Expression of cystathionine β-synthase and cystathionine γ-lyase in human pregnant myometrium and their roles in the control of uterine contractility

Background

Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H₂S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H₂S produced locally in the control of uterine contractility during labour is unknown.

Methodology/Principal Findings

Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H₂S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H₂S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10⁻⁷–10⁻² mol/L), a precursor of H₂S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10⁻³ mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.

Conclusion/Significance

H₂S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H₂S synthetic enzymes is down-regulated during labour, suggesting that H₂S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.     

Cystathionine β-synthase and cystathionine γ-lyase double gene transfer ameliorate homocysteine-mediated mesangial inflammation through hydrogen sulfide generation

Elevated level of homocysteine (Hcy) induces chronic inflammation in vascular bed, including glomerulus, and promotes glomerulosclerosis. In this study we investigated in vitro mechanism of Hcy-mediated monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) induction and determined the regulatory role of hydrogen sulfide (H?S) to ameliorate inflammation. Mouse glomerular mesangial cells (MCs) were incubated with Hcy (75 μM) and supplemented with vehicle or with H?S (30 μM, in the form of NaHS). Inflammatory molecules MCP-1 and MIP-2 were measured by ELISA. Cellular capability to generate H?S was measured by colorimetric chemical method. To enhance endogenous production of H?S and better clearance of Hcy, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) genes were delivered to the cells. Oxidative NAD(P)H p47(phox) was measured by Western blot analysis and immunostaining. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH?-terminal kinase (JNK1/2) were measured by Western blot analysis. Our results demonstrated that Hcy upregulated inflammatory molecules MCP-1 and MIP-2, whereas endogenous production of H?S was attenuated. H?S treatment as well as CBS and CSE doubly cDNA overexpression markedly reduced Hcy-induced upregulation of MCP-1 and MIP-2. Hcy-induced upregulation of oxidative p47(phox) was attenuated by H?S supplementation and CBS/CSE overexpression as well. In addition to that we also detected Hcy-induced MCP-1 and MIP-2 induction was through phosphorylation of ERK1/2 and JNK1/2. Either H?S supplementation or CBS and CSE doubly cDNA overexpression attenuated Hcy-induced phosphorylation of these two signaling molecules and diminished MCP-1 and MIP-2 expressions. Similar results were obtained by inhibition of ERK1/2 and JNK1/2 using pharmacological and small interferring RNA (siRNA) blockers. We conclude that H?S plays a regulatory role in Hcy-induced mesangial inflammation and that ERK1/2 and JNK1/2 are two signaling pathways involved this process.     

Exploration of structure–function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~ 25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80 °C, compared to 66 °C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.     

Cystathionine β-synthase in structural elements of the human brain and spinal cord

By means of the immunohistochemical method, the presence and distribution of cystathionine β-synthase (CBS) was studied in nerve cells of the spinal cord and brainstem nuclei in eight men aged 18–44 years who had died as a result of causes not connected with damage to the central nervous system. CBS-positive neurons are revealed in all studied brain parts, in which their content varied in different nuclei from 0.9 to 17%. Large cells of motor nuclei more often had high and very high density of the reaction product deposition. In sensory nuclei the high portion was of small neurons with low intensity of the enzymatic reaction.     

Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase

BackgroundRegulatory cystathionine β-synthase (CBS) domains are ubiquitous in proteins, yet their mechanism of regulation remains largely obscure. Inorganic pyrophosphatase which contains regulatory CBS domains as internal inhibitors (CBS-PPase) is activated by ATP and inhibited by AMP and ADP; nucleotide binding to CBS domains and substrate binding to catalytic domains demonstrate positive co-operativity.Methods: Here, we explore the ability of an AMP analogue (cAMP) and four compounds that mimic the constituent parts of the AMP molecule (adenine, adenosine, phosphate, and fructose-1-phosphate) to bind and alter the activity of CBS-PPase from the bacterium Desulfitobacterium hafniense.ResultsAdenine, adenosine and cAMP activated CBS-PPase several-fold whereas fructose-1-phosphate inhibited it. Adenine and adenosine binding to dimeric CBS-PPase exhibited high positive co-operativity and markedly increased substrate binding co-operativity. Phosphate bound to CBS-PPase competitively with respect to a fluorescent AMP analogue.ConclusionsProtein interactions with the adenine moiety of AMP induce partial release of the internal inhibition and determine nucleotide-binding co-operativity, whereas interactions with the phosphate group potentiate the internal inhibition and decrease active-site co-operativity. The ribose moiety appears to enhance the activation effect of adenine and suppress its contribution to both types of co-operativity.General significanceOur findings demonstrate for the first time that regulation of a CBS-protein (inhibition or activation) is determined by a balance of its interactions with different chemical groups of the nucleotide and can be reversed by their modification. Differential regulation by nucleotides is not uncommon among CBS-proteins, and our findings may thus have a wider significance.     

Molecular characterization of mimosinase and cystathionine β-lyase in the Mimosoideae subfamily member Mimosa pudica

Journal of Plant Research - Mimosinase degrades the non-protein amino acid mimosine and is thought to have evolved from cystathionine β-lyase (CBL) via gene duplication. However, no study has,...     

Characterization of the S289A,D mutants of yeast cystathionine β-synthase

Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue S289 of yeast CBS, predicted to form a hydrogen bond with the pyridine nitrogen of the PLP cofactor, was mutated to alanine and aspartate. The k_cat/K_m^l-Ser of the S289A mutant is reduced by a factor of ～ 800 and the β-replacement activity of the S289D mutant is undetectable. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D. These results demonstrate that residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the PLP cofactor. The reduction in activity of ytCBS-S289A suggests that ytCBS catalyzes the α,β-elimination of l-Ser via an E1cB mechanism.     

Purification and characterization of cystathionine β-synthase bearing a cobalt protoporphyrin

Human cystathionine β-synthase (CBS), a pivotal enzyme in the metabolism of homocysteine, is a pyridoxal-5′-phosphate-dependent enzyme that also contains heme, a second cofactor whose function is still unclear. One strategy for elucidation of heme function is its replacement with different metalloporphyrins or with porphyrins containing different substituent groups. This paper describes a novel expression approach and purification of cobalt CBS (CoCBS), which results in a high yield of fully active, high purity enzyme, in which heme is substituted by Co-protoporphyrin IX (CoPPIX). Metal content analysis showed that the enzyme contained 92% cobalt and 8% iron. CoCBS was indistinguishable from wild-type FeCBS in its activity, tetrameric oligomerization, PLP saturation and responsiveness to the allosteric activator, S-adenosyl-l-methionine. The observed biochemical and spectral characteristics of CoCBS provide further support for the suggestion that heme is involved in structural integrity and folding of this unusual enzyme.     

Over-expression of cystathionine γ-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine

Cystathionine γ-synthase (CGS, EC 4.2.99.9), the first committed enzyme in methionine biosynthesis, was over-expressed in Arabidopsis thaliana by introducing in the genome of this plant the coding sequence of the Arabidopsis enzyme under the control of the cauliflower mosaic virus 35S promoter. In order to target the recombinant protein to the chloroplast, the transgene included the sequence encoding the N-terminal transit peptide of Arabidopsis CGS. CGS activity and polypeptide were increased several fold in these plants. There was a markedly increased level of soluble methionine in the leaves of the transformed plants, up to 15-fold, indicating that CGS is a rate-limiting enzyme in this metabolic pathway. In addition, the transformed plants strongly over-accumulated S-methylmethionine, but not S-adenosylmethionine, in agreement with the view that S-methylmethionine corresponds to a storage form of labile methyl groups in plants and/or plays a role in preventing S-adenosylmethionine accumulation. The same strategy was used to increase the level of cystathionine β-lyase (CBL, EC 4.4.1.8), the second committed enzyme in methionine biosynthesis, in transformed A. thaliana. Despite an increase in both CBL activity and polypeptide in transformed Arabidopsis plants over-expressing Arabidopsis CBL, there was very little change in the contents of soluble methionine and S-methylmethionine, suggesting strongly that CBL is not rate limiting in the methionine biosynthetic pathway.     

Frequency of cystathionine β-synthase 844INS68 polymorphism in Southern Iran

Iranian population with an Indo-European origin is one of the oldest populations in the world. Historical evidence suggests the close similarity in the origin of Iranian, European and north Indian population. However, there are few anthropological and genetic evidences on this subject. This study, which is the first report from Iran, was performed to investigate the genetic origin of Iranian population using a polymorphism in Cystathionine beta synthase (CBS) gene known as 844INS68bp in this respect, genomic DNA was extracted from the whole blood of 480 healthy normal blood donors referred to Fars Blood Transfusion Center, using a salting out method. The fragment containing 844INS68bp was amplified, the normal fragment was 174 bp and the fragment containing the insertion was 242 bp in length. Results indicated that 418 (87.08%) out of 480 individuals had a normal (N/N) genotype, 59 (12.29%) individuals were heterozygote (N/I) and 3 (0.63%) had homozygote a mutated genotype (I/I). The total frequency of 844INS68bp allele was found 6.8% which is similar to with the reported in White Caucasians. Comparison of the genotype of this study with the polymorphism in other populations revealed that Southern Iranian population has a great similarity with other Caucasians populations’ especially South Italy and North America while differed from East Asian and African populations. These results are in agreement with the result of other studied polymorphisms. Therefore, despite the great admixture of Iranian population with the neighboring non-Caucasian populations during the time, Iranian population still share a genetic background with other Caucasian populations.     

A critical life-supporting role for cystathionine γ-lyase in the absence of dietary cysteine supply

This study examined the important relationship between cystathionine γ-lyase (CSE) functionality and cysteine supply for normal growth and life span. Mice with a targeted deletion of the CSE gene (CSE-KO) were fed a cysteine-limited diet and their growth and survival patterns as well as levels of cysteine, homocysteine, glutathione, and hydrogen sulfide (H2S) were measured. CSE-KO mice fed a cysteine-limited diet exhibited growth retardation; decreased levels of cysteine, glutathione, and H2S; and increased plasma homocysteine level. However, histological examinations of liver did not reveal any abnormality and plasma levels of aspartate aminotransferase, alanine aminotransferase, and albumin were normal in these animals. No CSE-KO mice survived after 12 weeks of feeding with the cysteine-limited diet. Supplementation of H2S to the CSE-KO mice failed to reverse the aforementioned abnormalities. On the other hand, supplementation of cysteine in the drinking water of the CSE-KO mice significantly increased plasma cysteine and glutathione levels. This eventually led to an increase in body weight and rescued the animals from death. In conclusion, CSE is critical for cysteine biosynthesis through the transsulfuration pathway and the combination of CSE deficiency and lack of dietary cysteine supply would threaten life sustainability.     

Conformational properties of nine purified cystathionine β-synthase mutants

Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS.     

Cystathionine β-synthase (CBS) domains 1 and 2 fulfill different roles in ionic strength sensing of the ATP-binding cassette (ABC) transporter OpuA

The cystathionine β-synthase module of OpuA in conjunction with an anionic membrane surface acts as a sensor of internal ionic strength, which allows the protein to respond to osmotic stress. We now show by chemical modification and cross-linking studies that CBS2-CBS2 interface residues are critical for transport activity and/or ionic regulation of transport, whereas CBS1 serves no functional role. We establish that Cys residues in CBS1, CBS2, and the nucleotide-binding domain are more accessible for cross-linking at high than low ionic strength, indicating that these domains undergo conformational changes when transiting between the active and inactive state. Structural analyses suggest that the cystathionine β-synthase module is largely unstructured. Moreover, we could substitute CBS1 by a linker and preserve ionic regulation of transport. These data suggest that CBS1 serves as a linker and the structured CBS2-CBS2 interface forms a hinge point for ionic strength-dependent rearrangements that are transmitted to the nucleotide-binding domain and thereby affect translocation activity.     

Assignment of the gene for cystathionine β-synthase to human chromosome 21 in somatic cell hybrids

Summary Among several established mouse, rat, and Chinese hamster cell lines that were screened for cystathionine -synthase (CBS) activity, mouse 3T3 and Chinese hamster Don fibroblasts were found to contain no detectable activity. Somatic cell hybrids between human fibroblasts KG-7 with normal CBS activity and Don/a23TK^- cells (series XXI) were examined for CBS activity and for human chromosome content. Only chromosome 21 cosegregated with CBS activity. Because the activities measured could represent either Chinese hamster or human gene products, we have prepared a new series of hybrids between Don/a23TK^- cells and mutant human fibroblasts from a patient with homocystinuria due to deficiency of functional CBS mRNA. None of these (series XXV) hybrids contained detectable CBS activity, although collectively all human chromosomes were represented. Our results suggest that the human gene for CBS, called CBS, and thus for the most common form of homocystinuria, is located on chromosome 21.     

Cystathionine γ-lyase contributes to selenomethionine detoxification and cytosolic glutathione peroxidase biosynthesis in mouse liver

We earlier found that seleno-l-methionine (L-SeMet) as a food source of selenium (Se) is directly converted to methylselenol (CH₃SeH), α-ketobutyrate, and ammonia by the mouse hepatic cystathionine γ-lyase. The purpose of this study was to clarify the biological role of cystathionine γ-lyase in Se detoxification and cytosolic glutathione peroxidase (cGPx) biosynthesis because another metabolic pathway to CH₃SeH via seleno-l-cystathionine and seleno-l-cysteine (l-SeCyH) from l-SeMet has been shown by several enzymatic reactions. When mice were treated with either toxic doses of l-SeMet or a Se-deficient diet, the cystathionine γ-lyase activity for l-SeMet was invariable, suggesting that this enzyme was effective in both detoxification and biotransformation of Se. Concerning Se biotransformation into cGPx, production of H₂Se as the possible precursor was not observed by the in vitro reaction of the liver cytosol with CH₃SeH. When l-SeMet was administered at the nutritional dose to mice fed a Se-deficient diet, levels of both cGPx mRNA and cGPx protein were significantly restored. This recovery was not comparatively suppressed by coadministration of periodate-oxidized adenosine, an inhibitor of S-adenosylhomo-cystenase, where the conversion of l-SeMet to l-SeCyH is inhibited. However, the recovery was strongly suppressed when propargylglycine, an inhibitor of cystathioine γ-lyase that catalyzes the α,γ-elimination reaction of both l-SeMet and seleno-l-cystathionine, was treated. These results suggest that cystathionine γ-lyase is a notable enzyme, in SeMet metabolism and that CH₃SeH produced by the enzymatic reaction is utilized for cGPx biosynthesis.     

Enhanced Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase During Acute Cholecystitis-Induced Gallbladder Inflammation

Background

Hydrogen sulfide (H₂S) has recently been shown to play an important role in the digestive system, but the role of endogenous H₂S produced locally in the gallbladder is unknown. The aim of this study was to investigate whether gallbladder possesses the enzymatic machinery to synthesize H₂S, and whether H₂S synthesis is changed in gallbladder inflammation during acute acalculous cholecystitis (AC).

Methods

Adult male guinea pigs underwent either a sham operation or common bile duct ligation (CBDL). One, two, or three days after CBDL, the animals were sacrificed separately. Hematoxylin and eosin-stained slides of gallbladder samples were scored for inflammation. H₂S production rate in gallbladder tissue from each group was determined; immunohistochemistry and western blotting were used to determine expression levels of the H₂S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gallbladder.

Results

There was a progressive inflammatory response after CBDL. Immunohistochemistry analysis showed that CBS and CSE were expressed in the gallbladder epithelium, muscular layer, and blood vessels and that the expression increased progressively with increasing inflammation following CBDL. The expression of CBS protein as well as the H₂S-production rate was significantly increased in the animals that underwent CBDL, compared to those that underwent the sham operation.

Conclusions

Both CBS and CSE are expressed in gallbladder tissues. The expression of these enzymes, as well as H₂S synthesis, was up-regulated in the context of inflammation during AC.