Cloning, characterization and sulfonamide inhibition studies of an α-carbonic anhydrase from the living fossil sponge Astrosclera willeyana.

The α-carbonic anhydrase (CA, EC 4.2.1.1) Astrosclerin-3 previously isolated from the living fossil sponge Astrosclera willeyana (Jackson et al., Science 2007, 316, 1893), was cloned, kinetically characterized and investigated for its inhibition properties with sulfonamides and sulfamates. Astrosclerin-3 has a high catalytic activity for the CO(2) hydration reaction to bicarbonate and protons (k(cat) of 9.0×10(5) s(-1) and k(cat)/K(m) of 1.1×10(8) M(-1) × s(-1)), and is inhibited by various aromatic/heterocyclic sulfonamides and sulfamates with inhibition constants in the range of 2.9 nM-8.85 µM. Astrosclerin, and the human isoform CA II, display similar kinetic properties and affinities for sulfonamide inhibitors, despite more than 550 million years of independent evolution. Because Astrosclerin-3 is involved in biocalcification, the inhibitors characterized here may be used to gain insights into such processes in other metazoans.

Anion inhibition studies of an α-carbonic anhydrase from the living fossil Astrosclera willeyana.

An α-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the living fossil sponge Astrosclera willeyana, Astrosclerin, was investigated for its inhibition profile with simple inorganic anions, complex anions and other small molecules known to interact with these zinc enzymes. Astrosclerin is a catalytically highly efficient enzyme, and is inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, and in the submillimolar range by a variety of anions including fluoride, chloride, cyanate, thiocyanate, cyanide, hydrogen sulfide, bisulfate, stannate, perosmate, divanadate, perrhenate, perruthenate, selenocyanide, trithiocarbonate, diethyldithiocarbamate and iminodisulfonate. Less efficient Astrosclerin inhibitors were sulfate, bromide, iodide, azide, bicarbonate, carbonate, tetraborate and perchlorate (K(I)s of 5.11-30.6mM) whereas tetrafluoroborate was not at all inhibitory. Because Astrosclerin is involved in calcification processes in vivo, its anion inhibition profile may be important for future studies designed to shed light on the physiologic functions of α-CAs in marine organisms.

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells.

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.

Induction of carbonic anhydrase IX by hypoxia and chemical disruption of oxygen sensing in rat fibroblasts and cardiomyocytes.

CA IX is an active transmembrane carbonic anhydrase isoform functionally implicated in cell adhesion and pH control. Human CA IX is strongly induced by hypoxia and frequently associated with various tumors. In this study, we investigated the expression of the rat CA IX in response to chronic hypoxia and to treatment with chemical compounds that disrupt oxygen sensing, including dimethyloxalylglycine, dimethylester succinate, diazoxide, and tempol. We brought the evidence that expression of CA IX is regulated by hypoxia and hypoxia-mimicking compounds in immortalized Rat2 fibroblasts and BP6 rat fibrosarcoma cells in a cell-type-specific manner. We also demonstrated, for the first time, that CA IX is expressed in hypoxic primary rat cardiomyocytes and in immortalized H9c2 cardiomyocytes exposed to physiological or chemical hypoxia and that CA IX expression is increased in hypoxic rat tissues in vivo. Our findings suggest that CA IX expression is not limited to cancer but may be also induced in other pathological situations associated with ischemia or metabolic disturbances leading to activation of the HIF pathway. These data support the view that rats can represent useful model for studies of CA IX as a component of endogenous protection mechanisms associated with hypoxia or perturbed oxygen sensing.

Hypoxia-inducible expression of the mouse carbonic anhydrase IX demonstrated by new monoclonal antibodies.

CA IX is a transmembrane carbonic anhydrase isoenzyme predominantly expressed in human tumors in response to hypoxia and functionally implicated in adaptation of tumor cells to hypoxic stress via control of pH and cell adhesion. Intense investigations of the human CA IX as a hypoxic marker and a therapeutic target have been facilitated by specific monoclonal antibodies. However, no such reagents existed for the mouse CA IX ortholog. We generated five new anti-mouse CA IX monoclonal antibodies AM1-4, AM4-3, AM27-4, AM34-7 and AM35-1 produced using CA IX-deficient mice. The antibodies are suitable for various immunodetection methods including immunoblotting and immunohistochemistry. Using these reagents we show that the mouse CA IX is expressed in three out of nine tested mouse cell lines, namely in L929, MEF and TSA and is regulated by hypoxia and cell density similarly to human CA IX. We also demonstrate that the mouse CA IX exhibits hypoxia-related expression pattern in multicellular spheroids and in tumor xenografts. Our results indicate the use of the mouse model as suitable for further studies of CA IX role in tumor development and for its pre-clinical investigations. The new monoclonal antibodies represent potent tools for accomplishment of these future studies.

Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus.

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.