Carbonic Anhydrase in Chlamydomonas reinhardtii II. Requirements for Carbonic Anhydrase Induction

Carbonic anhydrase (CA) activity in wild type cells of Chlamydomonasreinhardtii was low when cells were cultured under 2% CO₃ inthe light. When the gas phase was changed to air, CA activityincresaed as much as 20 fold over the next 24 hours. In contrast,CA activity did not change markedly in cells of the mutantspet 20-8 (PS II-negative), lip 10-2 (photophosphorylation-negative),and F60 (phosphoribulokinase-negative), when they were subjectedto the same induction regimen. DCMU (10^–5 M) and cydoheximide(3 µg/ml) severely inhibited the induction in wild typecells. No induction occured when CO₂ concentration was loweredin darkness. ³Present adress: Photoconversion Research Branch, Solar EnergyResearch Institute, Golden, Colorado 80401, USA. (Received June 7, 1982; Accepted December 25, 1982)     

Blue Light Induction of Carbonic Anhydrase Activity in Chlamydomonas reinhardtii

Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m². The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. ¹On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)     

Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO₂, but its synthesis was induced under low concentrations of CO₂ (air levels of CO₂). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO₂ to growth on air levels of CO₂, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.     

Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.     

Synthesis and Degradation of Carbonic Anhydrase in a Synchronized Culture of Chlamydomonas reinhardtii

The biosynthesis and degradation of carbonic anhydrase (CA;EC 4.3.1.1 [EC] ) was investigated during the course of synchronousculture of the unicellular green alga Chlamydomonas reinhardtii,carried out under a regime of 12 h of light and 12 h of darknesswith bubbling of ordinary air. The enzymatic activity increasedlinearly during the light phase. A coordinate increase in thelevel of the 35-kDa mature CA polypeptide was demonstrated byimmunostaining after poly-acrylamide gel electrophoresis andWestern blotting. Pulse-labeling with [¹⁴C]arginine followedby immunoprecipitation showed that the biosynthesis of the CApolypeptide is very active in the early light phase and rapidlydecreases after the middle of the light phase, indicating thatthe bio-synthetic activity does not reflect the quantity ofenzyme protein or the level of enzymatic activity. The 42-kDaprecursor but not the 35-kDa mature polypeptide was synthesizedin the dark. The 35-kDa polypeptide, pulse-labeled at the beginningof the light phase, was gradually degraded throughout the lightphase while it appeard to be stable in the dark. These resultssuggest that the messenger RNA coding for CA is present butits translation is limited in the dark. Normal translation ofmRNA and processing of the precursor to yield the holoenzymemay both require light. ¹Present address: Plant Laboratory, Kirin Brewery Co., Ltd.,Kitsuregawa-machi, Shioya-gun, Tochigi-ken, 329-314 Japan. (Received August 22, 1988; Accepted March 9, 1989)     

Salt-Induced Dissociation of Carbonic Anhydrase from Intact Cells of Chlamydomonas reinhardtii

The extracellular carbonic anhydrase of Chlamydomonas reinhardtii is dissociated from either intact or lysed cells by treatment with a 20 millimolar potassium phosphate buffer containing 0.4 molar KCI at pH 7.4. Electrophoretic analysis of proteins dissociated by the high salt treatment reveals that carbonic anhydrase comprises over 70% of the total released. These results suggest that the extracellular carbonic anhydrase in C. reinhardtii is bound to either the cell wall or plasma membrane through ionic interactions.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Release of Carbonic Anhydrase from the Cell Surface of Chlamydomonas reinhardtii by Trypsin

Treatment with trypsin of Chlamydomonas reinhardtii cells grownin ordinary air (low-CO₂ cells) caused almost complete releaseof carbonic anhydrase (CA) into the suspending medium, but didnot affect the shape and kinesis of the cells. These resultsindicate that most of the CA exists on the cell surface of low-CO₂cells. The released CA has the same molecular weight, specificactivity and susceptibility to various CA inhibitors as thatpurified from non-treated low-CO₂ cells. (Received August 24, 1985; Accepted November 20, 1985)     

Carbonic Anhydrase of Chlamydomonas reinhardtii. Cellular Distribution and Subunit Constitution in Two Strains

Cellular distribution and subunit constitution of carbonic anhydrase(CA) were compared between the CBSC strain of Chlamydomonasreinhardtii (purchased from Carolina Biological Suuply Co.)and C. reinhardtii C-9. CAs existed exclusively outside theplasma membrane inboth strains. Mol wts of the monomer and theholoenzyme from both strains were 35 kDa and 115–117 kDa,respectively. These results show that the CAs of both strainsare identical or verysimilar. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan. (Received June 29, 1987; Accepted October 9, 1987)     

Crystal Structure and Functional Characterization of Photosystem II-Associated Carbonic Anhydrase CAH3 in Chlamydomonas reinhardtii

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO₂ and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO₂ fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO₃^– increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO₃^– from CO₂. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO₂/HCO₃^– on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO₂ concentration.Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, which catalyze the interconversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻), a reaction that otherwise proceeds slowly at physiological pH. CAs belong to three evolutionary distinct classes, α, β, and γ, which share no significant amino acid sequence identity and are thought to be the result of convergent evolution (Hewett-Emmett and Tashian, 1996; Supuran, 2008; Ferry, 2010; Rowlett, 2010). Animals have only the α-CA type, but as multiple isoforms. By contrast, higher plants, algae, and cyanobacteria may contain members of all three CA families. In algae, CAs has been found in mitochondria and chloroplasts and in the cytoplasm and apoplasm.Many fresh-water and soil-living microalgae face limiting concentrations of inorganic carbon (Ci) in their environments. To overcome this, the green microalga Chlamydomonas reinhardtii, as well as most other unicellular algae and cyanobacteria, actively accumulate Ci inside the cells. This mechanism is known as the carbon-concentrating mechanism (CCM; Raven, 1997; Wang et al., 2011; Meyer and Griffiths, 2013). CCM allows the algae to maintain a high concentration of CO₂ around the carboxylating enzyme, Rubisco, even under limiting external Ci. The increased concentration of CO₂ in the chloroplast increases the CO₂/O₂ specificity for Rubisco that leads to a decreased oxygenation reaction, and hence carboxylation becomes more efficient.CCM can be induced in C. reinhardtii cultures by bubbling air containing CO₂ at ambient or concentrations (≤0.04%; Vance and Spalding, 2005). Full metabolic adaptation is usually reached within 10 to 12 h after transfer to air CO₂ conditions (Renberg et al., 2010). Already within the first few hours after induction, several genes are either up- or down-regulated (Miura et al., 2004; Yamano et al., 2008; Fang et al., 2012). Surprisingly, the global changes in protein expression do not correspond to those in the gene expression; only few proteins are either up- or down-regulated during CCM induction (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989). CAs are important components of the CCM. In C. reinhardtii, 12 genes are expressed that encode for CA isoforms (Moroney et al., 2011). Among the many genes that are significantly up-regulated during CCM induction, there is one encoding for an apoplastic CA (CrCAH1) and two encoding for mitochondrial CAs (CrCAH4 and CrCAH5; Fujiwara et al., 1990; Eriksson et al., 1996).An α-type CA (CrCAH3) located in the thylakoid lumen in C. reinhardtii has also been identified as important at low CO₂ levels (Karlsson et al., 1998). The sequence indicates that it is transported through the thylakoid membrane via the Twin Arg Translocation pathway (Albiniak et al., 2012). A mutant not expressing CrCAH3 (knockout of the cah3 gene) shows no or poor growth under air CO₂ levels (Spalding et al., 1983; Moroney et al., 1986) and has a severely impaired photosynthetic capacity under low Ci conditions. This mutant, called CrCIA3, has been a valuable tool for resolving the CrCAH3 function.It is also established that CrCAH3 is associated with PSII (Stemler, 1997; Villarejo et al., 2002; Blanco-Rivero et al., 2012). Using isolated PSII membranes from C. reinhardtii, Shutova et al. (2008) presented data suggesting that CrCAH3 is important for efficient water oxidation by facilitating the removal of protons that are produced when water is oxidized by PSII. This is in line with recent studies (Zaharieva et al., 2011; Klauss et al., 2012) showing that it is crucial to have alternating electron and proton removals from the oxygen-evolving complex (OEC) during the five-state catalytic cycle, i.e. the Kok cycle (Kok et al., 1970), of photosynthetic water oxidation. If proton removal is slow, this leads to less efficient O₂ production and consequently may lead to donor side photoinhibition (Minagawa et al., 1996). That HCO₃^– acts as a mobile proton carrier has been recently demonstrated for spinach (Spinacia oleracea) PSII membrane fragments using membrane inlet mass spectrometry (MIMS; Koroidov et al., 2014). These results show that PSII possesses a light- and HCO₃^–-dependent CO₂ production for up to 50% of the O₂ produced.Taken together, these data suggest that CrCAH3 plays an important role in regulating PSII reactions. In this work, we present further evidence for its function in PSII primary reactions, in particular at low Ci concentrations. We determined crystal structures of CrCAH3 at 2.6 to 2.7 Å resolution in complex with acetazolamide (AZM) or phosphate ions. Our results support a zinc-hydroxide catalytic mechanism of CrCAH3 similar to that of other α-CAs. CrCAH3 has, however, an activity optimum at lower pH values than CAs of the same type, which normally operate at pH 7.0 and higher (Demir et al., 2000). The activity optimum of CrCAH3 makes it more suitable for CO₂/HCO₃^– interconversion at the pH levels present in the thylakoid lumen under light exposure.     

Characterization of Carbonic Anhydrase Isozyme CA2, Which Is the CAH2 Gene Product,in Chlamydomonas reinhardtii

From high-CO₂ (5% CO₂) grown unicellular green alga, Chlamydomonas reinhardtii, carbonic anhydrase (CA) was isolated by affinity chromatography and characterized. Isolated CA was identified as an isozyme (CA2) which is the product from the second gene CAH2 by peptide sequencing. The CA2 was inactivated by dithiothreitol. This treatment caused dissociation of CA2 into the large (38 kDa) and small subunits (4243 Da). The molecular mass of the CA2 holoenzyme measured by low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC was 87.9 kDa. These results and gene structure indicate that CA2 is a heterotetramer consisting of two large and two small subunits linked by disulfide bonds like CA1, which is the CAH1 gene product. The speciffc activity of CA2 purified by anion-exchange HPLC was 3300 units per mg protein, which was approximately 1.6 times higher than that of CA1. Therefore, it was concluded that two structurally related isozymes, CA1 and CA2, are present in the wild type cells of C. reinhardtii and differentially regulated by the atmospheric CO₂ concentration.     

Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

Background

Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO₂ fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO₂ conditions.

Results/Conclusions

In the present work we demonstrate that after transfer to low CO₂, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO₂ conditions, the Cah3 activity increased about 5–6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO₂ conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO₂ conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO₂ the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.

Significance

This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.     

Carbonic Anhydrase Activity in Isolated Chloroplasts of Wild-Type and High-CO2-Dependent Mutants of Chlamydomonas reinhardtii as Studied by a New Assay

In an assay of carbonic anhydrase (CA), NAH14CO3 soltution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment. The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C. reinhardtii had 740 [plus or minus] 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 [plus or minus] 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 [plus or minus] 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw-15, believed to lack an internal CA, had 2.8 [plus or minus] 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 [plus or minus] 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 [mu]M inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80[deg]C for 10 min inhibited this CA activity by 90.8 [plus or minus] 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.