A Structure–Function Study of a Catalytic Antiidiotypic Antibody to the Human Erythrocyte Acetyl Cholinesterase

The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure–function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 × 10⁹ M^–1) was found by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited the esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites.     

A monoclonal antibody recognizing the FAD-binding site of 4-aminobenzoate hydroxylase from Agaricus bisporus

A monoclonal antibody against 4-aminobenzoate hydroxylase (EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, has been produced by the fusion of BALB/c mouse spleen cells immunized with the denatured enzyme and P3x63Ag8U1 myeloma cells in order to locate and characterize the catalytic site of the enzyme. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated its apoenzyme. The immunoprecipitation was inhibited in the presence of FAD, and the monoclonal antibody competitively inhibited the binding of FAD to the apoenzyme. The monoclonal antibody, therefore, recognizes the FAD-binding site of 4-aminobenzoate hydroxylase. Interestingly, it was shown that the monoclonal antibody was cross-reactive with FAD-dependent enzymes such as salicylate hydroxylase (EC 1.14.13.1) and D-amino acid oxidase (EC 1.4.3.3), and that it was specific for the FAD-binding sites of these enzymes. This fact suggests that these FAD-dependent enzymes have immunologically similar structures on their FAD-binding sites.     

Binding sites for A2 and A24 monoclonal antibodies on the alpha-latrotoxin molecule

alpha-Latrotoxin (alpha-LTX) binding sites to functionally active monoclonal antibodies (MA) A4 and A24 were localized using three approaches: hydrolysis of the toxin followed by the N-terminal sequencing of immunoreactive peptides; the study of antibody interaction with several recombinant alpha-LTX fragments; and Western immunoblotting of synthetic overlapping peptides (6-8 aa) whose structures correspond to that of the immunoreactive alpha-LTX fragment. It was shown that the MA A4 epitope is located within the F234-M294 protein fragment and that of MA A24 interacts with the fragment 347FDKDIT352.     

Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD], 20 microM) and low-affinity (KD, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.     

1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.     

Recognition sites of monoclonal antibodies A4 and A24 in the α-latrotoxin molecule

α-Latrotoxin (α-LTX) binding sites to functionally active monoclonal antibodies (MA) A4 and A24 were localized using three approaches: hydrolysis of the toxin followed by theN-terminal sequencing of immunoreactive peptides; the study of antibody interaction with several recombinant α-LTX fragments; Western immunoblotting of synthetic overlapping peptides (6–8 aa) whose structures correspond to that of the immunoreactive α-LTX fragment. It was shown that the MA A4 epitope is located within the F²³⁴-M²⁹⁴ protein fragment and that MA A24 interacts with the fragment³⁴⁷FDKDIT³⁵².     

Contact residues and predicted structure of the reovirus type 3-receptor interaction.

Sequence similarity between the reovirus type 3 hemagglutinin (HA3) and a anti-idiotypic monoclonal antibody (87.92.6) has been shown to define the site of interaction with a neutralizing (idiotypic) monoclonal antibody (9B.G5) and the cellular receptor for the virus. A synthetic peptide (VL peptide) derived from the anti-idiotypic sequence inhibits viral binding to the receptor. In this study, variants of the VL peptide were utilized to probe specific amino acid residues involved in binding the neutralizing antibody and the receptor. These studies indicate that the--OH groups of several residues are involved in contacting the reovirus type 3 receptor, including Tyr49, Ser50, Ser52, and Thr53 in the anti-idiotypic sequence, corresponding to Tyr326, Ser327, Ser329, and Ser325 in HA3, respectively. In contrast, only Ser50 of the anti-idiotypic sequence, corresponding to Ser327 of HA3, significantly altered neutralizing antibody binding. Additional studies implicate sialic acid as a potential reovirus type 3 receptor on some cells. This includes inhibition of binding of reovirus type 3 and 87.92.6 to L cells by heavily sialylated glycoproteins. Sialic acid was therefore utilized as a candidate receptor to analyze potential interaction schemes with HA3/87.92.6. Sequence similarity to other immunoglobulin structures with similar sequences allowed modeling of the three-dimensional structure of these epitopes. These structures, in combination with peptide studies, allow the development of a model of the interaction of these epitopes with sialic acid, which serves as a reovirus type 3 receptor. These models reveal that similar amino acid residues and side-chain geometries may be utilized by the reovirus type 3 and influenza hemagglutinins in their interactions with cell-surface receptors.     

Production of Monoclonal Antibodies to Guinea Pig Liver Transglutaminase

Monoclonal antibodies are now a powerful tool in biology and medicine. Transglutaminase has been implicated in diverse biological functions, and the characteristics of its catalytic action are suitable for applied enzymology. In this study, we produced hybridoma cells which synthesize monoclonal antibodies against guinea pig liver transglutaminase by fusing mouse myeloma cells with spleen cells of mouse immunized with the enzyme protein. Eight hybridoma clones (coded 2F, 4B, 7C, 8B, 8D, 8E, 9F and 11C) were selected to produce monoclonal antibodies. The subclass of IgG produced by clone 9F was IgG_2a and those from the seven other clones were all IgG₁ The 9F antibody inhibited transglutaminase activity, but the other antibodies did not. A solid-phase antibody-binding assay showed that of these antibodies, 8D antibody has the highest affinity to the antigen. Transglutaminase protein in crude liver extract was identified with Western blotting analysis using 8D antibody as the probe.     

Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa

A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.     

Modeling and structural analysis of evolutionarily diverse S8 family serine proteases

Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.     

The short‐chain oxidoreductase Q9HYA2 from Pseudomonas aeruginosa PAO1 contains an atypical catalytic center

The characteristic oxidation or reduction reaction mechanisms of short‐chain oxidoreductase (SCOR) enzymes involve a highly conserved Asp‐Ser‐Tyr‐Lys catalytic tetrad. The SCOR enzyme Q9HYA2 from the pathogenic bacterium Pseudomonas aeruginosa was recognized to possess an atypical catalytic tetrad composed of Lys118‐Ser146‐Thr159‐Arg163. Orthologs of Q9HYA2 containing the unusual catalytic tetrad along with conserved substrate and cofactor recognition residues were identified in 27 additional species, the majority of which are bacterial pathogens. However, this atypical catalytic tetrad was not represented within the Protein Data Bank. The crystal structures of unligated and NADPH‐complexed Q9HYA2 were determined at 2.3 Å resolution. Structural alignment to a polyketide ketoreductase (KR), a typical SCOR, demonstrated that Q9HYA2's Lys118, Ser146, and Arg163 superimposed upon the KR's catalytic Asp114, Ser144, and Lys161, respectively. However, only the backbone of Q9HYA2's Thr159 overlapped KR's catalytic Tyr157. The Thr159 hydroxyl in apo Q9HYA2 is poorly positioned for participating in catalysis. In the Q9HYA2–NADPH complex, the Thr159 side chain was modeled in two alternate rotamers, one of which is positioned to interact with other members of the tetrad and the bound cofactor. A chloride ion is bound at the position normally occupied by the catalytic tyrosine hydroxyl. The putative active site of Q9HYA2 contains a chemical moiety at each catalytically important position of a typical SCOR enzyme. This is the first observation of a SCOR protein with this alternate catalytic center that includes threonine replacing the catalytic tyrosine and an ion replacing the hydroxyl moiety of the catalytic tyrosine.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (Mldl). Structure-functional correlation

Based on three-dimensional model of the bifunctional enzyme Destabilase-Lysozyme (mlDL-Ds2) in complex with trimer of N-acetylglucosoamine (NAG)3 the functional role of the stereochemically based group of amino acids (Glu14, Asp26, Ser 29, Ser31, Lys38, His92), in manifestation of glycosidase and isopeptidase activities has been elucidated. By method of site-directed mutagenesis it has been shown that mlDL glycosidase active site includes catalytic Glu14 and Asp26, and isopeptidase site functions as Ser/Lys dyad presented by catalytic residues Lys38 and Ser29. Thus, among the invertebrate lysozymes mlDL presents first example of the bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

Immunopurification and characterization of a 40-kD 1-aminocyclopropane-1-carboxylic acid N-malonyltransferase from mung bean seedling hypocotyls.

1-Aminocyclopropane-1-carboxylic acid (ACC) N-malonyltransferase catalyzes the transfer of the malonyl group from malonyl coenzyme A to ACC to form malonyl ACC. Using partially purified ACC N-malonyltransferase from the hypocotyls of mung bean (Vigna radiata) seedlings, we produced two mouse monoclonal antibodies (1H5 and 2G3) to this enzyme. These antibodies bind to sites other than the active site of the enzyme because monoclonal antibody-bound ACC N-malonyltransferase still exhibits full catalytic activity. A monoclonal antibody column was constructed using 1H5 and protein G Sepharose. The ACC N-malonyltransferase purified from this monoclonal antibody column has a molecular mass of 40 kD, which is different from that reported previously. The enzyme has a higher electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of the reducing agent dithiothreitol. The optimum temperature of this 40-kD ACC N-malonyltransferase is 45 degrees C and the apparent Kms for ACC and malonyl coenzyme A are 66.7 and 40 microns, respectively.     

Epitope of the catalytic subunit of viscumin, exposed on its interaction with a lipid bilayer

It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, pp. 63-66). The interaction of the synthetic F101-T105 and A96-T105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1H NMR spectroscopy. The results of this study demonstrated that only the A96-T105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen-antibody complex and registered on the resonances of the free peptide and exchanging between the free state and the antibody-bound state was analyzed; the mab_TA7 antigen determinant (H99-T105) was identified; and its conformation and orientation within the complex with the antibody were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Reverse micelles,a system for antibody-catalysed reactions

This work presents some aspects of the application of catalytic antibodies in water-in-oil microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane. The monoclonal antibody (mAb) 9A8 used in this study is a fully characterised acetylcholinesterase-like antibody produced by the anti-idiotypic approach. The effect of various parameters, such as, the size and the concentration of reverse micelles, as well as the concentration and the nature of substrates on abzyme catalytic activity were investigated.     

Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G4 enzyme

Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.     

Epitope mapping of the monoclonal antibody FAC2 on the apoprotein of Cpa-1 in photosystem II

Using a combination of cyanogen bromide cleavage and endoproteinase digestion we have shown that the putative epitope for the monoclonal antibody FAC2 lies in the region 360Pro(-391)Ser on the apoprotein of CPa-1. This region lies entirely within the large extrinsic loop of this protein. We have shown previously that the epitope of FAC2 becomes exposed in oxygen-evolving membranes upon treatment with alkaline Tris which releases all four of the manganese associated with the oxygen-evolving site of photosystem II. The epitope is not exposed, however, after CaCl(2) treatment and exposure to low concentrations of chloride, conditions which lead to the release of two of the four manganeses associated with the oxygen-evolving site. These results suggest that, upon release of the chloride-insensitive manganese from photosystem II membranes, a conformational change occurs which leads to the exposure of 360Pro(-391)Ser on CPa-1 to the monoclonal antibody FAC2.     

Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium

Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.