strength. REFERENCES 1. STAFFAN, S. (1972) Endocrinology 91,1300-1306. 2. LOEWENSTEIN, J. E., AND WOLLMAN, S. H. (1967) Endocrin&gy 81, 1086-1090. 3. HAEBERLI, A., MILLAR, K. F., AND WOLLMAN, S. H. (1978) Erw!owinolog~ 102, 1511-1519. 4. KAELLIS, E., AND GOLDSMITH, E. D. (1965) Acta

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