cl?// calcium (1995) 17, 14-20 0 Pearson Professional Ltd 1995

Thapsigargin protects human erythrocyte Ca*+-ATPase from proteolysis

C.O. BEWAJI and A.P. DAWSON

School of Biological Sciences, University of East Anglia, Norwich, UK

Abstract - The effect of thapsigargin on the activation by partial proteolysis of the plasma membrane Ca2+-ATPase was studied in intact human erythrocyte membranes and in the purified enzyme. The enzyme was maximally activated in the absence of thapsigargin within 1 min of exposure to trypsin. However, in the presence of thapsigargin maximal activation was achieved only after 5 min trypsin digestion. Thapsigargin did not alter the pattern of proteolysis as revealed by SDS-PAGE of the tryptic fragments, although it slowed down the rate of appearance of the fragments. Thapsigargin also enhanced the activation of the enzyme by calmodulin. These findings suggest that, although thapsigargin at low concentrations has no effect on the catalytic activity of the Ca2+-ATPase in vitro in the absence of calmodulin, it could interfere with its regulation in vivo.

A Ca2+-activated, Mg2+-dependent ATPase (Ca2+, Mg2+-ATPase) operates as a calcium pump in the plasma membrane of red cells [l,Z]. This enzyme belongs to the P-class of ATPases and is the largest of all P-type ion pumps [3]. Recent cloning work has confirmed that it is a single polypeptide with a molecular weight of about 134 kD [4]; its kinetic and enzymatic properties are generally believed to be different from those of the sarco-endoplasmic re- ticulum (SERCA) types of Ca*+-ATPases [4,5].

A number of investigators have reported that thapsigargin (TG), a plant-derived sesquiterpene lac- tone, is a potent and specific inhibitor of calcium transport (and Ca2+-ATPase) by the SERCA family of Ca*+ pumps [6-IO] but does not inhibit the plasma membrane Ca*+ pumps (PMCA) or other ion-motive ATPases [IO]. One major difference be-

tween the properties of the PMCA and SERCA types of Ca2+-ATPases is that the former is acti- vated by calmodulin, acidic phospholipids and by limited proteolysis [ 111. The calmodulin-binding domain of the erythrocyte Ca2+-ATPase is at the C- terminal region of the enzyme and is cleaved off during proteolysis [ 121.

In order to examine further whether or not TG interacts with the plasma membrane Ca2+-ATPase (but without inhibiting its catalytic activity), we have studied the effects of TG on some of the acti- vating treatments of the human erythrocyte Ca*+- ATPase. We report here that the enzyme is resistant to proteolysis in the presence of TG and is hence more slowly activated by this treatment. However, TG enhances the activation of the enzyme by cal- modulin. It therefore seems that this PMCA type

14

THAPSIGARGIN AND ERYTHROCYTE Ca’+-ATPase

enzyme has a binding site for TG, although, unlike the SERCA enzymes, occupation of the binding site does not inhibit the catalytic activity.

Materials and methods

Materials

ATP (vanadate-free) and dithiothreitol were from Boehringer-Mannheim, UK Ltd. Phosphati- dylcholine, Triton X-100, sodium dodecyl sulphate, thapsigargin, trypsin and soya bean trypsin inhibitor were purchased from Sigma Chemical Co., Poole, Dorset, UK. All other reagents were of analytical grade.

Preparation of erythrocyte ghost membranes and purification of Ca2+-ATPase

Haemoglobin- and calmodulin-free ghost mem- branes were isolated from recently outdated human erythrocytes and the Ca*+-ATPase was purified

A 0.6

8 m 0.1 1

? n +/W-i

0 II ,

0 5 10 15

Time (min)

15

from these membranes, in the presence of phosphati- dylcholine, essentially as described by Niggli et al. [1 l] except that protease inhibitors were omitted from the buffers.

Assays

Protein concentrations were determined by the method of Lowry et al. [13] using bovine serum al- bumin as standard. In samples of the purified AT- Pase which contain interfering materials such as Tri- ton X-100, the protein was first precipitated with de- oxycholate and trichloroacetic acid and determined as described by Bensadoun and Weinstein [14]. ATPase activity was assayed by measuring the re- lease of inorganic phosphate from ATP as pre- viously described [15]. The assay contained, in a volume of 1.0 ml, 130 mM KCl, 60 mM HEPES pH 7.4, 4 mM MgCl2, 10 pM CaC12 (or 2 mM EGTA) and approx. 400 pg ghost protein. The reaction was started by the addition of 1 mM ATP and stopped after 30 or 60 min at 37°C by 0.5 ml of 5% TCA. Unless otherwise specified, calmodulin was not present.

B

0 5 IO 15

Time (mini

Fig. 1 Controlled proteolysis and activation of the human erythrocyte Ca’+-ATPase. 20 pl aliquots of erythrocyte ghost membranes (corresponding to 400 pg of protein) were exposed to trypsin at 25°C at a ratio of membrane protein to trypsin of (a) 20: 1 and (b) 10: 1, for the various times indicated, either in the absence (open symbols) or in the presence (filled symbols) of TG (250 nM) in an incubation volume of 0.1 ml. The reaction was stopped with 10 pl of 10 mg/ml soya bean trypsin inhibitor and the Ca’+-ATPase (circles) and Mg*+-ATPase (triangles) activities were assayed as described in Materials and methods. Data shown are typical of 3 determinations carried out in duplicate.

16

Controlled proteolysis of Ca2’-ATPase

Aliquots of the membrane-bound or purified Ca2+- ATPase (about 400 ug or 10 pg of protein, respec- tively) were incubated for various times with trypsin at a ratio of trypsin to ATPase ranging between 1: 10 and 1:25 (w/w). The standard incubation medium contained, in a volume of 0.1 ml, 130 mM KCl, 30 mM HEPES, pH 7.4 and 1 mM MgC12. The reac- tion was stopped with a lo-fold concentrated solu- tion of soya bean trypsin inhibitor (1 mg/ml final concentration). In some cases the reaction was stopped by adding a 3-fold concentrated electro- phoresis buffer, followed by 5 min of boiling. The mixture was then applied to 10% SDS polyacryla- mide slab gels. Other experimental conditions were as described in the captions to figures. The electro- phoretic system was as described by Laemmli [16] and the gels were stained with silver as described by Merril et al. [ 171.

Results

Time courses for the activation of the membrane- bound human erythrocyte Ca2+-ATPase with two trypsin concentrations in the presence and absence of TG (250 nM) is shown in Figure 1. In Figure la, in the absence of TG, the enzyme reaches maximum

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activation within 1 min of exposure to trypsin. How- ever, in the presence of TG, the enzyme is more slowly activated, reaching maximum activation only after 5 min of exposure to trypsin. In both cases, as the enzyme becomes further degraded, activity de- clines. A similar pattern is found at the higher tryp- sin concentration (Fig. lb), although the peak activ- ity reached is lower due to faster inactivation. At the higher trypsin concentration it appears that this inactivation phase is also slower in the presence of TG. Although the experiments shown in Figure 1 were carried out on ghosts prepared in the absence of proteolytic enzyme inhibitors, we have obtained very similar data using ghosts prepared in the presence of 0.1 mM phenylmethylsulphonylfluoride, 0.02% thimerosal and 1 yg/ml aprotinin followed by wash out of the inhibitors. The effect of TG on the rate of proteolysis can still be observed at a concen- tration as low as 100 nM TG (data not shown). In the experiment shown in Figure 2, proteolysis of the Ca2+-ATPase was carried out for 1 min or 2 min in the presence of various concentrations of TG and ATPase activities were measured thereafter. TG re- duces the magnitude of the activation of the enzyme in a concentration-dependent manner, the protective effect being nearly maximal at 250 nM TG. In con- trol experiments, using 0.5% fluorescein isothio- cyanate-labelled casein as a fluorescent trypsin sub- strate according to the procedure of Twining [18],

TO Concentration ()IM)

Fig. 2 Effect of TG on the activation of human erythrocyte Ca*’ -ATPase. 200 pg of erythrocyte ghost membranes were exposed to 10 pg of trypsin for I min (filled squares) or 2 min (open squares) in the presence of various concentrations of TG in a total volume of 0.1 ml. The reaction was stopped with 10 p1 of soya bean trypsin inhibitor (10 mg/ml) and ATPase activity was measured as described in Materials and methods and expressed as a percentage increase in the activity of the unproteolysed enzyme. The data shown are typical of 3 determinations.

THAPSIGARGIN AND ERYTHROCYTE Ca’+-ATPase 17

KDa

Fig. 3 Effect of TG on the trypsin fragmentation of the purified human erythrocyte Cazf-ATPase. 50 pl aliquots of Triton X-100 solubilised and purified CazC-ATPase (about 10 pg protein) were exposed to 1 pg trypsin for 0, 0.5, 1, 1.5, 2, 3, 4, 5, 8, 10 and 20 min (lanes l-l 1, respectively) in the absence (a) and in the presence (b) of TG (250 nM). The reaction was stopped by the addition of 25 pl of 3-fold concentrated electrophoresis sample buffer, followed by 5 min of boiling. 40 pl aliquots of the mixture were then applied to 10% SDS polyacrylamide slab gels. The gels were stained by the silver impregnation technique. The data shown are from 1 of 4 similar experiments.

we found that TG (2.50 nM) does not inhibit trypsin. omitted to prevent interference with trypsin. In the Figure 3 shows the tryptic digestion pattern of absence of TG, the ‘intact’ enzyme, Mr 134 kD, is

the purified enzyme when subjected to electro- degraded within 30 s of exposure to trypsin. This is phoresis on 10% polyacrylamide slab gels. The accompanied by the appearance of a 90 kD polypep- purified enzyme appears as a doublet due to the ac- tide, which is also degraded on further exposure to tivity of endogenous proteases. The inclusion of trypsin to products with Mr of 81, 76 and sub- protease inhibitors in the purification buffer was sequently 34 and 27 kD. The time of appearance of

18 CELL CALCIUM

Table Effect of thapsigargin on the activation of human erythrocyte membrane Ca*+-ATPase by calmodulin.

Additions ATPase nctivity (pmol/mg protein/h)

-TG +TG

EGTA (2 mM) 0.10 f 0.01 0.10 f 0.01 CaCl2 (10 PM) 0.31 + 0.06 0.32 If: 0.05 CaC1.7 + CaM 0.85 zkO.04 1.32 f 0.07

ATPase activity was determined as described in the Materials and methods section. Values represent the means + SD for 4 independent determinations. When present, calmodulin (CaM) concentration was 120 nM and TG was 250 nM.

the 76 kD band corresponds to the point in Figure 1 at which the activity of the enzyme begins to de- cline. In the presence of TG, the intact enzyme is more slowly degraded and the 81 kD and 76 kD polypeptides persist at longer digestion times.

The Table shows the effect of TG on the interac- tion between the Ca2+-ATPase and calmodulin. TG substantially enhances (by about 50%) the activation of the enzyme by calmodulin.

Discussion

It is now well established that the plasma membrane calcium pump, which is responsible for the extru- sion of Ca2+ from the cytosol, can be activated by a number of treatments: by calmodulin; by acidic phospholipids (particularly polyphosphoinositides) and long chain polyunsaturated fatty acids; by limited proteolysis; or by a phosphorylation cata- lysed by CAMP-dependent protein kinase. These treatments result in an increase in both the Ca2’ af- finity and maximal velocity of the enzyme.

A number of investigators have raised the ques- tion of whether or not the various activating treat- ments act by the same or by different mechanisms. Enyedi et al. [19] have shown that acidic lipids are more effective than calmodulin in increasing the Ca2+-affinity of the enzyme. They suggested that the activation of the enzyme by these lipids was of physiological importance when the free cytoplasmic

Ca2’ level decreased to levels where calmodulin ac- tivation became insignificant.

Previous studies have shown that the rate of trypsin proteolysis of the erythrocyte Ca2+-ATPase is influenced by various effecters of the enzyme such as calmodulin, Ca2+, Mg2+ and ATP [20,21]. These studies provided evidence that the enzyme undergoes conformational changes during the cata- lytic cycle which may expose or hide trypsin-sensi- tive peptide bonds. It has also been established that the erythrocyte Ca2+-ATPase shifts reversibly be- tween two states: the so-called calmodulin-deficient A-state and the more active calmodulin-saturated B- state with high Ca2’ affinity [22]. The results presented in the present study show that TG de- creases the rate of proteolysis (and activation) of the erythrocyte Ca2+-ATPase by trypsin in a manner that does not involve direct inhibition of trypsin. This suggests that the trypsin-dependent transition from the low Ca2+-affinity (A-state) to the high Ca2’ affinity (B-state) is slow in the presence of TG. An alternative explanation is that the binding of TG to the enzyme could result in a conforma- tional rearrangement in the enzyme molecule which makes otherwise trypsin sensitive bonds inaccessible to trypsin. In either case, it is clear that the erythro- cyte Ca2+-ATPase has a binding site which can be occupied by TG, although there is very little effect of the latter on the catalytic activity of the enzyme in the absence of calmodulin. This is in agreement with the previously reported lack of effect of TG on the transport of Ca2+ by plasma membrane vesicles [6]. However, using calmodulin-containing erythro- cyte membranes, Foder et al. [26] showed that TG activated Ca2+ pumping and also that there was a small effect of TG on ATPase activity. Binding of TG to the Ca2+-ATPase could, therefore, affect the regulation of the enzyme by calmodulin (or en- dogenous proteases) in vivo.

More recent studies on controlled proteolysis of the Ca2+-ATPase have led to the proposal that the stimulation by calmodulin and acidic lipids may in- volve independent domains of the enzyme molecule [23-251. Proteolytic removal of the calmodulin- binding region (at the C-terminal end) results in the activation of the enzyme in a calmodulin-like man- ner, as well as loss of calmodulin sensitivity. Fur- ther removal of 44 amino acids from the residual

THAPSIGARGIN AND ERYTHROCYTE Ca*+-ATPase 19

polypeptide results in a further activation of the enzyme similar to that produced by acidic lipids. It has, therefore, been suggested that the calmodulin binding region (C region) and the region containing the 44 amino acids (G region) confer conformational restraints on the enzyme molecule, which are re- lieved by the removal of these regions by prote- olysis or by the binding of the natural activators to these regions. It is very likely that binding of TG to the enzyme tends to impede removal of either the C or G regions or both. We cannot conclude from our present results which region of the ATPase molecule contains the TG binding site. We, however, can rule out the possibility that TG binds at the same point as calmodulin since the binding of TG to the enzyme does not prevent (or inhibit) its activation by calmodulin. Indeed, since the enzyme is acti- vated by TG in the presence of calmodulin, it is clear that both must bind simultaneously and the re- sulting conformation must be different from that in the presence of calmodulin alone. Furthermore, we have found that activation of the purified erythro- cyte Ca*+-ATPase by acidic phospholipids (phos- phatidyl inositol) is unaffected by TG (data not shown). This could either be because TG does not bind under these circumstances or that the confor- mational transition caused by acidic phospholipids over-rides that produced by TG.

5.

6.

7.

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Kijima Y. Ogunbunmi E. Fleischer S. (1991) Drug action of thapsigargin on the Ca2+ pump protein of sarcoplasmic reticulum. J. Biol. Chem., 266, 22912-22918. Papp B. Enyedi A. Kovacs T. et al. (1991) Demonstration of 2 forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles. J. Biol. Chem., 266, 14593-14596. Niggli V. Adunyah ES. Carafoli E. (1981) Acidic phospholipids, unsaturated fatty-acids, and limited proteolysis mimic the effect of caimodulin on the purified erytbrocyte Ca’+-ATPase. J. Biol. Chem., 256, 8588-8592. James P. Vorherr T. Krebs J. et al. (1989) Modulation of erythrocyte Ca‘+-ATPase by selective calpain cleavage of the calmodulin-binding domain. J. Biol. Chem., 264, 8289-8296.

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Acknowledgements

We thank the Wellcome Trust for financial support and Judith Nuttall for technical assistance.

15. Bewaji CO. Bababunmi EA. (1987) Further characterization of the membrane-bound (Ca*+, Mg**)-ATPase from porcine erythrocytes. Int. J. Biochem., 19,721-724.

16. Laemmli UK. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680-685.

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Please send reprint requests to : Dr Alan P. Dawson, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

Received : I I May 1994 Revised : 8 August 1994 Accepted : 6 September 1994