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Protistology 2 (2), 123-129 (2001) PPOtlStOlO&y
Heat shock protein of 70 kDa in Amoeba proteus
Yulia I. Podlipaeva
Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
Summary
Heat shock protein of 70 kDa (HSP70) was revealed in the cells of Amoeba proteus strain Da by immunoblotting with the use of poly- and monoclonal anti-HSP70 antibodies. This protein can bind ATP and GTP according to ATP-agarose chromatography data. The untreated cells contained surprisingly high level of HSP70. The one hour heating of the amoeba culture at 37 °C did not cause the induction of HSP70, but resulted in pronounced decrease of its level in 4 hrs after the heat shock. This phenomenon might be connected with the utilization of HSP70 reserves in stress-conditions to protect sensitive protein structures of the cell. After using the monoclonal anti-HSP70 antibodies the increase in the HSP70 content was revealed in 4 hrs after one hour heating of amoeba cells at 32 °C - the temperature close to the borders of temperature tolerant range (TTR) of Da amoeba strain. It looks like the temperatures causing the induction of HSPs depend upon the strain (species) TTR, presumably even indicating its natural borders.
Key words: Amoeba proteus, heat shock protein
Introduction
Protozoans, like other organisms, may employ biochemical and molecular responses to minimize the damage caused by inadequate (stress) temperature conditions. Among them, the synthesis of heat shock proteins (HSPs) is the most important. In protozoans the heat shock proteins, belonging to various HSP families in accordance to their molecular weight were revealed. The most prominent stress protein is a member of heat shock 70 kDa family - HSP70. This type ofmolecular chaperones is presented in protozoans by the same main classes as it is in other eukaryotic organisms, i.e. by proteins located in cytosol, endoplasmic reticulum and in organelles - mitochondria or chloroplasts.
HSPs70 are comprehensively studied in parasitic protozoans, in heteroxenic trypanosomatid parasites (Trypanosoma and Leishmania) in particular, because during their life cycles these parasites are exposed to
major temperature differences when moving from invertebrate (ectothermal) vectors to mammalian (endothermal) hosts. Usually after temperature shifts parasites undergo a classical heat shock response, i.e. the transcriptional activation of heat shock genes and heat shock proteins synthesis (Polla, 1991; Wallace et al., 1992). It was shown, for example, that in Trypanosoma cruzi the HSP70 level at 37 °C was 4fold higher than at 28 °C (Requena et al., 1992). Interestingly, after the experimental temperature shift from 22 to 37 °C the synthesis of HSPs belonging to different families, HSP70 among them, was observed in heteroxenic trypanosomatid Phytomonas characias
— parasite of plants which lacks endothermal host in its life-cycle (Sanchez-Moreno et al., 1997). It is worth mentioning that such biochemical response of plant-insect inhabiting Phytomonas may be presumed to be an atavistic feature pointing to the existence of common ancestors of insect-plant trypanosomatids and Trypanosoma - parasites of mammals. The latter
© 2001 by Russia, Protistology
1 2
Figs 1, 2. Blots after electroblotting and SDS-electrophoresis of untreated strain Da amoebae homogenates and homogenates of amoebae, heated at 37 °C during 1 hr. The blots are treated with polyclonal anti-HSP70 antibodies and stained with secondary antibodies conjugated with peroxydase (1) and alkaline phosphatase (2); K — untreated, control amoebae; 0, 2, 3, 4, 6, 24 — homogenates prepared from the amoeba cells in various time periods (0, 2, 3, 4, 6, and 24 hrs, respectively) after the heat shock; on the start of every lane 10 |ig of protein; a - molecular marker of 66 kDa.
appear to be at the base of trypanosomatid evolutionary tree (see: Maslov et al., 2001).
Besides the universal phenomenon of increased synthesis in response to a temperature shift, the parasite HSPs seem to play unique role in differentiation of life cycle stages - for example in the transformation of promastigotes to amastigotes in Leishmania major (Van der Ploeg et al., 1985). Expression of high levels of HSP70 was also observed during a short period of conversion of bradizoites to tachyzoites in the course of the life cycle of Toxoplasma gondii (Silva et al., 1998).
One more characteristic of parasite HSP70 is that it may serve as an antigen, causing the immune response of the host as it was shown for HSP70 of Trypanosoma cruzi (Requena et al., 1993). The 70 kDa protein from trophozoites of the pathogenic Entamoeba histolytica strain which caused the immune response in a group of patients with invasive amoebiasis had about 70% sequence identity with human HSP70 (Ortner et al., 1992).
Heat shock protein of 70 kDa is a highly conserved protein and is thus a potentially reliable phylogenetic marker to solve some phylogenetic problems (Germot and Philippe, 1999). Inter alia, the protein product of HSP70 gene normally functions in mitochondria and the identification of HSP70 gene in amitochondriate microsporidia allowed to realize that these protozoans initially contained mitochondria and to correct their
M 1 2 3 4 5 6
.
it
position on the phylogenetic tree (Hirt et al., 1997; Peyretaillade et al., 1998).
Contrary to parasites, HSPs70 are studied more poorly in free-living protozoans, Chlamydomonas reinhardtii (Drzymalla et al., 1996), Tetrahymena thermophila (McMullin and Hallberg, 1986), T. pyriformis (Fink and Zeuthen, 1980), Paramecium tetraurelia, Euplotes aediculatus (Budin and Philippe,
1998) and Naegleria gruberi (Walsh, 1980) among them. As far as we know there is only one work devoted to heat shock proteins of free-living freshwater amoebae
- Amoeba proteus and A. borokensis. The synthesis of a new class of proteins which might be classified as heat shock proteins was detected in amoeba cells after the 10 °C-increase of temperature of surrounding medium (Kalinina et al., 1988).
The present work is an initial approach to reveal and study HSP70-related protein in the A. proteus cells by the method of immunoblotting, thus trying to compare its level in shocked and untreated cells and to evaluate the dynamics, if any, of HSP70 expression.
Materials and methods
Da strain (clone) of Amoeba proteus from the amoeba culture collection maintained in the Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of Sciences, was used
x Fig. 3. SDS-electrophoresis of the fractions obtained after the ATP-agarose chromatography of amoeba Da homogenates. 1-6 - №№ of the lanes; M — molecular markers (a -66 kDa); 1-4 - 5mM GTP/TM eluate; 5-6 — 3mM ATP/TM eluate; 12% PAAG, Coomassie brilliant blue.
K 28 32 37
Fig. 4. Blots after electroblotting and SDS-electrophoresis of untreated strain Da amoebae homogenates and homogenates prepared from amoebae in 4 hrs after their heating at different temperatures during 1 hr. The blot is treated with polyclonal anti-HSP70 antibodies and stained with secondary antibodies conjugated with alkaline phosphatase; K—untreated, control amoebae; 28, 32, 37 — homogenates prepared in 4 hrs from amoebae, heated at different temperatures (28, 32 u 37 °C, respectively); a -66 kDa.
in this study. This strain originated from Southgampton University, UK, and was received in the Laboratory in 1970. The temperature optimum of multiplication (TOM) for clone Da is 22 °C (Sopina, 1986).
The amoebae were cultivated according to the method of Prescott and Carrier (1964) in Koch-dishes or minicristallizers 30-50 mm in diameter and fed with Tetrahymena pyriformis GL every 48 hrs. (Yudin, 1990). Before the experiments amoebae starved for 72 hrs.
Amoebae, cultivated at 22 °C were treated during 1h at 28, 32 or 37 °C. Then the portions of cells were separated from the major culture volume immediately after the treatment (0h) and after some time - different in different experiments. Every portion, as well as control, untreated amoebae, was precipitated with a low speed (1000 rpm) centrifuge and then homogenized with a teflon pestle in a glass homogenizer. Buffer for extraction contained 20 mM Tris HCl, pH 7.5, 20 mM NaCl, 0.1mM EDTA, 0.5 mM dithio-threitol (DTT) and 0.2 mM phenylsulfonylfluoride (PMSF). The volumes ratio between precipitated cells and the buffer was 1:1. All homogenates, including that of untreated amoeba cells, were placed into the refregerator (-20 °C). Then the homogenates were defrozen and simultaneously centrifuged in high speed centrifuge (15000 rpm) for 45 min at 4°C. Supernatant was examined by SDS-electrophoresis in 7, 10 and 12% PAA gels in Tris-glycine system (Laemmli, 1970). To prepare the sample, 3 supernatant aliquot was mixed with 1 aliquotes of fourfold Laemmli buffer (1% SDS, 5% b-mercaptoethanol, 10% glycerine), the mixture was incubated in water bath at 100 °C for 3-4
min. Electrophoresis was carried out in gel slabs 120 x 90 x 0.8 mm firstly at I = 10 - 12 mA (1-1.5 hrs), and then at I = 20 - 25 mA (2-2.5 hrs). After the electrophoresis gels were fixed by the formaldehyde, ethanol and acetic acid mixture, stained with 0.25% solution of Coomassie blue and differentiated in 7% acetic acid.
To reveal HSPs the proteins were transferred to the nitrocellulose immediately after the electrophoresis by the method of electroblotting (Towbinet al., 1979). We used non-commercial rabbit polyclonal anti-HSP70 antibodies which were obtained against cattle heat shock proteins, were specific for inducible form of HSP70, and exhibited the cross-reactivity with HSPs ofdifferent animal species (Margulis et al., 1991). These antibodies were kindly provided by Dr. A.Kinev (Institute of Cytology, RAS). We also used monoclonal anti-cattleHSP70 antibodies directed against the inducible form of HSP70 family; antibodies had been provided de bonne grece by Dr. B. Margulis (Institute of Cytology, RAS). Zones ofbinding with anti-HSP70 antibodies were stained with antibodies (AT II), conjugated with alkaline phosphatase (Sigma), or peroxydase (Merck) by fermentative reaction. To identify the molecular masses of polypeptides, high molecular weight markers (Sigma) were used; zones, corresponded to the positions of the markers in the blots were stained by Ponceau (Sigma).
To start the purification procedure of the amoebae heat shock protein of HSP70 family, we prepared the homogenate by mixing 1 aliquote ofthe dense precipitate of amoeba cells with 3 aliquotes of extraction buffer (20 mM Tris HCl, pH 7.5, 20 mM NaCl, 0.1 mM EDTA,
0.5 mM DTT, 0.2 mM PMSF and 0.5% nonidet-40). Amoebae were homogenized on the ice and then the homogenate was centrifuged at 14000 rpm for 30 min at 4 °C. After removing the supernatant one more portion (1 aliquote) of the buffer was added to the precipitate, centrifugation was repeated and supernatants, obtained after both centrifugations were united.
Primarily the supernatant was loaded over the HSP70-sepharose column to isolate HSP70 binding proteins (Margulis and Wfelsh, 1991), then the fraction eluted from the column was loaded over a 1.02 ml ATP-agarose column previously equilibrated by TM buffer (20 mM Tris-HCl, pH 7.5 and 5 mM MgCl2). Then TM/1M NaCl, TM/0.05% tween-20, TM/ 5 mM GTP and TM/3 mM ATP eluates were sequentially washed from the column. After the chromatography the proteins of different fractions were examined by SDS-PAAG electrophoresis in 10 and 12% gels, the gels were stained by Coomassie.
M K+ 28 32 37+
12 34 56789 10 11
H
— 1
Fig. 5. “Calibrating” SDS-electrophoresis of untreated strain Da amoebae homogenates and homogenates prepared from amoebae in 4 hrs after their heating at different temperatures during 1 hr.
1-11 - №№ of the lanes; M — molecular markers (a - 66 kDa); K — untreated, control amoebae; 28, 32, 37 — homogenates prepared in 4 hrs from amoebae, heated at different temperatures (28, 32 h 37 °C, respectively);
+ - comparable protein amounts on the lanes;
10% PAAG; Coomassie brilliant blue.
Results and Discussion
Amoebae of Da strain were cultured at 22 °C, then heated at 37 °C (sublethal temperature for this strain) during 1 hr and the homogenates were prepared from the cells immediately after the temperature treatment, and in various time periods after it. It is seen in Figs 1 and 2, that the zone, which corresponds to the polypeptide, situated somehow above the molecular marker of 66 kDa, is well-pronounced on the blots. W account the protein revealed in such a way to be the heat shock protein, belonging to HSP70 family. We did not find the dependence of protein amount on the time, passed after the temperature shock. The differences in staining intensity of zones of HSP70 localization in control and treated amoebae also were not revealed in both types of experiments.
According to ATP-agarose chromatography data 70 kDa protein from amoebae can bind ATP and GTP. It appeared that 70 kDa protein occurs in practically pure form in the TM/GTP eluate (Fig. 3, lanes 1-4), and in the TM/ATP eluate. The latter also contains polypeptide of molecular weight about 50 kDa (Fig. 3, lanes 5-6).
The affinity to nucleotides is the characteristic feature of both inducible and constitutive forms of HSP70 family heat shock proteins (Welch and Feramisco, 1985). The fact that 70 kDa protein from amoebae shares this feature confirms that this protein belongs to HSP70 family (Podlipaeva and Kinev, 1996).
Other series of experiments were carried out to find more adequate temperature for heat treatment. By the electrophoresis in one gel we examined the homogenates
of untreated (control) amoebae and homogenates, obtained from amoebae in 4 hrs after they had been heated at 28, 32 and 37 °C during 1hr (Fig. 4). In parallel to electroblotting the stained gels with the same set of samples were examined to reveal the possible reasons of differences in bands intensity on the blots and to calibrate the protein amount in different samples (Fig. 5). It is seen that in the sample, from the amoebae after 37 °C-heat shock, the HSP70 level is considerably lower, than in control sample (Fig. 4, lanes “K” and “37”). The amounts of the protein in the start gels are comparable (Fig. 5, lanes 2 h 10). Unfortunately we can not reliably examine the HSP70 levels after 28- and 32 °C-heat treatment because, as seen from the Fig. 5 the amount of total protein in the start gels is higher in both experimental samples (lanes 4 and 7) in comparison with that in the control one (lane 2).
We consider the temperature tolerant range (TTR) of the amoeba strain as a diapason of temperatures which are appropriate for the continued (more than two months) cultivation of the amoebae without pronounced degradation of the culture. The temperature 28 °C lays on the border of the strain Da TTR and was earlier described as causing a slight decrease in cloning efficiency (Sopina, 1986) and altering some enzyme characteristics in the course of amoebae temperature acclimation (Podlipaeva, 2000). The attempts to cultivate Da strain amoebae at 32 °C have never been carried out, but their cultivation at 31 °C had shown that the maintenance of the culture is possible for no more than 13-14 days, the efficiency of cloning being undeviatingly declined (Podlipaeva, unpublished). So, the temperature 32 °C surely lays outside the strain Da TTR, but not far from its borders.
Thus, it has been demonstrated that unheated cells of Amoeba proteus (strain Da) constitutively contained relatively high level of HSP70, revealed by Western blotting with polyclonal antibodies, directed to inducible form of cattle HSP70 (Figs 1, 2, 4). None of the temperatures applied have caused the reliable increase of HSP70 level in 4 hrs after heating; moreover, the treatment of the culture by 37 °C resulted in pronounced decrease of HSP70 level (Fig. 4).
One more experiment was directed to evaluate the HSP70 content in unheated control amoeba cells and in the cells after 1 hr heating at 32 and 37 °C, loading minimal and thoroughly-calibrated amounts of protein into the start gel for electroblotting and using monoclonal anti-HSP70 AT-I for blot treatment. It appeared that the HSP70 level just after the heating at 32 and 37 °C was comparable with that of control cells (Fig. 6, lanes “32°, 0”, “37°, 0 and “K”), in 4 hrs after 37 °C-heat shock its level decreased, as it was demonstrated by polyclonal AT-I blot treatments (Fig. 6, lanes “K” and “37°, 4”), and the increase in the HSP70 content
(HSP70 induction) was revealed in 4 hrs after heating of amoeba cells at 32 °C (Fig. 6, lanes “32°, 4” and “K”).
From the results given above it is noticeable that the HSP70 content in the untreated amoeba cells is relatively high. This fact is of undoubted interest and presumes researches in this field to be continued. The considerable amounts of constitutively contained inducible form of HSP70 were described, for example, for some human tissues and were shown to play an important role in rehabilitation of cardiosurgery patients (Demidov et al.,
1999). There is an opinion that the higher the level of hsp70 in the cells of an organism in the non-stress conditions is, the more reliable adaptive mechanisms of this organism are; some data point to the level of hsp70 content as to a characteristic of populations, positively correlating with temperature conditions oftheir habitats (see: Margulis and Guzhova 2000).
There are some literary data allowing to connect the constitutive HSP70 level with the thermotolerance (thermoresistance) of the cells (Ulmasov et al., 1992; Li, 1989). In the work devoted to heat shock proteins in two Amoeba species — A. proteus and A. borokensis -the 35S-methionine incorporation was used to reveal and evaluate heat shock proteins in amoeba cells (Kalinina et al., 1988). The gradual temperature shift from 22 to 32 °C resulted in de novo HSP70 synthesis, the rate of its synthesis being the same in both species, nevertheless these species differ distinctly in their thermoresistance. The scheme of experiments applied in this work did not allow the constitutive HSP70 level evaluation in the untreated amoeba cells. Taking into account a lot of data concerning thermoresistance of protozoan cells (various Amoeba proteus strains among them) and the role these data had played in the development of the concept of temperature adaptations in Protozoa (Sukhanova, 1968; Sopina, 1968; Poljansky, 1973, etc.), it would be useful to study whether the relation exists between the level of constitutive HSP70 and thermoresistance of the cells of various amoeba strains.
When discussing the HSP70 decrease at 37 °C, it is worth mentioning that the lower HSP70 level (Figs 4, 6) does not reflect the inhibition of total protein synthesis at this temperature (Fig. 5, lanes 2, 10). As the HSP70 studies in amoeba cells are now at the very beginning, it is difficult to explain this fact in proper way, but bearing in mind that 37 °C is sublethal temperature for Da strain amoebae, it may be presumed that under stress conditions the amoeba cells probably utilize the reserves of HSP70 to protect sensitive protein cell structures. If the utilization ofHSP70 prevails over its synthesis, it may be reflected on the blots as HSP70 content diminution. The influence of sublethal and lethal temperatures on the various mammalian cell lines and the quantitative differences of their HSP70 level after the extreme heat
32° K 37° 37°
4 0 4
Fig. 6. Blots after electroblotting and SDS-electrophoresis of untreated strain Da amoebae homogenates and homogenates prepared from amoebae after their heating at 32 and 37 °C during 1 hr. The blot is treated with monoclonal anti-HSP70 antibodies and stained with secondary antibodies conjugated with alkaline phosphatase; K — untreated, control amoebae; 32°0, 32°4, 37°0, 37°4 — homogenates of amoebae heated at 32 h 37 °C, respectively and prepared immediately (0) after heat shock and in 4 hrs (4) after the shock; ® - molecular marker 66 kDa; on the start of every lane - 7 mg of protein.
shock was shown to depend upon the cell metabolic state in normal conditions (Margulis et al., 1991).
Increasing of the HSP70 level at the temperature close to the TTR border (Podlipaeva, Gromov, 1998), and the HSP70 synthesis de novo at the same temperature 32 °C (Kalinina et al., 1989), make us presume the extreme temperatures, belonging to tolerant range, to be the points that begin to cause the induction of major heat shock protein HSP70, thus indicating the natural borders ofthe strain (species) temperature tolerant range.
Acknowledgements
I am very thankful to D.B.Gromov, A.V.Kinev and
B.A.Margulis for their help in the initiation and continuation of this work, to R.G.Chalikov for the help with the illustrations and to A.L.Yudin for critical reading the manuscript.
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Address for correspondence: Yulia I. Podlipaeva. Institute of Cytology, Russian Academy ofSciences, 4 Tikhoretsky Ave., St. Petersburg 194064, Russia. E-mail: yulia@cit.ras.spb.ru; julia@SP4588.spb.edu
The manuscript is presented by A.L.Yudin
