Bioengineered protein phosphatase 2A

Commentary

Bioengineered 4:2, 72–77; March/April 2013; © 2013 Landes Bioscience

Bioengineered protein phosphatase 2A Juan A. Rubiolo,1,2,* Henar López-Alonso,2 Amparo Alfonso,2 Félix V. Vega,1 Mercedes Rodríguez Vieytes1 and Luis M. Botana 2,* Departamento de Fisiología; Facultad Veterinaria; Lugo, Spain; 2Departamento de Farmacología; Facultad Veterinaria; Lugo, Spain

1

H

Keywords: recombinant PP2AC, insect larvae, harmful algal blooms, eutrophication, natural toxin detection Submitted: 08/31/12 Revised: 10/04/12 Accepted: 10/04/12 http://dx.doi.org/10.4161/bioe.22461 *Correspondence to: Juan Andrés Rubiolo and Luis M. Botana; Email: [email protected] and [email protected] Comment on: Rubiolo JA, López-Alonso H, Alfonso A, Vega FV, Vieytes MR, Botana LM. Characterization and activity determination of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae. Appl Biochem Biotechnol 2012; 167:918-28; PMID:22639363; http://dx.doi.org/10.1007/ s12010-012-9737-1

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armful algal blooms caused by phytoplankton can occur in all aquatic environments. Some of the algae present in these blooms are capable of producing extremely potent toxins. Due to climate change and eutrophication, harmful algal blooms are increasing on a global scale. One kind of toxin producing algae are those that produce okadaic acid, its derivatives (dinophysistoxin-1 and 2), and microcystins. These toxins are potent inhibitors of protein phosphatase 2A, so this protein is used to detect the mentioned toxins in natural samples. Originally protein phosphatase 2A purified from animal tissues was used, but enzyme activity and stability fluctuations prevented the use of the enzyme in detection kits. Expression of the enzyme as a recombinant protein provided a solution to this problem. For this purpose, several strategies have been followed. We evaluated the activity, specificity and stability of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae and showed that this expression system can be a reliable source of high quantities of stable enzyme. Harmful Algal Blooms Climate Change and Eutrophication Harmful algal blooms (HABs) are caused by single-celled algae known as phytoplankton, such as dinoflagellates, diatoms and cyanobacteria. These blooms can occur in all aquatic environments. One common example are the “red tides” observed in the marine environment, were the organisms produce a change in the

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water color. The harm produced by the HABs to the environment can be either through overgrowth, with the consequent inhibition of the growth of other species, and/or by the production of extremely potent natural toxins. Since the 1750s the global atmospheric concentration of carbon dioxide, methane, and nitrous oxides have increased markedly as a result of human activities. They now exceed pre-industrial values as determined from ice cores spanning many hundreds of thousands of years.1 These increases in greenhouse gas concentrations will produce, among others, global temperature changes, acidification, changes in the structure of the upper ocean, intensification/weakening of upwelling winds, and changes to the timing and volume of freshwater runoff into coastal marine waters. Indeed, actual data show that these changes might be underway.2 Over the past decade, several studies have suggested possible relationships between climate and the magnitude, frequency and duration of HABs.3-7 Nowadays it is generally accepted that HABs are increasing in frequency, intensity, and duration in all aquatic environments on a global scale.8,9 Harmful algae (HA) can be found in the ocean or in freshwater. Some toxins produced by marine and freshwater phytoplankton can be acutely lethal. In the ocean, the most prominent toxin producing algae known so far are the following: diatoms from the genus Pseudo nitzschia responsible for amnesic shellfish poisoning, species of dinoflagellates from the genera Alexandrium, Pyrodinium and Gymnodinium that cause

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Update on need

Figure 1. Structure of PP2A (PDB ID: 3dw8,38). The quaternary protein structure is shown, composed of the catalytic subunit Cα, the 65 kDa regulatory subunit Aα, and the 55 kDa regulatory subunit Bα. The two manganese atoms present in the catalytic subunit are also shown.

the paralytic shellfish poisoning, Karenia responsible for the neurotoxic shellfish poisoning, Dinophysis and Prorocentrum responsible for the diarrheic shellfish poisoning (DSP) and Gambierdiscus responsible for the ciguatera fish poisoning. In freshwater, the most important HABs are caused by certain species of cyanobacteria from the genera Anabaena, Microcystis, and Apyanizomenon.10 The toxins, small non-peptides, are some of the most powerful natural substances known.11 In the marine and freshwater systems, humans and animals can get exposed to HA toxins by eating contaminated fish or shellfish, drinking contaminated water, inhaling contaminated aerosol, or by contacting contaminated water. With increasing worldwide seafood consumption and trade, as well as international tourism, these diseases are expanding beyond their traditional geographic boundaries producing serious consequences on human health and industry. It was estimated that at least US$ 449,291,987 were spent on dealing with the known HABs from 1987 to 1992 in public health, commercial fishery, recreation/tourism and monitoring/management in the US alone.12

and Planktothrix, produce toxins [okadaic acid (OA), dinophysis toxin-1 and -2 (DTX-1 and -2) and microcystins] that are potent inhibitors of protein phosphatases 1, 2A and 2B (PP1, PP2A and PP2B). Of the three phosphatases, PP2A is the most strongly inhibited.13,14 The toxins from these organisms, are responsible of the diarrheic shellfish poisoning (DSP) and can produce liver damage in humans and animals.15,16 They are globally widespread and their blooms are predicted to increase, as a consequence of natural or anthropogenic eutrophication (enhanced phytoplankton growth due excess supply of nutrients).11 Diarrheic toxins and microcystins pose an important threat for human and animal health, and are also responsible for important fish and shellfish industry loses. As previously mentioned, the blooms of toxin producing organisms is predicted to increase, so the development of rapid, sensitive, and inexpensive methods to monitor the DSP toxins and microcystins occurrence in water and contaminated shellfish is needed, in order to manage the health and economic risk posed by these toxins.

Diarrheic and Hepatotoxic Toxins

PP2A as a Tool for Toxin Detection

Among the previously mentioned organisms, Dinophysis, Prorocentrum, Microcystis

Based on the PP2A inhibitory capacity of OA, DTXs and microcystin, initially

assays for determining OA shell-fish contamination were developed using enzymes purified from animal tissues.17,18 These methods have not been widely used due to fluctuations in enzyme quality. One of the sources of these fluctuations is the enzyme quaternary structure that can change during purification, and differs between different tissues. The PP2A (Fig. 1) is a trimmer consisting of a 36 kDa catalytic subunit (PP2AC), and two regulatory subunits, A and B. The core enzyme consists of the catalytic subunit and the regulatory subunit A (PP2A D). Two isoforms are known of subunits A (Aα and Aβ) and C (Cα and Cβ). Subunit B associate to the core enzyme and regulates the enzyme localization and specific activities, and several isoforms have been identified.19 PP2A has been purified in both, dimeric and trimeric forms,20,21 while purification procedures have been applied to obtain trimeric PP2A without the presence of PP2A D.22 This indicates that depending the purification procedure, different forms of the enzyme can be obtained. Besides this, there are other draw backs in using PP2A purified from animal tissues; when purified from muscle, kilos of tissue are needed and the purification process involves several chromatographic steps (4 to 9, depending the purification procedure),22 making the process expensive and time consuming, even more if large quantities of enzyme are needed to use in multiple assays. These problems, observed when purifying PP2A from animal tissues gives an idea of the fluctuations in enzymatic stability and composition that makes at least complicated, the use of this type of PP2A in assays for toxin detection. In order for an enzyme to be used in a microplate assay, high purity, stability, and sensitivity are essential. So, to satisfy these needs, recombinant PP2A has been produced in different hosts. In general, the first choice for the expression of recombinant proteins is E. coli, but no successful expression of active human PP2A has been reported yet. On the contrary, this enzyme in its active form, has been successfully expressed in yeast,23,24 mammalian cells,25 insect cells26-28 and insect larvae.29 In yeast and mammalian cells, only low quantities of active enzyme were produced while a high

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Commentary

Commentary

Table 1. Comparison of purification results for PP2A obtained from different sources, showing the enzyme quantity, activity and inhibition by OA Starting tissue, cells, larvae

Source

Enzyme composition

Purified protein (mg)

Activitya (μmol/ min/mg protein)

OA IC50 (nM)b

References

2.4

119 ± 8

0.32

39–41

0.1

190

0.66

24

N.R.

27

PP2AC Rabbit muscle

2.5 kg of muscle

PP2AD PP2AD+regulatory sub-unit B

Pichia pastoris

1 L of culture

PP2ACα

Sf9 cells

1 × 109 cells

PR65/A PR55α/Bα

0.15

1025 ± 229c 1153 ± 76c

PR55β/Bβ e

High Five cells

1 × 10 cells

PP2ACα

1–2

180 ± 30.4d

N.R.

28

d

4 × 10 cells

PP2ACα

0.5

83.7 ± 2.51

0.37

26

High Five cellse

4 × 108 cells

PP2ACβ

~0.5

81.8 ± 9.92

0.39

26

High Five cells

4 × 10 cells

d

PP2AD(Aα/cα)

N.R.

105.3 ± 3.75

0.47

26

High Five cellse

4 × 108 cellse

PP2AD(Aα/cβ)

N.R.

73.9 ± 3.71

0.44

26

Trichoplusia ni

1 g insect larvae

PP2ACα

0.25

94 ± 7.1

0.28

29

High Five cells

e

9

8

8

Determined by the p-NPP method. bDetermined by the p-NPP method. cDetermined by myosin light chain assay. dDetermined with 32P-labeled peptide (LRRASVA; kemptide). eInsect cell line (official name: BTI-TN-5B1–4).a a

expression level was achieved in insect cells and insect larvae (Table 1). In both, insect cells and larvae, purification is relatively easy involving only one chromatographic step allowing the production of high quantities of active PP2Ac. In insect larvae, after generating the baculovirus carrying the PP2ACα sequence for larvae infection, the expression system provides a reliable and cheap source of the enzyme. As shown in Table 1, several forms of recombinant PP2A have been obtained from different hosts. While studies of enzyme function and structure might require the dimeric of trimeric forms of the enzyme, for PP2A production in order to be used in toxin detection assays, the expression of the monomeric catalytic subunit is preferred. An important advantage of recombinant PP2AC expression is the activity consistency of the polypeptide obtained. The recovery of high quantities of pure recombinant PP2AC after a couple of purification steps, gives this approach an important advantage. Also, there is a reduced loss of protein activity due to the lower purification steps used. Both systems, insect cell driven expression and insect larvae expression, produce high quantities of the catalytic subunit of PP2A with high activity and with inhibition by OA similar to that observed for the enzyme

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purified from animal tissues (Table 1). The insect larvae expression system has the advantage of being cheaper, considering that infected larvae can be grown on an artificial wheat germ diet. One of the most important problems that arise after purification is the enzyme stability, which is crucial, when the intended use is to produce microplate assays for the detection of toxins. As determined by the 4-morpholine umbelliferone (4-MUP) method, performed as previously described,29 there is no activity decrease in PP2ACα obtained from insect larvae when stored at -20°C in 50 mM Tris-HCL, pH 7.2 for two months.29 In the same conditions, there is a loss in enzyme activity of approximately 30% after 12 mo of storage (Fig. 2). When the enzyme was stored at -20°C dissolved in 50 mM TRIS-HCl, pH 7.2 and 50% glycerol, there was a dramatic decrease in enzymatic activity in the first two months of storage that progressed to almost a complete lack of activity after a year (Fig. 2). These results indicate that even when high quantities of active recombinant PP2ACα can be obtained from insect larvae, several aspects of the enzyme stability, when intended for long-term storage, could be improved. Studies of the dissolution of the enzyme in the presence of manganese, which is

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part of the enzyme (see Fig. 1), could be performed. Also, if needed, genetic engineering in order to introduce mutations that would render a more stable enzyme after recombinant expression could be attempted, with the introduction of disulfide bonds or residues that increase protein stability in solution without activity loss. The PoPMuSiC (Prediction of Protein Mutant Stability Changes) server30 for the prediction of protein stability changes after mutation, shows that several mutations could produce a more stable protein in solution (Fig. 3). In the insect larvae expression system this could be done relatively easy given that after introducing point mutations that would lower the folding free energy of the polypeptide in the PP2AC cDNA, it could be used to produce baculovirus to infect insect larvae which would produce the new recombinant enzyme. At the same time enzyme activity evaluation is rapid and easy, so several recombinant enzymes could be produced to assay which are the polypeptide modifications that, while maintaining enzymatic activity, increase the stability of the protein. As previously mentioned, microplate assays for the detection of OA, DTXs and microcystins have been designed that employs PP2A purified from animal tissues.17,18 However, the enzyme purity and

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PP2AC

in the case of OA, DTXs and microcystins, the use of recombinant PP2AC represents a clear advantage when trying to develop a microplate assay. The expression of PP2ACα in insect larvae system provides a stable, rapid and cheap source of PP2ACα to fulfill this task. Disclosure of Potential Conflicts of Interest

References 1.

2.

Figure 2. Activity of PP2ACα purified from insect larvae. Activity was determined after 1, 2 or 12 mo after purification by the 4-MUP method. The enzyme was stored in 50 mM TRIS-HCl, pH 7.2, or in 50 mM TRIS-HCl pH 7.2 and 50% glycerol. The enzyme retained 100% of its activity after 2 mo, when stored in 50 mM TRIS-HCl, pH 7.2, and lost approximately 30% of its activity after a year in the same conditions. On the other hand, there was a decrease in enzymatic activity after 1 mo when the PP2ACα was dissolved in 50 mM TRIS-HCl pH 7.2 and 50% glycerol. These decrease in enzyme activity was more pronounced after 2 mo and almost no activity was observed after one year. AUF, arbitrary fluorescence units; MIN, minutes.

stability do not meet the quality to be used in kits. Recently a method for the detection of DSPs (OA and DTX 1) has been reported showing that the recombinant human PP2ACα can be used in inhibition assays for the detection of OA in shellfish.31 The assay using the human recombinant protein was shown to be more sensitive than native PP2A. This, together with higher stability and purity make the use of recombinant PP2AC more suitable in the production of kits for the detection of OA, DTXs and microcystins. Another problem to deal with, when attempting to detect diarrheic and hepatotoxic toxins in natural samples, is that other toxins are usually present in the lipophilic fraction besides OA, DTXs and microcystins. These toxins, which include pectenotoxins, yessotoxins and azaspiracids, do not inhibit PP2A and it was recommended not to include them in the DSP toxin group.32 This fact makes the use of PP2A suitable for the detection of OA, DTXs and microcystins without the interference of other toxins present at the same time.

The use of PP2A for toxin detection is not circumscribed to OA, DTXs and microcystins given that several other toxins have been shown to inhibit PP2A. These are calyculin-A,33 tautomycin,34 nodularin,35 cantharadin36 and fostriecin.37 So, as a consequence of the variety of molecules interacting with it, the recombinant PP2ACα produced in insect larvae can become a valuable tool for use in harmful toxin screening, and qualitative indicative tests for toxic compounds associated with micro-algal aquatic blooms, prior to liquid chromatography with mass spectrometry (LC-MS) detection.

3.

4.

5.

6.

7.

8.

Conclusion Harmful algal blooms are nowadays a serious health and industrial problem around the world. These blooms are expected to appear in new geographical regions and to be more frequent and intense in the following years, due to the increased eutrophication produced mainly by human activities. There is an urgent need for rapid methods of toxin detection, and

9.

10.

IPCC. Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. In: Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt K, et al., eds. Summary for Policymakers. Cambridge, United Kingdom and New York, NY, USA: Cambridge University Press, 2007. Bindoff N, Willebrand J, Artale V, Cazenave A, Gregory J, Gulev S, et al. Observations: Oceanic Climate Change and Sea Level. In: Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt K, et al., eds. Climate Change 2007: The Physical Science Basis Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge, United Kingdom and New York, NY, USA: Cambridge University Press, 2007. Dale B, Edwards M, Reid PC. Climate Change and Harmful Algal Blooms Ecology of Harmful Algae. In: Granéli E, Turner JT, eds.: Springer Berlin Heidelberg, 2006:367-78. Edwards M, Johns D, Leterme S, Svendsen E, Richardson A. Regional climate change and harmful algal blooms in the northeast Atlantic. Limnol Oceanogr 2006; 51:820-9; http://dx.doi. org/10.4319/lo.2006.51.2.0820. Epstein PR, Oceanic USN. Programs AAOoG, Aeronautics USN, Administration S, Ecological H, et al. Marine ecosystems, emerging diseases as indicators of change: health of the oceans from Labrador to Venezuela. Boston: The Center for Health and the Global Environment, Harvard Medical School, Oliver Wendell Holmes Society, 1998. Hayes M, Bonaventura J, Mitchell T, Prospero J, Shinn E, Van Dolah F, et al. How are climate and marine biological outbreaks functionally linked? Hydrobiologia 2001; 460:213-20; http://dx.doi. org/10.1023/A:1013121503937. Trainer V, Eberhart B-T, Wekell J, Adams N, Hanson L, Cox F, et al. Paralytic shellfish toxins in Puget Sound, Washington State. J Shellfish Res 2003; 21:213-23. Van Dolah FM. Marine algal toxins: origins, health effects, and their increased occurrence. Environ Health Perspect 2000; 108(Suppl 1):133-41; PMID:10698729; http://dx.doi.org/10.1289/ ehp.00108s1133. Glibert P, Anderson D, Gentien P, Granéli E, Sellner K. The global, complex phenomena of harmful algal blooms. Oceanography (Wash DC) 2005; 18:13647; http://dx.doi.org/10.5670/oceanog.2005.49. Kite-Powell HL, Fleming LE, Backer LC, Faustman EM, Hoagland P, Tsuchiya A, et al. Linking the oceans to public health: current efforts and future directions. Environ Health 2008; 7(Suppl 2):S6; PMID:19025677; http://dx.doi.org/10.1186/1476069X-7-S2-S6.


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No potential conflicts of interest were disclosed.

©2013 Landes Bioscience. Do not distribute Figure 3. Plot of the sequence optimality score (sum of negative ΔΔGs) for each position in the PP2ACα sequence obtained with the PoPMuSiC (Prediction of Protein Mutant Stability Changes) server. The ΔΔG predicts the change in folding free energy upon mutation. ΔΔG values are given in kcal/mol. Red, Helices; blue, β-strands; green, turns and coils. The predicted stability changes for each possible mutation, and the sequence optimality score computing the sum of negative ΔΔGs for each position in the sequence can be accessed at the PoPMuSiC server introducing the PDB ID: 2ie4.

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23. Evans DR, Myles T, Hofsteenge J, Hemmings BA. Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms. J Biol Chem 1999; 274:24038-46; PMID:10446173; http://dx.doi. org/10.1074/jbc.274.34.24038. 24. Swiatek W, Sugajska E, Lankiewicz L, Hemmings BA, Zolnierowicz S. Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris. Eur J Biochem 2000; 267:5209-16; PMID:10931206; http://dx.doi. org/10.1046/j.1432-1327.2000.01591.x. 25. Wadzinski BE, Eisfelder BJ, Peruski LF Jr., Mumby MC, Johnson GL. NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells. J Biol Chem 1992; 267:16883-8; PMID:1380955. 26. Ikehara T, Shinjo F, Ikehara S, Imamura S, Yasumoto T. Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta. Protein Expr Purif 2006; 45:150-6; PMID:16039140; http://dx.doi. org/10.1016/j.pep.2005.06.002. 27. Kamibayashi C, Estes R, Lickteig RL, Yang SI, Craft C, Mumby MC. Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. J Biol Chem 1994; 269:20139-48; PMID:8051102. 28. Myles T, Schmidt K, Evans DR, Cron P, Hemmings BA. Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells. Biochem J 2001; 357:225-32; PMID:11415453; http://dx.doi.org/10.1042/02646021:3570225. 29. Rubiolo JA, López-Alonso H, Alfonso A, Vega FV, Vieytes MR, Botana LM. Characterization and activity determination of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae. Appl Biochem Biotechnol 2012; 167:91828; PMID:22639363; http://dx.doi.org/10.1007/ s12010-012-9737-1. 30. Dehouck Y, Kwasigroch JM, Gilis D, Rooman M. PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics 2011; 12:151; PMID:21569468; http://dx.doi.org/10.1186/14712105-12-151. 31. Ikehara T, Imamura S, Yoshino A, Yasumoto T. PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish. Toxins (Basel) 2010; 2:195-204; PMID:22069554; http://dx.doi.org/10.3390/toxins2010195.

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