Protonography, a technique applicable for the analysis of η-carbonic anhydrase activity

Abstract

Protonography, a sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) technique derived from zymography was recently reported by our group to be an effective, cheap and reproducible technique for evidencing catalytically active α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as the bovine red blood cell isoform bCA or the bacterial enzyme from Vibrio cholerae, VchCA. CA activity was also observed on the protonogram of a cellular extract of Escherichia coli, evidencing the presence of one or more β-class such enzymes. Here we show that protonography can also be applied to the recently discovered η-CA family using the Plasmodium falciparum enzyme PfCA as an example. The protonogram of PfCA clearly showed catalytically active η-CA with a specific band at 22.0 kDa, which was quite distinct from the band of the red blood cell bovine enzyme bCA, which was observed at 28.8 kDa. The different migration pattern of α- and η-CAs might be a useful tool to detect Plasmodium falciparum in infected human red blood cells by an easy, routine inexpensive technique.

• η-Class enzyme
• carbonic anhydrase
• hydratase activity
• metalloenzymes
• protonography

Introduction

Carbonic anhydrases (CAs, CA, EC 4.2.1.1) catalyze a simple reaction in all life domains: the carbon dioxide hydration to bicarbonate and protons: 1–4. These enzymes are thus involved in many physiologic processes, such as photosynthesis, respiration, CO₂ transport, as well as metabolism of xenobiotics (e.g. cyanate in Escherichia coli)1–7. Six different, genetically distinct CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs8–11. The α-, β-, δ-and η-CAs use Zn(II) ions at the active site, the γ-CAs are probably Fe(II) enzymes (but they are active also with bound Zn(II) or Co(II) ions), whereas the ζ-class uses Cd(II) or Zn(II) to perform the physiologic reaction catalysis5^,11–28. Many representatives of all these enzyme classes have been crystallized and characterized in detail, except δ- and η-CAs. The three-dimensional analysis of CAs present a substantial variability between the different classes: the overall shape of the molecules, the protein folding patterns as well as the oligomeric organization of the six genetic CA families are very much different5^,11–28. For example, α-CAs are normally monomers and rarely dimmers; β-CAs are dimers, tetramers or octamers; γ-CAs are trimers, whereas the δ- and ζ-CAs are less well understood. The only ζ-CA crystallized so far has three slightly different active sites on the same polypeptide chain, whereas X-ray crystal structures of δ- and η-CAs are not available so far12–14^,29–35.

Many classes of CA inhibitors (CAIs) are known to date: the metal complexing anions and the unsubstituted sulfonamides which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere, generating trigonal–bipyramidal species are the classical, are the most frequently investigated ones15^,36–52. In addition to them, coumarins, phenols, dithiocarbamates and xanthates as well as polyamines, are new classes of CAIs reported in the last years by these groups9^,53–63.

The η-CA class, recently discovered in our laboratories, was identified in the genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, and in that of other Plasmodia. The enzyme identified in P. falciparum was indicated with the cryptonym PfCA11. We demonstrated that PfCA has significant CO₂ hydrase activity, with a k_cat of 1.4 × 10⁵s⁻¹ and a k_cat/K_M of 5.4 × 10⁶M⁻¹ s⁻¹. Moreover, through an extensive inhibitory study we detected numerous effective inhibitors such as inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, phenylboronic/arsonic acids and sulfonamides11.

Recently, our group reported a new technique to assay CA activity, named “protonography”, similar to zymography and derived from it. However, unlike zymography, protonography allows the detection of the activity of α-CAs on a sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) gel, as showed in our original work27. The technique is based on monitoring the pH variation in the gel (protonogram) as a consequence of the CA-catalyzed conversion of CO₂ to bicarbonate and protons. The protonogram is then stained with bromothymol blue, which is an indicator of pH variations. This dye appears blue in its deprotonated form, while its color changes to yellow in the protonated form. Thus, the production of hydrogen ions during the CO₂ hydration reaction, due to the hydratase activity of the enzyme lowers the pH of the solution/gel until the color transition point of the dye is reached (pH 6.8). The yellow band on the gel corresponds to the CA position on the protonogram27. The time required for the color change is inversely related to the quantity of carbonic anhydrase, which is responsible for dropping the pH from 8.2 to the transition point of the dye in a control buffer. The success of this technique is that the hydratase activity of the α-CAs was reversibly inhibited by SDS during electrophoresis and recovered by incubating the gel in aqueous Triton X-100.

In the present paper, we demonstrate that the protonography technique can be used to detect the η-CA activity of PfCA on a SDS-PAGE gel also, allowing us to prove that this technique may be extended to enzymes belonging to other CA classes than the α one.

Materials and methods

Mammalian bCA

Bovine α-CA (bCA) was supplied by Sigma-Aldrich (St. Louis, MO) as a crude extract from bovine erythrocytes (lyophilized powder).

Expression and purification of PfCA

A truncated form of the P. falciparum CA gene (PlasmoDB: PF3D7_1140000) encoding for the η-CA, PfCA (amino acid residues from 211 to 445)64 and contained into the expression vector pET-43.1a, was kindly donated by Prof. Sally-Ann Poulsen, Eskitis Institute, Griffith University, Brisbane, Australia. Arctic Express competent cells (Agilent Technologies, La Jolla, CA) were transformed with pET43.1a/PfCA expression vector. Several transformant colonies were picked and grown overnight in LB containing 20 μg/ml of gentamicin and 100 μg/ml of ampicillin for selecting the expression plasmid at 37 °C. Then, cells were grown without antibiotic selection at 30 °C and induced at an A₆₀₀ of 0.6–1 with 1 mM isopropylthiogalactopyranoside (IPTG). Zn(SO₄) was added after 30 min. After additional growth for 16 h at 20 °C, cells were harvested, resuspended in 10 mM buffer Tris/HCl, pH 8.3 and disrupted by sonication at 4 °C. Following centrifugation, soluble protein extract was loaded onto a His-select HF nickel affinity gel column (GE Healthcare), then washed with five column volumes with His binding buffer (20 mM NaH₂PO₄, 500 mM NaCl, 20 mM Imidazole, pH 8). The His-tagged protein was eluted from the column with elution buffer (20 mM NaH₂PO₄, 500 mM NaCl, 250 mM Imidazole, pH 8). The purified recombinant PfCA was dialyzed over-night against 10 mM buffer Tris/HCl, pH 8.3.

CA assay

An applied photophysics stopped-flow instrument was used for assaying the CA catalyzed CO₂ hydration activity65. Phenol red (at 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm with 10 mM Hepes (pH 7.5) as buffer and 0.1 M NaClO₄ (for maintaining constant ionic strength), at 20 °C, following the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s (the uncatalyzed reaction needs around 60–100 s in the assay conditions, whereas the catalyzed ones are of around 6–10 s). The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Enzyme concentrations in the assay system were 9.1 nM for bCA and 8.6 nM for VchCA.

Protonography

SDS-PAGE was performed as described by Laemmli66. Briefly, wells of 12% SDS-gel were loaded with bCA or PfCA mixed with Laemmli loading buffer without 2-mercaptoethanol and without boiling the samples, in order to avoid protein denaturation. The gel was run at 180 V until the dye front ran off the gel27. Following the electrophoresis, the 12% SDS-gel was subject to protonography to detect the bCA and PfCA hydratase activity on the gel as described by Capasso and coworkers27.

Results and discussion

Recombinant PfCA was recovered in the soluble fraction of the Arctic Express cell extract after sonication and centrifugation as describes in the “Experimental section”. PfCA was produced as fusion proteins containing an N-terminal His-Tag coding sequence. The recombinant fusion protein was purified by affinity chromatography on a column of HisTrap FF, prepacked with precharged Ni Sepharose 6 Fast Flow (GE Healthcare, Umeå, Sweden). Chromatography was performed on FPLC System. The (His)6-tagged PfCA protein eluted at 250 mM imidazole was recovered at approximately 90% purity.

A phylogenetic tree was constructed for showing that the Plasmodia CAs form a distinct group noticeably different from α-CAs (Figure 1). The CA class, organism, accession numbers and cryptonyms of the amino acid sequences used to construct the phylogenetic tree are reported in Table 1.

Figure 1. Phylogenetic tree constructed using amino acid sequences of the α- and η-CAs from selected prokaryotic and eukaryotic species. The tree was generated using the program PhyML 3.0 (Montpellier (France) Bioinformatic platform). Class, organisms, accession numbers and cryptonyms of the sequences used in the alignment have been indicated in Table 1.

Table 1. CA class, organism, accession numbers and cryptonyms of the amino acid sequences used to construct the phylogenetic tree.

CA class	Organism	Accession number	Cryptonym
Alpha (α)	Homo sapiens, isoform II	AAH11949.1	HumCAII_alpha
	Homo sapiens, isoform I	NP_001158302.1	HumCAI_alpha
	Sulfurihydrogenibium yellowstonense YO3AOP1	ACD66216.1	SspCA_alpha
	Streptococcus salivarius	EIC81445.1	SsalCA_alpha
	Neisseria gonorrhoeae	CAA72038.1	NgonCA_alpha
Eta (η)	Plasmodium falciparum 3D7	PF3D7_1140000	PfCA_eta
	Plasmodium reichenowi	CDO65199.1	Preich_eta
	Plasmodium falciparum Palo Alto/Uganda	ETW54987.1	PfIT_eta

A stopped-flow CO₂ hydrase assay has been used to measure the catalytic activity of PfCA, which was compared with those of other CAs belonging to a different CA classes. Table 2 shows the catalytic activity of these enzymes for the CO₂ hydration reaction. It may be observed that PfCA has kinetic parameters quite similar to those of the β- and γ-CAs.

Table 2. Kinetic parameters for CO₂ hydration reaction catalyzed by PfCA and other CA classes at 20 °C and pH 7.5.

Enzyme	Class	Activity level	kcat (s^−1)	kcat/Km (M^−1 s^−1)	KI (nM)
bCA II^a	α	very high	1.62 × 10^6	1.3 × 10^8	15
PgiCAb^b	β	high	2.80 × 10^5	7.7 × 10^7	214
PgiCA^c	γ	high	4.10 × 10^5	5.4 × 10^7	324
PfCA^d	η	high	1.40 × 10^5	5.4 × 10^6	170

K_I for acetazolamide was determined for all CAs considered.

^aCommercial enzyme supplied by Sigma, stopped flow CO₂ hydrase assay method, this work.

^bFrom references [18, 21, 24]; ^cfrom [19, 25, 28, 49]; ^dfrom [11].

To investigate the hydratase activity of η-CA on the SDS-Page gel, 300 ng of PfCA or bCA were loaded on the gel. PfCA molecular weight estimated by SDS-PAGE carried out without 2-mercaptoethanol and without boiling the samples was 22.0 kDa, while bCA showed a mass of 28.8 kDa (Figure 2). A subunit molecular mass of 30 kDa was calculated on the basis of PfCA and bCA amino acid sequences. Molecular mass difference between the theoretical molecular weights and those determined by SDS-Page is due to the fact that SDS-Page was carried out without 2-mercaptoethanol and without boiling samples to avoid protein denaturation, which is essential to follow the protonography (as explained in the “Materials and methods” section).

Figure 2. SDS-PAGE of bCA and PfCA. The gel was stained with Coomassie blue and run under denaturing but non-reducing conditions (see text for details). Gels were loaded with 300 ng/well of bCA and PfCA.

The protonography monitors the pH variation in the gel (protonogram) as a function of the catalyzed conversion of CO₂ to bicarbonate and protons. The protonogram is stained with bromothymol blue as pH indicator. Thus, it monitors the production of hydrogen ions during the CO₂ hydration reaction, due to the bCA or PfCA hydratase activity, which lowered the pH of the solution until the color transition point of the dye has been reached (pH 6.8). The protonogram obtained using the η-CA (PfCA) and the α-CA (bCA) is shown in Figure 3. The yellow band corresponds to the individual CA position on the gel. The hydratase activity of η-CA can be reversibly inhibited by SDS during electrophoresis and recovered by incubating the gel in aqueous Triton X-100, as already demonstrated for α-CAs27.

Figure 3. Protonogram of bCA and PfCA. The yellow bands on blue background correspond to the bCA and PfCA activity, which determine the drop of pH from 8.2 to the transition point of the dye (pH 6.8). Yellow bands were obtained with an incubation time of 15 s.

It is interesting to note a difference in the migration of bCA and PfCA on the SDS-PAGE and protonogram carried out without 2-mercaptoethanol and without boiling the samples, although both proteins possess a theoretical molecular weight of 30 kDa. This is probably due to the very different shape of the two proteins which although having the same molecular weight, belong to two different CA classes which is another proof of the relevant difference between the α and η-CA genetic families.

We also hypothesize that the different migration pattern of α-CA (bCA) and η-CA (PfCA) might be a useful tool to detect the presence Plasmodium falciparum, the agent responsible for malaria, in infected human red blood cells. In fact, the η-CA class is produced by the Plasmodium falciparum proliferation and is absent in the non-infected human erythrocytes, which are characterized by the presence of high amounts of α-CAs (hCA I and hCA II). Work is in progress to test this hypothesis.

Conclusions

Protonography, an SDS-PAGE technique similar to zymography was recently reported by our group to be an effective, cheap and reproducible technique for evidencing catalytically active α-CA isoforms, such as the bovine red blood cell isoform bCA or the bacterial enzyme from Vibrio cholerae, VchCA27. In the original paper we also observed CA activity on the protonogram of a cellular extract of E. coli, evidencing the presence of one or more β-class such enzymes. Here we show that protonography can be applied to the recently discovered η-CA family, by using the P. falciparum enzyme PfCA as an example. The protonogram of PfCA clearly showed catalytically active η-CA with a specific band at 22.0 kDa, which was quite distinct from the band of the red blood cell bovine enzyme bCA, which was observed at 28.8 kDa. The different migration pattern of α- and η-CA might be a useful tool to detect Plasmodium falciparum in infected human red blood cells.

Declaration of interest

The authors declare no conflict of interest.