Thyrotropin interaction with high-density lipoproteins

Biochimica et Biophysics Acta 835 (1985) 191-200

Elsevier

191

BBA 51943

Thyrotropin interaction with high-density lipoproteins

Maurizio Bifulco a,c, Harry A. Saroff b, Leonard D. Kohn a and Salvatore M. Aloj a,c, *

Sections on a Biochemistty of Cell Regulation, b Pharmacology, Luboratoty of Biochemical Pharmacology, NIADDK, NIH, Bethesda, MD 20205 (U.S.A.), and’ Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Dipartimento di

Biologia e Patologia Cellulare e Molecolare, Universitd di Napoli, via S. Pansini 5, 80131 Napoli (Italy)

(Received December 13th, 1984)

Key words: Thyrotropin; HDL; Apolipoprotein A-I; Fluorescence polarization

Human high-density lipoproteins (HDL), but not other lipoprotein classes, bind bovine thyrotropin (TSH) with moderately high affinity. Binding of 1251-labeled HDL to TSH has been measured in a solid-phase assay; it is saturable and can be displaced by unlabeled HDL but not by other lipoproteins or bovine serum albumin. The interaction of HDL with TSH has been studied by fluorescence spectroscopy: HDL specifically modifies the fluorescence properties of the biologically active dansyl derivative (DNS, (5-dimethyl- aminonaphtalene-1-sulfonyl) chloride) of TSH (DNS-TSH) causing a 12 nm shift to lower wavelength of the emission maximum, a two-fold increase of the quantum yield and a significant increase of fluorescence polarization. The primary site of TSH binding on the HDL particle is likely to be located on its protein moieties, since other lipoprotein classes, which share similar lipids with HDL, do not bind TSH. ‘251-labeled apolipoprotein A-I binds TSH in the solid-phase assay and titration of DNS-TSH with apolipoprotein A-I causes perturbations nearly identical to those observed with intact HDL. One HDL particle has at least 12 binding sites for TSH with an association constant, K = 10’ M -’ whereas one apolipoprotein A-I molecule binds one or two TSH molecules with an association constant slightly lower than that for HDL (K = lo6 M -‘). The lipid moieties of HDL also appears to be perturbed by the interaction with TSH.

Introduction

Thyrotropin (TSH) is a glycoprotein hormone of M, 27500 which is produced by the anterior part of the hypophysis and secreted into the bloodstream. Its target organ is the thyroid gland whose structure and differentiated functions are strictly dependent upon the availability of the hormone. It is believed that the amount of TSH in

* To whom correspondence should be addressed at the Uni- versita di Napoli, Italy.

Abbreviations: LH, lutropin; FSH, follitropin; DNS, (S-di- methylaminc-naphthalene-1-sulfonyl) chloride; DPH, 1,6-di- phenyl-1,3,5_hexatriene.

the bloodstream is inversely related to the concentration of thyroid hormones. The effects of TSH on the thyroid are exerted through the interaction with specific plasma membrane receptors.

The development of a reproducible radiorecep- tor assay for circulating TSH has been hampered by the existence, in human sera, of a variety of components which inhibit the binding of TSH to its membrane receptor [l]. For example, it has generally been believed that normal immunoglobulins can inhibit TSH binding to plasma membrane preparations by competing for the TSH receptor site either by direct binding to the same receptor or by interacting with a site adjacent to the TSH receptor, thus decreasing its affin. .y for

0005-2760/85/$03.30 6 1985 Elsevier Science Publishers B.V. (Biomedical Division)

the hormone [2-31. In addition, the interaction of TSH with serum immunoglobulins has been pos- tulated [4] and it has been proposed that the formation of a TSH-immunoglobulin complex might be relevant for the pathogenesis of Graves’ disease. In a previous report [5] we showed also that normal human serum contains a protein component, which does not precipitate with the y- globulin fraction in ammonium sulphate, and which inhibits TSH binding to membrane receptors. Inhibition was the result of the interaction of this serum component with TSH. forming a complex which did not bind to the TSH receptor. The inhibitor appeared to bind also other glycoprotein hormones such as human chorionic gonadotropin (human CC) and lutropin (LH) f5].

In the course of our work aimed at the identification and characterization of the last described serum component(s), we have made the observation that human high-density lipoproteins (HDL) are able to bind TSH.

This report describes our study of the interaction of TSH with human HDL.

Materials and Methods

Human lipoproteins were prepared from normal human sera provided by the NIH Blood Bank. The ultracentrifugal flotation technique of Hatch and Lees [6] was used.

The lipoprotein fraction isolated between densities 1.063 and 1.21, referred to as HDL, was tested by immunoprecipitation using specific antisera against apolipoprotein A-I, apolipoprotein B (Calbiochem-Behring), against immunoglobulins G and serum albumin (Cappel), in either standard Ouchterlony or in radial immunodiffusion plates. A clear precipitate was seen only when the HDL preparation was reacted with anti-apolipoprotein A-I; no precipitate was observed when it was tested with anti-IgG serum and anti-albumin. Each HDL sample was kept at +4’C and was used within 1 week after preparation. Since the HDL is heterogeneous with respect to the molecular weight of lipoprotein particles, which range between 1.75 + lo5 and 4.0. lo5 [7-81, we have assumed an average molecular weight of 2.65 . lo5 for calcu- lations of HDL concentrations and an average 1 : 1 ratio of protein and lipid moieties for each

HDL particle is]. HDL were delipidated by solvent extraction and apolipoprotein A-I was purified by column chromatography [lo]. Gel electrophoresis of the purified apolipoprotein A-I using 10% poly- acrylamide, under denaturating conditions, in Tris-glycine (pH 8.6) and O.lsC;, sodium dodecyl sulphate gave rise to a single band with M, = 28 000. There were no detectable apolipoproteins A-II, E and C bands.

Bovine TSH was a purified preparation essentially free from TSH subunits and the p subunit of LH 111.121, with an activity of 25 & 4 IU/mg in

the McKenzie assay [13]. DNS. DPH, and pyrene were purchased from Sigma. Preparations of 12’1- labeled TSH were obtained as previously described [11,14], ‘251-labeled HDL and r2’1-labeled apolipoprotein A-1 were obtained using a similar procedure. All other chemicals were reagent grade. Glass double-distilled water was used throughout.

TSH concentrations were determined spectro- photometrically at 276.5 nm using E = 25 700 l/mol per cm 1151. Other protein concentration measurements were carried out according to established calorimetric procedures [16,17] using 5 x recrystalized serum albumin as the standard.

S&Q&use binding assay. Binding of ‘251- HDL and ‘~‘I-apolipoprotein A-f to TSH or “‘I- TSH to HDL was assessed in a solid-phase assay 1181 in which either unlabeled TSH or HDL are layered into individual wells of 96 wells Dynatech microtiter plates, precoated with 0.2 ml of poly-t- lysine (M, 70000 Sigma, 20 pg/ml H,O) for 1 h at room temperature. The poly-L-lysine was removed and 0.1 ml of either TSH or HDL, at the noted concentrations in 20 mM Tris-acetate (pH 7.2) were added to triplicate wells. Controls con- sisted of poly-L-lysine coated or uncoated wells incubated with 0.5% bovine serum albumin in the same buffer. After 3 h or more at room temperature, unadsorbed proteins were removed and each well was incubated with 0.5% bovine serum albumin wash solution in 20 mM Tris-acetate at pH 7.2 for 30 min at room temperature. The wash solution was removed and each well was incubated with SO ~1 of i2’I-HDL, 1~5~-apolipoprotein A-I or ‘251-TSH (approx. 2 - lo5 cpm) with or without unlabeled ligands in the same wash solution and with or without 0.15 M NaCl.

After 2 h at room temperature, the unbound

193

radiolabeled ligand was decanted and the wells were washed once; the radioactivity of each well was measured in a gamma-spectrometer (Beckman gamma 9000).

Dh?S ~~orescence studies. The DNS derivative of TSH was prepared as previously described [19]. The extent of labeling, i.e., the molar ratio DNS/TSH, was determined from absorption measurement at 276.5 nm for TSH and at 340 nm for the dye (using an E = 3300 l/mol per cm) [ZO].

Because the dye also absorbs at 276.5 nm and because the dye absorption at this wavelength is 1.2-fold its absorption at 340 nm the concentration of TSH is given by the equation:

TSH(mg/~) = [a-(1.2xp)]x1.07

In this equation (Y is the absorbance at 276.5 nm and B is the absorbance at 340 nm, and 1.07 is the ratio 27 500/25 700, i.e., TSH molecular weight/TSH molar extinction coefficient at 276.5 nm. Pola~ation of DNS fluorescence was measured as previously described [19].

Labeling of HDL with DPH and microviscosity measurement. DPH was dissolved in distilled tetrehydrofuran at 2 mM and stored in the dark at room temperature before dilution and use in each experiment. Labeling of HDL was accomplished by dissolving 5 ~1 of this solution in 2 ml of HDL (5 . lop6 M) in 20 mM T&acetate (pH 7.2) directly in the fluorescence cuvette, while the mix- ture was gently stirred under a stream of N, to facilitate the evaporation of tetr~ydrofur~. After 30 min at room temperature the sample was ready for fluorescence polarization measurements in a Perk&Elmer MPF44A fluorescence spectropho- tometer equipped with a thermostated cell holder and an automated fluorescence pollution acces- sory (C.N. Wood, Newtown, PA), with Polaroid filters on the excitation and emission beams. The excitation and emission monochromators were set at 358 and 427 nm, respectively. Microviscosities were derived from polarization values 1211.

Pyrene ~uorescence. Pyrene emission spectra were obtained at 25°C from samples of HDL in 20 mM T&-acetate (pH 7.4) titrated with repeated additions of small aliquots (l-2 ~1) of a 1 mM solution of recrystallized pyrene in dimethyl sulfoxide (DMSO) delivered from a H&lton

syringe. Excitation wavelength was at 338 nm with excitation and emission band width of 4 nm. Titra- tions were continued until the ratio of the excimer emission (470 nm fluorescence) to the monomer emission (393 nm fluorescence) was greater than 0.5. The temperature of the samples during all fluorescence measurements was controlled with a Lauda ka/R circulation bath and measured directly in the cuvette with a thermistor probe of an accuracy of O.l”C. The absorbance of all fluorescence samples was within 0.1 A at the excitation wavelength.

Analysis of fluorescence data. The binding parameters were calculated from fluorescence intensities in experiments in which DNS-TSH was titrated with either HDL or apolipoprotein A-I, using the equations and assumptions detailed as follows. For a single set of sites for the binding of TSH to HDL the ratio of bound to total TSH is

eI_(Kcn) +(l+ Xc)

where K is the intrinsic association constant, n is the number of binding sites on HDL, c, CT and cL are the concentrations of free TSH, total TSH and total HDL, respectively.

The ratio of the change in fluorescence on the binding of TSH to HDL compared to the maximum change in fluorescence is:

F F max

where F is the change in fluorescence for a given increment of HDL and F,, is the maximum change in fluorescence for a given total concentration of TSH. These two ratios may be equated on the added assumption that the change in fluorescence is directly proportional to the bound TSH at all levels of binding.

These two ratios plus the equation for the con- servation of mass for TSH:

develop the equation

F=F,, cL(Kcn)

c,(l+Kc) (1)

194

where

c = (1+ Kc,_n - KQ2+4Kc, I”0

-(l+ Kc,n- Kc,-)j/ZK (2)

Eqns. 1 and 2 were used in the non-linear curve-

fitting routine of Marquardt and Levenberg [22,23]

to evaluate the three parameters, F,,,. K and n.

Results

Binding studies The ability of human HDL to interact with

TSH has been tested in a solid-phase radioligand

assay in which either unlabeled TSH or HDL is

linked to a poly-L-lysine coated plastic support, and the binding of ‘251-labelled HDL, TSH or

apolipoprotein A-I are measured as a function of

the amount of unlabeled protein coupled to the

plastic. In Fig. 1 are reported the results obtained

in experiments in .which unlabeled TSH, at the

noted concentrations, is used to coat the poly-L-

lysine-treated microtiter plate (see Methods). In

panel A, it is shown that ‘251-HDL binding in-

creases, in a saturable fashion, as a function of the

amount of TSH used to coat the wells up to a

concentration of 2.5. IO-’ M. In the presence of

,TSH, M x 105 -LOG wxl

Fig. 1. Solid-phase radioligand assay for the binding of 1251-

labeled HDL (approx. lo5 cpm added) to TSH-coated wells (see Methods fro details). In panel A, the radioactivity bound

to the wells in the absence (0) or in the presence (0) of 0.15 M

NaCl is plotted as a function of increasing concentration of

TSH used to coat the poly-t-lysine-treated wells. In panel B it

is shown that unlabeled HDL (A) can displace ‘2SI-labeled

HDL bound, whereas LDL and VLDL (A) are ineffective. The

experimental points are means of three different determina-

tions with the vertical bars indicating the standard error. HDL

concentrations are based on an average molecular weight of

2.65.105. LDL and VLDL are at 2 mg protein per ml as

measured by the method of Markwell et al. [17].

0.15 M NaCI, the ‘*‘I-HDL bound was reduced at all concentrations of TSH. The specificity of the

interaction is suggested by the observation that

bound ‘251-HDL can be displaced by unlabeled

HDL but not by LDL or VLDL or bovine serum

albumin, which is present throughout the assay at

a concentration of 0.5%.

The order of the reagents can be reversed to

show that lz5 I-TSH can bind to HDL-coated wells, as it is shown in Fig. 2. Unlabeled HDL was used

to coat poly-L-lysine-treated wells and the binding

of ‘251-TSH was measured using procedures identi-

cal to those reported for Fig. 1. The data presented

in Fig. 2 have been corrected by subtracting the

counts (1500-2000) of lz5 I-TSH bound to the wells

in the absence of HDL and to wells coated with LDL at molar concentrations identical to those

used for HDL. In panel B it is shown that un-

labeled TSH and, even better, the dansylated de-

rivative of the hormone (DNS-TSH), displace 1251-

TSH bound to HDL. The dansylated derivative is

used in the fluorescence studies described below. Thyroglobulin and human IgG were ineffective in

displacing ‘251-TSH.

rtiDLl , M x 10’ -LOG [COLO LIGANOI

Fig. 2. Solid-phase radioligand assay for the binding of ‘251-

labeled TSH (approx. lo5 cpm added) to HDL-coated wells

(see Methods for details). In panel A, radioactivity of the wells

is plotted as a function of increasing concentrations of HDL

used to coat the poly-L-lysine-treated wells. In panel B it is

shown that both unlabeled TSH (A) and DNS-TSH (A) can. whereas human thyroglobulin (TG) and immunoglobulin G

(IgG) cannot, displace 1251-TSH bound to HDL-coated wells.

The experimental points are means of three different de-

terminations with the vertical bars indicating the standard

error. The data have been corrected by subtracting the counts

of “‘1-TSH bound to the wells (lXK-2000) in the absence of

HDL and to wells coated with LDL at concentrations identical

to those used for HDL. Protein concentrations are based on the

following molecular weights: HDL, 2.6S.105; TG, 6.6.105:

IgG, 1.5.105.

19.5

Phospholipids [24-261 and glycolipids [25] have been previously shown to interact with TSH and perturb the fluorescence of DNS-TSH [19,27]. However, since the ability to bind TSH is a specific property of HDL (LDL and VLDL do not interact with the hormone, see below), it is unlikely that the lipid portion of HDL contributes to the primary site of interaction, since the lipid moieties of lipoproteins differ quantitatively but not qualita- tively. Thus, the specificity of the interaction of TSH with HDL appears to reside primarily in the apolipoproteins. The role of lipids, nevertheless, can be important as they contribute significantly to the conformation and stability of native HDL [28-301. The question then asked was to what extent would the integrity of lipid moieties of HDL be relevant in determining its ability to bind TSH. To answer this question we have tested whether the apolipoprotein A-I, which is the most abundant apolipoprotein of HDL, could bind to TSH-coated wells. Binding of 12sI-apo~poprotein A-I to TSH was measured, following the same procedure as for HDL. The binding curve and the displacement with unlabeled apolipoprotein A-I was nearly identical to those of HDL (Fig. 3).

Fluorescence spectroscopy is a powerful technique which is widely used in studies of molecular interactions. To study the interaction of HDL with TSH we have used the dansylated derivative of TSH (DNS-TSH) which we have previously char-

[TSH] M ‘10’ -LcG[*PoA-I]

Fig. 3. Solid-phase radio&and assay for the binding of 12’1- labeled apohpoprotein A-I (ApoA-I) (approx. lo5 cpm added) to TSH-caoed wells (see Methods for details). In panel A, the radioactivity bound to the wells is plotted as function of increasing concentration of TSH used to coat the poly- L-lysine-treated wells. In panel B is shown that unlabeled apolipoprotein A-I (A) can displace “*‘I-apolipoprotein A-I bound, whereas thyroglobuhn (TG) (A) is ineffective. Other details are as in Fig. 1.

acterized [19]. This derivative has been extensively tested and shown to retain, essentially unaltered, the properties of native TSH notably, the ability: (i) to bind to the TSH membrane receptor, (ii) to be as active as native TSH in the McKenzie’s bioassay, and (iii) to stimulate adenylate cyclase activity of thyroid membrane preparations. For the studies to be described in this report, a homo- geneous preparation of DNS-TSH with a dye- hormone molar ratio 2.5 + 0.1: 1 has been used. This preparation in 20 mM T&-acetate, at pH 7.4, exhibits a fluorescence spectrum with a maximum centered at 519 nm (Fig. 4).

When DNS-TSH is titrated with each of the individual serum fractions of different density, the fluorescence spectrum appears unmodified with VLDL (d < 1.006); a modest increase in the intensity and a 4 nm shift to shorter wavelength in the emission maximum is observed with LDL (d =

I- I 1 I I I I I - DNS-TSH

-- DNS-TSH + HDi ----* DNS-TSH + SERUM PROTEINS d>1.21

-.- DNS-TSH + LDL . . . . . DNS-TSH + VLDL

\ .2 A

\ WDLl mph,

I I I I I I I

Fig. 4. Fluorescence emission spectra of DNS-TSH (10M6 M in 20 mM phosphate buffer, pH 7.2) in the absence and in presence of 0.15 mg/ml serum lipoproteins or the proteins with density z 1.21. The inset shows the effect of increasing concentration of HDL on the emission maximum of DNS-TSH. Excitation is at 340 nm.

1%

1.006-1.063) and with the fraction (d > 1.21) which is defined ‘bottom’, since it occurs at the bottom of the centrifuge tube of the last preparation step. The possibility had to be taken into account that TSH or other ~ycoprotein hormones, structurally similar to TSH, might be already bound to the lipoproteins. A radioimmunoassay for TSH, LH, FSH and human CG was carried out on each fraction isolated by flotation at different densities. TSH was detected in two out of ten preparations of HDL; in one serum sample containing a high level of LH (143 mIU/ml) and no immunoreactive TSH associated with HDL, a significant amount (7.3 mIU/ml) of LH was measured in the HDL fraction. Only those lipoprotein preparations in which no glycoprotein hormones could be detected were used in the fluorescence experiments. The HDL fraction (d = 1.063-l .21) causes a large increase of the fluorescence intensity of DNS-TSH and a 12 nm shift of the emission maximum to shorter wavelength. In the inset of Fig. 4, the emission maximum of DNS-TSH fluorescence is plotted as a function of HDL concentration; from the titration curve the effect appears to be saturable. To prove that the large change of DNS-TSH fluorescence caused by HDL was not the result of a direct perturbation of the dye alone, HDL was added to DNS-glycine and DNS-lysine at a concentration of 2 mg/ml. No significant change in the emission spectra of the two DNS derivatives was observed. The fluorescence of dansylated bovine serum albumin and didansyl cadaverine was not affected by HDL, whereas dansylated LH displayed, upon titration with HDL, a fluorescence perturbation similar to that observed with DNS-TSH (data not shown).

Titration of DNS-TSH in 20 mM phosphate (pH 7.2) with increasing amounts of apolipoprotein A-l caused a concentration-dependent increase of DNS fluorescence intensity {Fig. 7, top). The fluorescence spectrum of DNS-TSH with apolipoprotein A-I is nearly superimposable with that exhibited with HDL (data not shown). Figs. 5 and 6 illustrate the data for the titrations with HDL and Fig. 7 gives the data for the titration with apolipoprotein A-I, The best-fit values for K and n were determined using the Marquardt- Levenberg, non-linear algorithm 122,231. This algorithm is an iterative procedure which requires

3o r ---.-- ---

Fig. 5. Titration of 3.4.10W7 M DNS-TSH with HDL. Upper panel: 0, experimental fluorescence intensities; the curve is calculated with Eqs. (1) and (2) and parameters, log k = 6.80, &,&top) = 28.7, n = 21.7 (variance = 0.076). Lower panel: sum of squares of differences (SSQ) between calculated and observed values determined from best-fits with eqs. (1) and (2) for the fixed parameters given on the abscissae with the other parameters allowed to vary to give a best fit. Each curve derived by connecting values of SSQ from 30 equally spaced values of the fixed parameter. (Value of SSQ at minimum = 1.45).

Fig. 6. Titration of 3.4. 1Om6 M DNS-TSH with HDL. Descrip- tion of panels the same as those for Fig. 5. Log k = 8.01, F = 26.5, n = 11.7 (variance = 0.083, value of SSQ at rrzmum = 1.16).

197

convergence within linear constraints in the neighborhood of the minimum value of the sum of the squares of the observed and predicted values (least-squares). In order to evaluate results in non- linear fits, the sum of squares are explored as a function of variations in the sum of squares with the parameters as illustrated in Figs. 5 and 6 for TSH titrated with HDL. While the sum of squares show true minima, some of the minima are quite shallow. Thus, the titration at a concentration of 3.4 . 10h7 M in TSH (Fig. 5) shows a more reliable value for the binding constant than does the titration of TSH at 3.4 - lop6 M (Fig. 6). Conversely, the number of sites can be better evaluated at a concentration of 3.4. low6 M in TSH (Fig. 6). Thus, reasonable values for the binding of TSH to HDL are: log K= 7 and n = 12. The binding data for TSH and apolipoprotein A-I are illustrated in Fig. 7. Despite the data indicating that apolipoprotein A-I is a subunit of about l/5 the site of the protein moiety of HDL, the apolipoprotein

0 1.6 3.2 4.0 6.4

[APO-AIM x 10’

14.5 8.8

7.6

; 11.0

i--,iu

6.4

7.5 5.2

4.0 5 6 7 0.8 1.6 2.4

LOG K No. SITES

Fig. 7. Titration of 6.7.10-’ M DNS-TSH with apolipoprotein A-I. Upper panel: 0, experimental fluorescence intensities; the curve is calculated with log k = 6.12, F,, = 39.9, n = 1.36 (variance = 0.30). Lower panel: same as those for Fig. 5 except that equally spaced points are marked with a filled symbol. (Value of SSQ at minimum = 4.58).

still binds from one to two TSH molecules, with an association constant smaller than that of HDL by l/2 to 1 log unit. These data suggest that the sites binding TSH on apolipoprotein A-I and HDL are similar.

Polarization of fluorescence In order to ascertain whether the fluorescence

changes observed with DNS-TSH, upon titration with HDL, resulted from a change in relaxation time or a perturbation of the dansyl chromophore, fluorescence polarization measurements were carried out as a function of HDL concentration. As shown in Fig. 8, the polarization of fluorescence of DNS-TSH increases upon addition of HDL. Since the polarization increases with the quantum yield, the interaction should reflect the formation of a species of greater relaxation time and consequently of larger molecular size; in fact, if the observed fluorescence changes were the result of a simple perturbation of the dansyl fluorochrome, as the excited lifetime of the latter increases, the polarization of fluorescence should have decreased [31,32].

The dependence of the interaction of HDL with TSH on charge interaction is suggested by the observation that changes of fluorescence and fluorescence polarization are sensitive to the ionic strength of the medium. Indeed, in the presence of 0.15 M NaCl (Fig. 8, panel A) the fluorescence changes are greatly reduced, although not com-

b ’ ’ ’

L / I I I I I , I 0 ;:o, mgirnl

.4 Jo (NaCI, ml,”

Fig. 8. Effect of NaCl on the perturbation of fluorescence (0, 0) and polarization of fluorescence (0) of DNS-TSH. In panel A, 10d6 M DNS-TSH in 20 mM phosphate buffer (pH 7.2) in the presence and in the absence of 0.15 M NaCl is titrated with HDL (mg/ml refers to the protein moiety alone). In panel B it is shown that the fluorescence perturbation of DNS-TSH in- duced by HDL can be reversed by NaCl. In both panels excitation and emission wavelengths are 340 nm and 510 nm, respectively.

198

pletely eliminated, and both fluorescence and fluorescence polarization changes can be reversed with NaCl (Fig. 8, panel B).

TSH pertur~atia~ of HDL lipid core

The effect of TSH on the behavior of the fluo- rescent, lipophylic probes, DPH and pyrene, which partition preferentially into the lipid core of lipoproteins [33,34] has been studied. The rate of formation of excited state dimers (excimers) between two pyrene molecules, one in the ground state and the other in an excited state, is directly proportional to the concentration of the probe and is limited by its diffusion through the lipid phase [3X36].

Pyrene excimer fluorescence has a maximum at 470 nm, whereas the peak intensities of monomer fluorescence occur at 375, 384, and 393 nm. The effect of TSH on the ratios of emission intensities J470(excimer)/l,,,(monomer) of pyrene in HDL is shown in Fig. 9. If the rate of excimer formation is diffusion limited, the ratio 1d70/Zj93 is directly proportional to the diffusion coefficient of pyrene. We did not measure the actual diffusion coeffi- cients of pyrene, which would have required the knowledge of pyrene lifetime, the partition water/HDL of the probe, and the volume of the HDL particle. However, the difference of the two slopes, in the absence or in the presence of TSH, is

5 10 15

[PYRENEj M x lff

Fig. 9. Dependence of excimer/monomer fluorescence ratios on the concentration of pyrene added to HDL (4. lo-’ M in 20 mM T&-acetate buffer, pH 7.2) in the absence (a) and in presence (0) of 1.6.10-” M TSH. The inset shows the effect of titration with TSH on the lipid microviscosity (?j = poises) as measured by fluorescence polarization of HDL containing DPH probe.

sufficiently large to warrant the conclusion that the microenvironment of the probe is significantly different under the two experimental conditions. Two interpretations of the data are equally plausi- ble; either TSH decreases the viscosity of HDL thus increasing pyrene diffusion rate, or TSH im- poses a conformational change associated with a reduced volume of the HDL particle which in turn would increase the collision frequency of the probe. However, the first inte~retation is also supported by the observation that titration of HDL, containing DPH probe, with TSH, at 25’C, causes a hormone concentration-dependent decrease of lipid microviscosity (inset, Fig. 9). Indeed, a volume reduction would be expected to affect viscosity in the opposite direction.

Discussion

In a past report [5] we described the presence, in normal human serum, of a protein component capable of inhibiting TSH binding to its membrane receptor, through the interaction with the hormone rather than the receptor. We also noted that this binding inhibitor was not an immunog- lob&n [5].

In this study, the interactions of serum lipoproteins with TSH have been evaluated. The choice of lipoproteins, as likely binding species, was reasonable, since lipoproteins contain polar lipids which are known to interact with TSH [19,27]. This approach led to the identification of the ~gh-density lipoprot~n as a TSN binding species. The results obtained suggest that the ability to bind TSH is a property of HDL, since other lipoproteins, and the fraction with density greater than 1.21, which includes all other serum proteins, failed to show any binding. The possibility, however, that different techniques could demonstrate additional TSH binding components cannot be ruled out.

Specific binding of TSH by human serum gIobulins has been reported, based on experiments in which a~onium sulphate was used to copre- cipitate globulins and i3’I-labeled TSH f47. The same report showed that TSH migrated with immunoglobulin G when studied by paper electrophoresis [4]. Since the HDL preparations used in the present study were free from immunoglobulin

contamination, the possibility that the effect observed could be due to IgGs can be ruled out. The relationship of HDL to the serum inhibitor described in our previous study [S], as well as the physiological sig~fic~ce of the interaction with TSH, is under investigation. The possibility that TSH might be bound to HDL in human serum is suggested by the observation that immunoreactive TSH could be detected in two out of ten preparations of HDL isolated by flotation. This finding was surprising in view of the very drastic conditions to which serum is exposed during the frac- tionation procedure (extremely high ionic strenght and centrifugal force) which are very unfavorable for the formation of the TSH-HDL complex. The physiolo~cal significance of this finding is a matter of further study.

The interaction of TSH with HDL and the absence of interaction with other lipoproteins can be explained if the primary binding site for the hormone is on the apolipoprotein portion which is unique to HDL. Al~ou~ apolipoprotein A-I, i.e., the major apolipoprotein of HDL, is also found to be present in VLDL, the lipid composition, structure or greater proportion of lipids to proteins in the latter, may prevent the interaction with the hormone. The following considerations support the hypothesis that TSH interaction with HDL occurs primarily with the apolipoprotein. Al- though the exact location of lipid and protein molecules in the HDL particle is still speculative, currently accepted models [37] propose that the helical portions of the apo~poproteins occupy the surface of the particle, which they share with the polar heads of phospholipids and the OH function of unesterified cholesterol. Side chains of non-polar amino acids, acyl chains of phospholipids, tri- acylglycerols, and cholesteryl esters fill the inner space forming an hydrophobic core. Since the interaction of HDL with TSH appears to be largely electrostatic, as suggested by the inhibitory effect of NaCl on the perturbation of DNS-TSH fluorescence and in the solid-phase binding assay, it probably takes place with the charged groups of the proteins at the surface of the lipoprotein. The polar lipids in HDL should not contribute to the binding sites, as the same phospholipids are also found in LDL which do not interact with TSH. Finally, fluorescence perturbations of DNS-TSH

199

and behavior in the solid-phase assay nearly identical to those caused by HDL are produced by apolipoprotein A-I.

Analysis of DNS-TSH fluorescence data sug- gests that there are at least 12 binding sites for TSH on each HDL particle of an average molecular weight of 2.65 * 105, while an upper limit cannot be derived with reasonable confidence. Similar treatment of the DNS-TSH fluorescence data, obtained with apo~poprotein A-I, yields one or two sites on the apolipoprotein molecule with a lower association constant than for HDL. This finding does not rule out the possibility that other apolipoproteins of HDL may also bind TSH; however, it is consistent with the proposed model which involves multiple, independent binding sites for TSH, located on the apolipoprotein portion of the HDL particle. Even if the primary site for interaction. can be assigned to the apolipoprotein, TSH binding to HDL is associated with a perturbation of its lipid moieties. This may be the result of a rearrangement of proteins and lipid molecules or may be related to the insertion of part of the TSH molecule within the lipid core of HDL. The change of the lipid core of the HDL particle is toward a more fluid state.

References

1

2

3

4 5

6 7 8

9

10

11

12

13 14

Beall, G.N., Chopra, I.J., Solomon, D.H. and Kruger, S.R. (1978) J. Clin. Endocrinol. Metab. 47, 967-973 McKenzie, J.M., Zakarija, M. and Sato. A. (1978) J. Clin. Endocrinol. Metab. 7, 31-45 Rees Smith, B. (1981) in Receptor Regulation (Lefkowitz, R.J., ed), pp. 217-244, Chapman and Hall, London Biro, J. (1981) J. Endocrinol. 88, 339-349 Lee, G., Aloj, S.M., Beguinot, F. and Kahn, L.D. (1977) J. Biol. Chem. 252, 7967-7970 Hatch, F.T. and Lees, R.S. (1968) Adv. Lipid Res. 6, 1-68 Hazelwood, R.N. (1958) J. Am. Chem. Sot. 80,2152-2156 Scanu, A.M. and Granda, J.L. (1966) Biochemistry, 5, 446-455 Kostner, G.M. (1981) in High Density Lipoproteins (Day, C.F., ed.), pp. l-42, Marcel Decker, New York Scanu, A.M., Toth, J., Edelstein, C., Koga, S. and Stiller, E. (1969) Biochemistry 8, 3309-3316 Winand, R.J. and Kahn, L.D. (1970) J. Biol. Chem. 245, 967-975 Tate, R.L., Schwartz, HI., Holmes, J.M., Winand, R.J. and Kahn, L.D. (1975) J. Biol. Chem. 250, 6509-6515 McKenzie, J.M. (1958) Endocrinology 63 372-381 Kahn, L.D. and Winand, R.J. (1971) J. Biol. Chem. 246, 6570-6575

I5 I&am. K.C., AIoJ. S.M. and Edelhoch, H. (1974) Arch.

Biochem. Biophys. 163. 589-599

16 Lowry, O.H.. Rosebrough. N.J.. Fax, A.L. and Randall,

R.J. (1951) J. Biol. Chem. 193, 265-275

17 Markwell, M.A.K., Haas. SM., Bieber, L.L. and Tolbert,

N.E. (1978) Anal. Biochem. 87. 206-210

18 Yavin, E., Yavin, Z., Schneider, M.D. and Kohn, L.D.

(1981) Proc. Natl. Acad. Sci. USA 78, 3180-3184

19 Aloj, S.M., Lee, G., Consiglio, E., Formisano, S., Minton.

A.P. and Kohn, L.D. (1979) J. Biol. Chem. 254, 9030-9039

20 Horton, H.R. and Kohsland, D.E.. Jr. (1967) Methods

Enzymol. 11, 556-565

21 Shinitzky, M. and Barenholtz, Y. (1978) Biochim. Biophys.

Acta 515, 367-394

22 Marqaurdt, D.W. (1963) J. Siam. 11, 431-441

23 Levenberg, K. (1944) Quart. Appl. Math. 2, 164-168

24 Bloom, R.J. and Elwood, J.C. (1981) Clin. Biochem. 14(3),

119-125

25 Dawson, G., Kruski. A.W. and Scanu, A.M. (1976) J. Lipid

Res. 17, 125-131

26 Scanu, A.M. (1966) J. Lipid Res. 7. 295-306

27 Aloj, SM., Lee, G., Grollman, E.F., Beguinot, F., Consiglio,

E. and Kohn, L.D. (1979) J. Biol. Chem. 254. 9040-9049

28 Palumbo, G. and Edelhoch, H. (1977) J. Biol. Chem. 252,

3684-3688

29 Reynolds, J.A. (1976) J. Biol. Chem. 251, 6013-6015

30 Gwynne, J., Palumbo, G.. Brewer, H.B., Jr. and Edelhoch.

H. (1975) J. Biol. Chem. 250. 7300-7306

31 Werber, G. (1952) Biochem. J. 51, 155-167

32 Perrin, F. (1926) J. Phys. Radium. 7, 390-401

33 Jonas, A. and Joung, R.W. (1975) B&hem. Biophys. Res.

Commun. 66, 651-657

34 Charlton, SC. and Smith, L.C. (1982) Biochemistry 21.

4023-4030

35 Galla, H.J. and Hartmann, W. (1980) Chem. Phys. Lipids

21. 1999219

36 Vanderkooi, J.M. and Callis, J.B. (1974) Biochemistry 13.

4000-4006

37 Verdery, R.B. and Nichols. A.V. (1975) Chem. Phys. Lipids

14. 123-124

Thyrotropin interaction with high-density lipoproteins

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