Extracellular Carbonic Anhydrase Research Articles

Effects of HCO3− and CO2 conversion rates on carbon assimilation strategies in marine microalgae: Implication by stable carbon isotope analysis of fatty acids

Marine microalgae are an essential component of marine plankton and critical primary producers, playing a vital role in marine ecosystems. The seawater carbonate system is a dynamic equilibrium system, and changes in any component can alter the carbonate balance. In CO2-concentrating mechanisms (CCMs), carbonic anhydrase (CA) regulates CO2 concentration by catalyzing the interconversion between CO2 and HCO3−. Therefore, limiting the activity of extracellular carbonic anhydrase (exCA) alters the rate at which carbonate equilibrium is reached and further affects the carbon assimilation process in microalgae. In this study, two different microalgae, Phaeodactylum tricornutum and Nannochloropsis oceanica, were selected to investigate the effects of changes in the carbonate system on photosynthetic carbon assimilation in microalgae by inhibiting exCA activity with acetazolamide (AZ). Inhibition of exCA activity reduces specific growth rates and photosynthetic efficiency of microalgae. The total alkalinity, HCO3− concentration, and CO2 concentration of the cultures increased with the decrease of pH, but the changes of the ribulose 1,5- bisphosphate carboxylase/oxygenase (Rubisco) activities of the two microalgae were different. In addition, the two microalgae possessed different lipid and carbohydrate synthesis strategies, but both restricted triacylglycerol (TAG) synthesis. Meanwhile, the microalgal cells had to utilize more 13CO2 when HCO3− and CO2 conversion rates were limited and restricted. This led to the continuous accumulation of 13C in fatty acids and the elevation of δ13CFAs. In conclusion, our study provides a new perspective on the role of microalgae in correlating carbonate changes with photosynthetic carbon assimilation strategies under mechanistic constraints on inorganic carbon utilization.

The requirement for external carbonic anhydrase in diatoms is influenced by the supply and demand for dissolved inorganic carbon.

Photosynthesis by marine diatoms contributes significantly to the global carbon cycle. Due to the low concentration of CO2 in seawater, many diatoms use extracellular carbonic anhydrase (eCA) to enhance the supply of CO2 to the cell surface. While much research has investigated how the requirement for eCA is influenced by changes in CO2 availability, little is known about how eCA contributes to CO2 supply following changes in the demand for carbon. We therefore examined how changes in photosynthetic rate influence the requirement for eCA in three centric diatoms. Modeling of cell surface carbonate chemistry indicated that diffusive CO2 supply to the cell surface was greatly reduced in large diatoms at higher photosynthetic rates. Laboratory experiments demonstrated a trend of an increasing requirement for eCA with increasing photosynthetic rate that was most pronounced in the larger species, supporting the findings of the cellular modeling. Microelectrode measurements of cell surface pH and O2 demonstrated that individual cells exhibited an increased contribution of eCA to photosynthesis at higher irradiances. Our data demonstrate that changes in carbon demand strongly influence the requirement for eCA in diatoms. Cell size and photosynthetic rate will therefore be key determinants of the mode of dissolved inorganic carbon uptake.

Carbonic anhydrases reduce the acidity of the tumor microenvironment, promote immune infiltration, decelerate tumor growth, and improve survival in ErbB2/HER2-enriched breast cancer

BackgroundCarbonic anhydrases catalyze CO2/HCO3– buffer reactions with implications for effective H+ mobility, pH dynamics, and cellular acid–base sensing. Yet, the integrated consequences of carbonic anhydrases for cancer and stromal cell functions, their interactions, and patient prognosis are not yet clear.MethodsWe combine (a) bioinformatic analyses of human proteomic data and bulk and single-cell transcriptomic data coupled to clinicopathologic and prognostic information; (b) ex vivo experimental studies of gene expression in breast tissue based on quantitative reverse transcription and polymerase chain reactions, intracellular and extracellular pH recordings based on fluorescence confocal microscopy, and immunohistochemical protein identification in human and murine breast cancer biopsies; and (c) in vivo tumor size measurements, pH-sensitive microelectrode recordings, and microdialysis-based metabolite analyses in mice with experimentally induced breast carcinomas.ResultsCarbonic anhydrases—particularly the extracellular isoforms CA4, CA6, CA9, CA12, and CA14—undergo potent expression changes during human and murine breast carcinogenesis. In patients with basal-like/triple-negative breast cancer, elevated expression of the extracellular carbonic anhydrases negatively predicts survival, whereas, surprisingly, the extracellular carbonic anhydrases positively predict patient survival in HER2/ErbB2-enriched breast cancer. Carbonic anhydrase inhibition attenuates cellular net acid extrusion and extracellular H+ elimination from diffusion-restricted to peripheral and well-perfused regions of human and murine breast cancer tissue. Supplied in vivo, the carbonic anhydrase inhibitor acetazolamide acidifies the microenvironment of ErbB2-induced murine breast carcinomas, limits tumor immune infiltration (CD3+ T cells, CD19+ B cells, F4/80+ macrophages), lowers inflammatory cytokine (Il1a, Il1b, Il6) and transcription factor (Nfkb1) expression, and accelerates tumor growth. Supporting the immunomodulatory influences of carbonic anhydrases, patient survival benefits associated with high extracellular carbonic anhydrase expression in HER2-enriched breast carcinomas depend on the tumor inflammatory profile. Acetazolamide lowers lactate levels in breast tissue and blood without influencing breast tumor perfusion, suggesting that carbonic anhydrase inhibition lowers fermentative glycolysis.ConclusionsWe conclude that carbonic anhydrases (a) elevate pH in breast carcinomas by accelerating net H+ elimination from cancer cells and across the interstitial space and (b) raise immune infiltration and inflammation in ErbB2/HER2-driven breast carcinomas, restricting tumor growth and improving patient survival.

Different photosynthetic inorganic carbon utilization strategies in the heteroblastic leaves of an aquatic plant Ottelia ovalifolia.

The leaves of the heteroblastic aquatic plant Ottelia ovalifolia faces submerged and aerial environments during its life history. However, the acclimation of the submerged leaves and floating leaves to these two environments in morphology, physiology, and biochemistry remain unclear. In the present study, we investigated the acclimation of the CO2-concentrating mechanisms in these two types of leaves. We found that the submerged leaves were longer, narrower, and thinner than the floating leaves, which increased the specific surface area of the leaves and lead to better absorption of the inorganic carbon underwater. Meanwhile, the floating leaves absorbed atmospheric CO2 directly through the stomata to acclimate to the aerial environment. Both the leaf types had the ability to use , but the capacity in submerged leaves was stronger than that in floating leaves. The extracellular carbonic anhydrase and anion exchanger were responsible for the use in both types of leaves. The higher ratio of chlorophyll a/b and content of anthocyanin in floating leaves than that in submerged leaves indicated that the acclimation of aerial and submerged photosynthesis depended on changes in the photosynthetic pigments. Based on the stable carbon isotope ratio, key enzyme activities of the C4 pathway indicated that submerged leaves might have the ability to perform C4 metabolism while floating leaves only performed C3 metabolism. In summary, O. ovalifolia acclimates to submerged and aerial environments through changes in morphology, physiology, and biochemistry during different growth stages.

Enhalus acoroides efficiently alleviate ocean acidification by shifting modes of inorganic carbon uptake and increasing photosynthesis when pH drops

Ocean acidification (OA) is causing increasing ecological damage, so it is worthwhile to find efficient and environmentally friendly ways to mitigate OA. The mechanism of inorganic carbon (Ci) absorption and the ability to mitigate OA of the tropical seagrass Enhalus acoroides were investigated in this study. At 2.2 mM Ci concentration, its CO2 fixation efficiency increased to 81.89 t CO2/year/Ha under pH 6.5 from 27.59 t CO2/year/Ha at pH 8.2, and even reached 88.11 t CO2/year/Ha at pH 6.5 with unlimited Ci availability, made possible by three pathways for Ci utilization, which included absorbing CO2 directly, transforming HCO3− into CO2 through extracellular carbonic anhydrase, and absorbing HCO3− directly by anion-exchange protein then transforming it to CO2 through intracellular carbonic anhydrase, as verified by inhibitor addition experiments. The carbon fixation rate increased with decreasing pH, suggesting a greater CO2 absorbing capacity for E. acoroides under acidic conditions, which further demonstrates the possibility of mitigating OA and increasing carbon fixation through conserving and restoring E. acoroides meadows. Due to the strong carbon absorption capacity of E. acoroides, it is very important to strengthen the artificial restoration of E. acoroides seagrass meadows in the environmental management of the coastline.

Effect of different mixed light-emitting diode light wavelengths on CO2 absorption from biogas and nutrient removal from biogas slurry by microalgae and fungi induced using strigolactone and endophytic bacteria.

In this study, biogas and biogas slurry were simultaneously treated using two symbiotic systems: Chlorella vulgaris-Ganoderma lucidum-S395-2 (endophytic bacteria) and Scenedesmus obliquus-G. lucidum-S395-2. The influence of different mixed illumination (red and blue) intensity ratios on the algal symbionts' extracellular carbonic anhydrase activities was investigated, as well as the rates of microalgal growth and photosynthesis. The treatment performance was simultaneously assessed in terms of the efficiency of organic matter or nutrient removal and the level of CO2 absorption. The results indicated that red-blue light combinations with an intensity ratio of 5:5 were optimal. When comparing the performance of the two symbiotic systems, the C. vulgaris-G. lucidum-S395-2 symbiont co-culture system achieved significantly improved photosynthetic rates, biomass growth, and treatment effects. Under the optimal treatment conditions, the organic matter and nutrient removal rates were 81.06% ± 7.06% for chemical oxygen demand, 82.32% ± 7.18% for total nitrogen, and 82.98% ± 7.26% for total phosphorus. In addition, the rate of CO2 removal from biogas was 63.38% ± 5.35%. PRACTITIONER POINTS: The red and blue light intensity ratio of 5:5 showed the best removal performance. C. vulgaris-G. lucidum-S395-2 system obtained the best photosynthetic performance. The carbonic anhydrase activity had positive effects on CO2 removal performance.

Effects of nutrients on the utilization pattern of photosynthetic inorganic carbon of the tropical <italic>Thalassia hemprichii</italic>

<p indent="0mm">Studies on the utilization strategy of photosynthetic inorganic carbon of seagrass can reveal its ecological adaptation mechanism. <italic>Thalassia hemprichii</italic>, a widely distributed seagrass species in the Indo-Pacific and Atlantic coastal seas, needs to evolve several utilization patterns of inorganic carbon to adapt different environments and meet its growth, development and reproduction. However, the effect of nutrient on the photosynthetic inorganic carbon utilization strategy in seagrass is still unknown and further research is urgently needed. Therefore, using oxygen electrode technology, chlorophyll fluorescence technology, and non-invasive micro-test technology, this study examined the similarity and difference in the utilization patterns of photosynthetic inorganic carbon of <italic>T</italic>.<italic> hemprichii </italic>from two typical tropical seagrass beds (Tanmen, Hainan Island, and Yongxing Island, Xisha), that were affected by varying degrees of human activity. Experiments showed that the photosynthetic oxygen release rate, maximum relative electron transfer rate, and half-saturated light intensity of <italic>T</italic>.<italic> hemprichii</italic> in Yongxing Island waters were 24.95%, 24.54%, and 28.73% higher than the corresponding in Tanmen, Hainan Island. This indicated that the growth status of <italic>T</italic>.<italic> hemprichii</italic> in the waters of Yongxing Island was better with higher photosynthetic efficiency. The addition of acetazolamide (AZ) and ethoxyzolamide (EZ) significantly reduced the photosynthetic oxygen release rate of <italic>T</italic>.<italic> hemprichii</italic>, with an insignificant difference in the inhibition between AZ and EZ. This indicated that it mainly absorbed HCO₃⁻ via extracellular carbonic anhydrase catalysis. Meanwhile, hydroxymethyl (Tris) inhibited the photosynthetic oxygen release of <italic>T</italic>.<italic> hemprichii</italic> in Tanmen by up to 100% and decreased the maximum relative electron transfer rate and the H⁺ inward flow rate, implying a synergistic H⁺/HCO₃⁻ transport mode. On the other hand, the rate of photosynthetic oxygen release and the maximum electron transfer rate of <italic>T</italic>.<italic> hemprichii</italic> in Xisha did not change significantly with the addition of AZ and EZ, while Tris buffer inhibited photosynthetic oxygen release by up to 100% and significantly reduced the maximum electron transfer rate. This suggested <italic>T</italic>.<italic> hemprichii </italic>in Xisha mainly relied on the H⁺/HCO₃⁻ synergistic transport. Therefore, <italic>T</italic>.<italic> hemprichii </italic>could uptake inorganic carbon in an environment with greater anthropogenic activity, larger nutrient load and lower light availability on Hainan Island by catalyzing HCO₃⁻ into CO₂ with extracellular carbonic anhydrase and the H⁺/HCO₃⁻ synergistic transport. In contrast, <italic>T</italic>.<italic> hemprichii</italic> in an environment with less anthropogenic activity, lower nutrient loading, and greater light availability, relied chiefly on H⁺/HCO₃⁻ synergistic transport. Facing a relatively unfavorable high-nutrient and low-light growth environment, <italic>T</italic>.<italic> hemprichii</italic> could increase the photosynthetic inorganic carbon utilization strategy (such as the activity of extracellular carbonic anhydrase) to improve its utilization ability of inorganic carbon to overcome stress and growth limitation. This suggested that <italic>T</italic>.<italic> hemprichii</italic> could adjust its inorganic carbon utilization patterns according to its geographical environment. With the continuous development of technology, it is urgent to study the utilization mechanism of inorganic carbon of seagrass by applying seagrass omics technology and stable isotope tracing to deepen the understanding of the carbon sink function of seagrass.

Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells

Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.

The roles of plasma accessible and cytosolic carbonic anhydrases in bicarbonate (HCO3-) excretion in Pacific hagfish (Eptatretus stoutii).

Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO2). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO2, hagfish can mount a rapid elevation of plasma HCO3- concentration (hypercarbia). Once PCO2 is restored, hagfish quickly excrete their HCO3- load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO3- dehydration into CO2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO2/HCO3- clearance from the plasma at the gills of E. stoutii, under control and high PCO2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO3- excretion by measuring in situ [14C]-HCO3- fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [14C]-HCO3- efflux was measured at the gills of fish exposed to control, hypercapnic (48h) and recovery from hypercapnia conditions (6h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO3- flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO3- fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO3- flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO2 conditions. In summary, our data suggests that there are additional pathways for HCO3- excretion at the gills of hagfish that are independent of plasma-accessible CA.

In vitro and in vivo roles of cyanobacterial carbonic anhydrase as a biomarker for monitoring antibiotics

Environmental biomarkers represent an emerging tool in environmental monitoring by measuring variations in cellular or molecular exposure to chemical pollutants. Carbonic anhydrase, a widespread enzyme in organisms, has the potential to be utilized as a biomarker because of its sensitive activity to chemical pollutants. Here, we report the first extracellular carbonic anhydrase (EcaA) from cyanobacteria as a biomarker for monitoring antibiotics. A recombinant microbial factory that is capable of heterologously overexpressing EcaA was constructed, and the purified enzyme exhibited superior performance in monitoring various antibiotics in vitro. The IC50 values of the four selected antibiotics, ciprofloxacin, spectinomycin, tetracycline and ampicillin, were 1.45 ± 0.61, 10.40 ± 0.34, 18.92 ± 2.42 and 59.73 ± 2.56 μM, respectively. The feasibility of EcaA as a biomarker for monitoring antibiotics in vivo was also confirmed. Growth of wild-type cyanobacteria was more inhibited by ciprofloxacin and tetracycline than an EcaA-null mutant, demonstrating that EcaA responded physiologically to the two antibiotics, thus causing growth defects. Our results enable advanced development and optimization of carbonic anhydrase as a biomarker to monitor antibiotics in vitro and in vivo.

Strategies of carbon use and photosynthetic performance of the two seaweeds Gracilaria chouae and Gracilariopsis lemaneiformis under different conditions of the carbonate system

The use of Gracilaria chouae and Gracilariopsis lemaneiformis in aquaculture has recently been rapidly expanding owing to their bio- and eco-functions on coastal areas. However, high density cultures of these commercial seaweeds can lead to rapid changes in carbonate system even on a very short-term basis, which, in turn, effectively affects the physiology of these commercial seaweeds. To explore the regulation of mechanism of carbon use of G. chouae and Gp. lemaneiformis in different carbonate systems, this study exposed two seaweeds to natural seawater (control), and treatments of high CO2 and normal HCO3− concentrations (HC-NB), and natural CO2 and low HCO3− concentrations (NC-LB) to determine the net photosynthetic rate, pH drift, rapid light curves and chlorophyll fluorescence measurements. The results showed that net photosynthetic rates, non-photochemical quenching (NPQ), potential mechanism of relative electron transfer (rETRm) and the ability of both seaweeds to tolerate high light (Ik) increased significantly in the HC-NB treatment. Gp. lemaneiformis had significantly higher rates of photosynthesis and more dramatic changes in pH in all the treatments. Thalli were treated with an inhibitor of extracellular carbonic ahhydrase acetazolamide (AZ) and an anion exchange protein inhibitor 4,4′-disocyanostilbene-2,2′-disulfonate (DIDS), AZ was the primary inhibitor of HCO3− used for both seaweeds during first 2 h. However, after 24 h, DIDS and AZ were the primary inhibitors of G. chouae in all the treatments, and DIDS was the main inhibitor of Gp. lemaneiformis in the control and HC-NB treatments. In conclusion, the photosynthetic ability of G. chouae is greater in carbonate system manipulated seawaters and is more effective at metabolizing HCO3− by extracellular carbonic anhydrase at high pH values. The direct HCO3− use by the anion exchange transporter could be induced at high pH values and low [HCO3−] for both seaweeds. This study will provide a theoretical basis for seaweed aquaculture.

A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

The effects of two sized polystyrene nanoplastics on the growth, physiological functions, and toxin production of Alexandrium tamarense

Micro- and nano-plastics (MNPs) are increasingly prevalent pollutants in marine ecosystems and result in various deleterious effects on marine organisms. There have been studies evaluated the toxic effects of MNPs on marine microalgae, but few of them focused on the effects of MNPs on dinoflagellate species and their toxins production, which could have significant implications on human health and ecological safety in coastal areas. In this study, the common harmful algal blooms-causing dinoflagellate Alexandrium tamarense was exposed to 0.1 and 1 μm sized polystyrene nanoplastics (NPs) to investigate the responding patterns of population growth, multiple physiological functions, as well as the intracellular paralytic shellfish toxins (PSTs) productions. The results indicated the population growth, photosynthetic parameters, nutrients (nitrate and phosphate) uptake rates and extracellular carbonic anhydrase activities (CAext) were all inhibited by the two sized NPs, accompanied by the prolonged and more aggregated microalgal cells under the observation of scanning electron microscope (SEM), and the inhibition effects were more severe under 1 μm sized NPs than 0.1 μm sized NPs. Finally, we found the intracellular PSTs contents increased 73.59% exposed to 0.1 μm sized NPs while decreased 85.50% exposed to 1 μm sized NPs comparing the controls at 96 h, without significant changes of relative compositions. These results provided evidence that MNPs were toxic to A. tamarense and affected their intracellular PSTs productions within 96 h, which is critical to consider when evaluating the potential risks of MNPs in marine ecosystems.

Concurrent enhancement of CO2 fixation and productivities of omega-3 fatty acids and astaxanthin in Haematococcus pluvialis culture via calcium-mediated homeoviscous adaptation and biomineralization

Haematococcus pluvialis has attracted interest as a bio-platform for producing omega-3 fatty acids (ω-3 FA) and astaxanthin that have a great potential as anti-inflammatory drugs. This study aimed to concurrently enhance the CO2 fixation and the productivities of ω-3 FA and astaxanthin, which have been difficult to achieve because of the dissimilar culture methods for each goal, via calcium-mediated homeoviscous adaptation and biomineralization. As a result of 3 mM of Ca2+ addition, ω-3 FA content was improved by 31% due to Ca2+-induced homeoviscous adaptation. Biomineralization was promoted by the extracellular carbonic anhydrase, which resulted in 46.3% improvement in CO2 fixation. CaCO3 from the biomineralization was beneficially re-used in the H. pluvialis culture and triggered 178- and 522-fold increased biomass productivity and astaxanthin content, respectively, thanks to its anisotropic nature. The Ca2+-based productivity enhancement strategy was applied to large-scale culture which resulted improvement in overall bioprocess performance.

The Role of Extracellular Carbonic Anhydrase in Biogeochemical Cycling: Recent Advances and Climate Change Responses.

Climate change has been predicted to influence the marine phytoplankton community and its carbon acquisition strategy. Extracellular carbonic anhydrase (eCA) is a zinc metalloenzyme that catalyses the relatively slow interconversion between HCO3− and CO2. Early results indicated that sub-nanomolar levels of eCA at the sea surface were sufficient to enhance the oceanic uptake rate of CO2 on a global scale by 15%, an addition of 0.37 Pg C year−1. Despite its central role in the marine carbon cycle, only in recent years have new analytical techniques allowed the first quantifications of eCA and its activity in the oceans. This opens up new research areas in the field of marine biogeochemistry and climate change. Light and suitable pH conditions, as well as growth stage, are crucial factors in eCA expression. Previous studies showed that phytoplankton eCA activity and concentrations are affected by environmental stressors such as ocean acidification and UV radiation as well as changing light conditions. For this reason, eCA is suggested as a biochemical indicator in biomonitoring programmes and could be used for future response prediction studies in changing oceans. This review aims to identify the current knowledge and gaps where new research efforts should be focused to better determine the potential feedback of phytoplankton via eCA in the marine carbon cycle in changing oceans.

Modulation of acid extrusion and acid loading in neurons and astrocytes by RPTPγ and RPTPζ

Receptor protein tyrosine phosphatases γ and ζ (RPTPγ & RPTPζ) are transmembrane signaling proteins that, as monomers, catalyze dephosphorylation of tyrosine residues on cytosolic targets. RPTPγ and RPTPζ are unusual in having an extracellular carbonic-anhydrase–like domain (CALD). Carbonic anhydrases (CAs) catalyze the reaction, CO₂ + H₂O ⇌ HCO3– + H⁺, but CALDs are enzymatically inactive. Previous studies on renal proximal tubules (PTs) strongly suggest that RPTPγ is a dual extracellular CO₂/HCO₃⁻ sensor. RPTPγ—expressed on the PT basal (i.e., blood-side) membrane—is essential for modulating acid-base transport in response to changes in basolateral [CO₂] and [HCO₃⁻]. RPTPγ and RPTPζ are also present in the central nervous system (CNS), including mouse hippocampus (HC). RPTPγ is expressed primarily in neurons and RPTPζ, in astrocytes. Here, we investigate whether RPTPγ and RPTPζ in brain, like RPTPγ in kidney, play roles in sensing extracellular [CO₂]ₒ and [HCO₃⁻]ₒ and altering acid-base transport. We hypothesize that activation/termination of the intracellular phosphatase activity of these RPTPs regulates initial steps in signaling cascades that defend cells against intracellular pH (pHi) decreases triggered by metabolic acidosis (MAc, caused by ↓ [HCO₃⁻]ₒ) or respiratory acidosis (RAc, caused by ↑ [CO₂]ₒ). We propose that the RPTPs respond to ↑[CO₂]ₒ or ↓[HCO₃⁻]ₒ by promoting a shift from dimers to monomers, thereby dis-inhibiting the activity of the intracellular phosphatase domains. The ultimate result of the RPTP dis-inhibition would be increased acid-extrusion rate (JE) or decreasedacid-loading rate (JL), both of which would minimize the fall of pHi during a first exposure to MAc or RAc, and may promote adaptation during a second exposure. Immunocytochemistry of mixed HC neuron-astrocyte cultures from wild-type (WT) mice using a primary antibody against the RPTPγ fibronectin III domain shows that RPTPγ colocalizes with MAP2 in HC neurons but not with GFAP in HC astrocytes . RT-PCR cloning from mixed HC cultures identifies specific RPTPγ and RPTPζ variants. Presumably it is the neurons that express two identified RPTPγ variants: the full-length protein, encoded by all 30 ptprg exons (NM_008981), and a second newly-detected variant that corresponds to the hypothetical assembly X1 (XM_006517956). The HC co-cultures also express six RPTPζ variants: Three RPTPζ variants (V) have previously been validated in mice: VR3 (NM_001081306), VR4 (NM_001311064), and VR5 (NM_001361349). Three others were previously only reported as hypothetical assemblies: X2 (XM_006505013), X3 (XM_006505014), and X4 (XM_006505015). To assess the influence of RPTPζ on pHi homeostasis, we loaded cells with the pH-sensitive dye BCECF, digitally monitored dye fluorescence, and computed pHi of cells subjected to acid-base disturbances. In MAc-MAc protocols, the effects of the knockout (KO) of RPTPζ are unremarkable in both neurons and astrocytes. In RAc-RAc protocols, the KO has unremarkable effects on neurons. However, in astrocytes the RPTPζ-KO markedly increases both the rates and the magnitudes of pHi decrease during both RAc pulses. Thus, RPTPζ may stabilize pHi in astrocytes subjected to RAc.

Effects of exogenous GR24 on biogas upgrading and nutrient removal by co-culturing microalgae with fungi under mixed LED light wavelengths

To realize the synchronous purification of raw biogas and biogas slurry, the algal-fungal symbiont pellets were cultivated by supplementing strigolactone (GR24) under different mixed LED light wavelengths. The optimal light intensity was proved to be red and blue in the ratio of 5:5. The symbionts treated with 10−9 M GR24 had the highest growth rate and mean daily productivity. The extracellular carbonic anhydrase activity and the content of chlorophyll were also affected by GR24 concentrations and mixed light wavelengths. With the induction of 10−9 M GR24, the maximum removal efficiency of chemical oxygen demand, total nitrogen, and total phosphorus reached 76.35 ± 6.87%, 78.77 ± 7.13% and 79.49 ± 7.43%, respectively. Besides, the CO2 removal efficiency reached 59.32 ± 5.19% when the concentration of GR24 was 10−7 M. This work will be beneficial for large-scale biogas slurry purification and biogas upgrading using co-cultivation of microalgae and fungi.

Active Blood Acidification Greatly Enhances Oxygen Supply to the Teleost Retina

Most ray-finned fishes possess a unique system for retinal oxygen delivery, where intracellular acidification of red blood cells (RBCs) in the retinal capillaries induces oxygen offloading from pH-sensitive hemoglobin. A countercurrent vascular network, the choroid rete mirabile, posterior to the retina augments this “oxygen-secretory” mechanism and confers retinal PO2 values that can exceed 1300 mmHg in some species. We hypothesized that the choroid rete mirabile exerts an active role in intracellular RBC acidification to facilitate retinal oxygen secretion. We show the presence and enzymatic activities of extracellular carbonic anhydrase (CA) and vacuolar-type H+-ATPase (VHA) in the lumen of the choroid rete mirabile. These data are consistent with a pathway, where VHA secretes H+ into the choroid rete mirabile lumen, which combines with HCO3– to form CO2 (catalyzed by extracellular CA) that rapidly diffuses into the RBC facilitating hemoglobin oxygen offloading. Next, we validated the model in vivo by injecting an extracellular CA inhibitor (C18) into the dorsal aorta of anesthetized rainbow trout with a PO2 electrode implanted into the retina. Here, the inhibition of extracellular CA resulted in a rapid reduction in retinal PO2 from 517 to 226 mmHg within minutes. These data strongly support the proposed model for RBC acidification in the choroid rete mirabile. Further, in vivo data show that this mechanism primarily augments oxygen diffusion to the inner retinal segments involved in signal-processing and that the evolution of this pathway was tightly associated with the morphological expansion of this part of the retina. Together, these data suggest that the evolutionary origin of retinal oxygen secretion in the early history of the ray-finned fishes was associated with a greatly enhanced capacity for visual processing, potentially permitting improved visual prey detection and the colonization of novel environments.

Proton Transport in Cancer Cells: The Role of Carbonic Anhydrases.

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H+ is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H+/HCO3−-coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined “transport metabolon”. While transport metabolons built with HCO3−-coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

Ultraviolet Radiation Stimulates Activity of CO2 Concentrating Mechanisms in a Bloom-Forming Diatom Under Reduced CO2 Availability.

The diatom Skeletonema costatum is cosmopolitan and forms algal blooms in coastal waters, being exposed to varying levels of solar UV radiation (UVR) and reduced levels of carbon dioxide (CO2). While reduced CO2 availability is known to enhance CO2 concentrating mechanisms (CCMs) in this diatom and others, little is known on the effects of UV on microalgal CCMs, especially when CO2 levels fluctuate in coastal waters. Here, we show that S. costatum upregulated its CCMs in response to UVR (295–395 nm), especially to UVA (320–395 nm) in the presence and absence of photosynthetically active radiation (PAR). The intensity rise of UVA and/or UVR alone resulted in an increase of the activity of extracellular carbonic anhydrase (CAe); and the addition of UVA enhanced the activity of CCMs-related CAe by 23–27% when PAR levels were low. Such UV-stimulated CCMs activity was only significant at the reduced CO2 level (3.4 μmol L−1). In addition, UVA alone drove active HCO3− uptake although it was not as obvious as CAe activity, another evidence for its role in enhancing CCMs activity. In parallel, the addition of UVA enhanced photosynthetic carbon fixation only at the lower CO2 level compared to PAR alone. In the absence of PAR, carbon fixation increased linearly with increased intensities of UVA or UVR regardless of the CO2 levels. These findings imply that during S. costatum blooming period when CO2 and PAR availability becomes lower, solar UVR (mainly UVA) helps to upregulate its CCMs and thus carbon fixation, enabling its success of frequent algal blooms.