Expression of hsp60 in the Rotifer Plationus patalus in Response to Metal Exposure

Karina Castillo, Martha Rios,
Judith Rios, M.S. and Dr. Walsh
Department of Biological Sciences

University of Texas El Paso

There are different ways in which organisms respond when they are under stressful conditions. Stressful conditions may include extremes in temperature or pH, exposure to toxic compounds as well as a variety of other factors. Stress may illicit evolutionary, behavioral, and physiological responses. Organisms  show evolutionary responses when they show long term adaptations to their environment. For example, some lizard species (Cnemidophorus) have evolved   function best at very high temperatures. Behavioral responses arise when organisms look for an environment that best fits their needs such as migration through the water column to avoid predation.  There are two major types of physiological responses external and internal responses. External responses, such as sweating, are observed when organisms unaccustomed to high temperatures are exposed to them.  Internal responses include induction of transcription and translation of specialized proteins such as heat shock proteins.

The heat shock protein 60 (hsp60) belongs to a family of chaperons. “Hsp60, which is called also GroEL, is a chaperon because it works along with the protein hsp10 (GroES) to promote efficient folding of proteins in the mitochondria and chloroplasts of eukaryotes” (1; Figure 1).

            GroEl is composed of fourteen identical 60 kDa subunits arranged in two stacked rings, and GroES is composed of seven identical 10 kDa subunits.  When there is an unfolded protein it will enter to the opening of the GroEl and it binds to the hydrophobic surface. Then the GroEs binds to the GroEL creating a chamber to protect the unfolded protein from aggregating with other unfolded proteins. The interior surface changes from hydrophobic to hydrophilic  through an adenosine triphosphate (ATP) mediated process.  The hydrophilic surface is the one that supports the folding of unfolded proteins (1,2).       Many organisms  synthesis hsp60 under stressful conditions (3).  The species Brachionus plicatilis, from the phylum Rotifera, is one of the organisms in which hsp60 is present (4). The phylum Rotifera is composed of invertebrate microorganisms, ranging in size from 40mm to 2500mm. Most rotifers live in freshwater habitats. They are very important to the aquatic ecosystem because they are at the base of the aquatic food web.  Rotifers feed on algae and bacteria.  Then they convert this energy into usable food for higher trophic levels of the food web. There are about 2000 different species of rotifers. Yet, all of them have two characteristics in common: the corona and the mastax. The corona is a ciliated region that rotifers use for locomotion and food gathering (Figure 2a). The mastax is a muscular pharynx that contains a set of hard jaws called trophy (Figure 2b).

            Another common characteristic that monogonont rotifers have is their special way of reproduction. Rotifers reproduce asexually when the conditions are optimal.  However, under stressful conditions, they can switch from asexual to sexual reproduction.  Females that reproduce asexually in optimal conditions are named amictic females. Under stressful conditions, amictic females will produce mictic females. These mictic females will begin to produce eggs by meiosis.  These haploid eggs develop into males that can fertilize amictic females and produce dormant diploid female offspring (resting eggs). These resting eggs do not hatch unless conditions return to optimal (5). A more immediate response to stress in rotifers is the induction of hsp60 (6). The induction of stress proteins due to elevated levels of Copper (Cu) and tributylin has been observed in other rotiferan species such as Brachiounus plicatilis (5). The purpose of the present study is to determine whether heavy metals (Copper, Arsenic, and Zinc) found in the Rio Grande are capable of inducing hsp60 in Plationus patulus, which is one of the species found in the Rio Grande. The study will be focused on two specific questions: 1) Do single metals induce hsp60 in rotifers? 2) Do mix metal solutions induce hsp60 in rotifers?

These questions leads to our hypothesis H0: metals will not induce hsp60 singly or in combination.  Ha: metals will induce hsp60 singly or in combination. 

Materials/Methods

            Prior to determining the induction of hsp60 by metals in rotifers, the   amount of protein found in 100 and 200 rotifers was quantified in order to define the number of rotifers required for good enzyme detection. Total protein was quantified following a modified Lowry procedure (Sigma Diagnostic Protein Assay Kit). Rotifer samples (0.5ml of 100 and 200 rotifers) were diluted to 1ml with  deionized distilled water. Protein calibration standards for 50, 100, 200, 300, and 400mg/ml were prepared by diluting specific amounts of a 400mg/ml protein standard up to 1ml with double distilled water. Calibration standards, samples and a calibration blank (1 ml of deionized distilled water) were simultaneously prepared. Deoxicholate (DOC)  and Tricholoroacetic Acid  (TCA) solutions (0.1ml of each) were added to the standard, blank, and samples. Solutions were centrifuged to pellet the precipitates.  Then supernatants were decanted and blotted away. Pellets were dissolved in 1.0mL of Lowry Reagent Solution and transferred to labeled tubes. Centrifuge tubes were rinsed with 1mL water and 1 ml of the contents then transferred to the label tubes. Then they were mixed and kept at room temperature for 20 minutes.  With rapid and immediate mixing, 0.5mL of Folin & Ciocalteu’s Phenol Reagent Working Solution was added to each tube.  The color was developed in 30 minutes. Calibration standards and samples absorbance was measured at 750nm in a Beckman DU 640 spectrophotometer. Least-Squares method for deriving calibration curves was used to determine the amount of total protein in rotifer samples. This method requires the calculation of the square sum of “x” (Ssx), “y” (Ssy) and “xy” (Ssxy) to determine the slope (m) and intercept (b) using the formulas in Table 1.1. The amount of total protein was estimated by substituting the variables on the straight line equation (y=mx+b).

Hsp60 induction in rotifers was determined by exposing rotifers to single and combined metal solutions. 200 rotifers were used for metal exposure experiments. Treatments included a control (rotifers  not exposure to metals),  three single metal exposure treatments (As, Cu, and Zn), and four metal combination treatments (As-Cu, As-Zn, Cu-Zn, As-Cu-Zn). 1600 rotifers, 200 per treatment, were placed into 8 small petri dishes (containing synthetic water) and fed with 1mL of algal suspension (Chlamydomonas reinhartti and Ankistrodesmus falcatus). The day of exposure, arsenic (50ppb), copper (50ppb), zinc (150ppb) and combination of these metals were added to each respective dish. Rotifers were exposed to single metals and combined metals for 1 hour. After exposure, the rotifers were placed in plastic vials for homogenization.

            Excess of synthetic water was removed for concentration of rotifers, and 300ml of Laemmi buffer were added. Each sample was homogenized for 30 seconds and placed on ice. All samples were denaturated in boiling water for four minutes. Gel electrophoresis was used to separate the proteins. For each gel 20ml of sample were loaded into 30% Acrylamide-8% bisacrylamide gel; 2ml of hsp60 standard and 2ml of prestained standard was loaded as positive controls for hsp60 and protein separation. Proteins were separated at 100v for one hour. Proteins were transferred from the gel to a nitrocellulose membrane for protein analysis (hsp60). The presence of hsp60 on the nitrocellulose membrane was determined through a Western blot procedure. The Western Blot is a technique used to identify specific proteins through antigen-antibody reactions. In order to identify the protein hsp60, a primary antibody (anti-hsp60) and a secondary antibody (goat anti-rabbit IgG alkaline phosphatase labeled) were used.  The anti-hsp60 antibody recognizes and binds hsp60; the secondary antibody recognizes and binds the primary antibody. Finally, the hsp60-primary-secondary antibody complex was developed using a alkaline phosphatase reaction with NBT/BCIP substrate. The alkaline phosphatase reaction was stopped with 5% acetic acid.

           

Results

            For the protein quantification the Ssx, Ssy and Ssxy were calculated with the concentration (x) and absorption (y) of the protein standard.  Table 1.1 shows the absorption, concentration and data required for the Least Square Method, as well as the Sum of Squares used to determine de total concentration of protein.  The following formulas were used to determine the intercept and slope:

Ssx=Sxi2 - (Sxi)2/N

Ssy=Sy2-(Syi)2/N

Ssxy= Sxiyi - Sxiyi /N

 where xi and yi are individual pairs of data for x and y, N is the data used in preparing the calibration curve, and x and < the average values. By substituting all variables on the straight lines equation, the total protein concentration of the samples was calculated. Total protein in 100 rotifers sample was 77.8mg/mL while for the 200 rotifers sample it was 457.2mg/mL. Since only 20ml of the 200 rotifers sample would be loaded in the gel, the total protein loaded would be approximately 9.1ml/mL.  To determine the induction of hsp60, four Western Blots were done.  Western Blot 1 had samples of rotifers exposed to single metals diluted 1/10 and 1/100.  Western Blot 2 had samples of rotifers exposed to combined metals diluted 1/10.  Western Blot 3 had samples of rotifers exposed to single and combined metals diluted 1/100. These dilutions were based on previous results of another Western Blot in which the dilutions were done to improve the visualization of hsp60 induction.  The three blots done with diluted hsp60, did not show any result.  The fourth blot had samples of rotifers exposed to single and combined metals (fig.3). In the combined metal samples there was a definite induction of hsp60. In rotifers exposed to single metals the bands for As and Zn looked similar to the control but, for the copper exposure, the band was thicker and darker than the control, indicating a clear induction of hsp60. The procedure was repeated for As and Zn exposure to verify that hsp60 was not induced.

The final Western Blot of single metals ( As, Zn and Cu)  showed an induction of hsp60 (fig.3).  The induction can be seen because the bands for single metals were darker and thicker compared to the control band.  Yet, the copper band was darker and thicker than the bands for As and Zn; therefore, it seems like rotifers exposed to copper induced more hsp60 than rotifers exposed to Zn and As.

 

Discussion

Based on the results from the protein quantification, 200 rotifers were used for the metal exposure experiment instead of 100 rotifers.  The reason why 200 rotifers were used for the experiment was to ensure there was enough protein to have measurable results.  The first three Blots (diluted samples of single and combined metals) did not show an induction of hsp60.  This may be because they did not have enough protein for visualization.  However, the more concentrated size standard and hsp60 standard were visible on the nitrocellulose membranes.  In previous experiments, the samples that were diluted showed an induction of hsp60 because the concentrations of metals during exposure time were different.  The blot that had samples of single and combined metals that were not diluted showed an induction of hsp60 because the synthesis of protein was increased in metal exposed treatments.

            In the last Western Blot, the blot that had samples of single metals showed an induction of hsp60 (fig. 4).  In this Blot the sample was not diluted.  Besides there were left spaces among each lane in order to see the difference in width and color between the control and rotifers exposed to metals.  In this Blot we were able to visualize a clear increase of induction of hsp60 in rotifers exposed to Cu, As, and Zn compared to the control.  Therefore, it  seems that As, Zn, and Cu at this concentrations (50ppb, 50ppb, 150ppb respectively) and time of exposure are stressors for rotifers; consequently, the protein was induced.

In addition, of these three metals (As, Zn, and Cu), it appears that rotifers exposed to copper synthesized more hsp60.  According to a research done by Rico Martinez et. al. (1998), copper can effectively reduce the species diversity of microinvertebrates, and terrestrial plants, and reduce plant biomass and algal crop.  They showed that aquaria exposed to Cu had their total zooplankton density drastically reduced, whereas the control showed no significant change. .In addition, this study showed that copper has lethal effects in the rotifer Brachiounus calyciflorus in concentration as low as 0.026 mg/L(7). The results of these studies support the fact that copper may induce hsp60 synthesis in rotifers.   This is a possible reason why rotifers exposed to copper induced more hsp60  under our experimental conditions (time and concentration).

Based on the experimental results, the protein hsp60 was more induced in rotifers exposed to single and combined metals than in rotifers that were not exposed to metals at all.

More studies are needed to optimize the detection of hsp60 induction in metal exposure treatments, including the increase of the number of rotifers used in the experiment, and changes on the exposure time. Differences in hsp60 induction due to metal type and concentration should be quantified using other methods, such as ELISA or fluorescent antibodies, to determine “ranges” of metal’s toxicity in rotifers.

 

Reference

1. Landry,.  Protein Interactions and Molecular Chaperones.

    http//www.Tulane.edu/~biochem/med/hsp.htm

2. Structure and Dynamics of the heat GroEl-binding GroES Mobile Loop

    Oxidizing Conditions exposure to toxic compounds

    http://www.tulane.edu/~biochem/sam/billboard.htm.

3. Campbell, A.N., Reece, J.B., Mitchell, G. L. 1999.  The structure and Function

    of Macromolecules. Biology. Fifth Edition

4. Cochrane, B.J., Irby, R.B., Snell, T.W. 1991. Effects of copper and 

    tributylin on stress protein abundance in the rotifer Brachiounus plicatilis

    Biochemistry Physiological 98C: 385-390.

5. Nogrady, T., Robert, L.W., Snell, T. W. 1993. Guides to the Identification of the

    Microinvertebrates of the Continental Waters of the World.  1: 5-41.

6. Morimoto, I. R., Tissieres, A., Georgopoulos, C. 1994. Progress and

    Perspectives on the Biology of Heat Schock Proteins and Molecular 

    Chaperones. p 9-20. The Biology of Heat Shock Proteins and Molecular 

    Chaperones.  Cold Spring Harbor Laboratory Press.

7. Rico-Martinez R., Perez-Legaspi A. I., Quintero-Diaz G. E., Rodriguez-Martinez M. G., Hernandez-Rodriguez, M. A., Zaragoza

    Almaraz J. E. 1998 Effects of copper addition to laboratory maintained microcosms of Presidente Calles Reservior

    organisms. Aquatic Ecosystem Health and Management 1:323-332

Table 1. Data required for protein quantification: standard and samples absorbance, and concentrations; square sums for Least square calibration curves method.

X Concentration

Y Abs.

X5

Y5

XY

50

.3964

2500

.1571

19.82

100

.6855

10000

.4699

68.55

200

1.1594

40000

1.3442

231.88

300

1.4577

90000

2.1249

437.31

400

1.6527

160000

2.7314

661.08

     77.75mg/ml                          

        0.1739*

   457.226mg/ml

        0.5488**

 

 

 

Ssx= 82000

Ssy= -4.3676

 

 

Ssxy=378.027

S :    1050     

       4.9553                 

       302500

     6.8275                 

        1418.64

x= 210

   <=1.07034

 *100 rotifer sample
** 200 rotifer sample

Fig.1. Representation of GroEs and GroEl chaperons (1).

Fig.2.  Rotifer specimens, the red circle indicates the two common characteristics of rotifers: a) Corona,   b) Mastax

                   

Fig. 3. Induction of Hsp60 observed in rotifers exposed to single and combined metals treatments: 1) high range standard protein ladder, 2) Hsp60 standard, 3) control, 4) As treatment, 5) Cu treatment, 6) Zn treatment, 7) As-Cu treatment, 8) As-Zn treatment, 9) Cu-Zn treatment, and 10) As-Cu-Zn treatment.

Fig. 4. Induction of Hsp60 observed in rotifers exposed to single metal treatments:1) high range standard protein ladder, 2) Hsp60 standard, 3) control, 4)As treatment, 5) Cu treatment, and 6) Zn treatment.


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