Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag.

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.     

A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.     

Aromatic l‐amino acid decarboxylase phosphorylation and activation by PKGIαin vitro

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α₁/β₁ and α₂/β₁ heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.     

Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site.     

Inhibition of deactivation of NO-sensitive guanylyl cyclase accounts for the sensitizing effect of YC-1

Many of the physiological effects of the signaling molecule nitric oxide are mediated by the stimulation of the NO-sensitive guanylyl cyclase. Activation of the enzyme is achieved by binding of NO to the prosthetic heme group of the enzyme and the initiation of conformational changes. So far, the rate of NO dissociation of the purified enzyme has only been determined spectrophotometrically, whereas the respective deactivation, i.e. the decline in enzymatic activity, has only been determined in cytosolic fractions and intact cells. Here, we report on the deactivation of purified NO-sensitive guanylyl cyclase determined after addition of the NO scavenger oxyhemoglobin or dilution. The deactivation rate corresponded to a half-life of the NO/guanylyl cyclase complex of approximately 4 s, which is in good agreement with the spectrophotometrically measured NO dissociation rate of the enzyme. The deactivation rate of the enzyme determined in platelets yielded a much shorter half-life indicating either partial damage of the enzyme during the purification procedure or the existence of endogenous deactivation accelerating factors. YC-1, a component causing sensitization of guanylyl cyclase toward NO, inhibited deactivation of guanylyl cyclase, resulting in an extremely prolonged half-life of the NO/guanylyl cyclase complex of more than 10 min. The deactivation of an ATP-utilizing guanylyl cyclase mutant was almost unaffected by YC-1, indicating the existence of a special structure within the catalytic domain required for YC-1 binding or for the transduction of the YC-1 effect. In contrast to the wild type enzyme, YC-1 did not increase NO sensitivity of this mutant, clearly establishing inhibition of deactivation as the underlying mechanism of the NO sensitizer YC-1.     

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.     

Nitric oxide activates the beta 2 subunit of soluble guanylyl cyclase in the absence of a second subunit.

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.     

Post-transcriptional regulation of soluble guanylyl cyclase expression in rat aorta

We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.     

Crystal structure of the signaling helix coiled-coil domain of the β1 subunit of the soluble guanylyl cyclase

Background  

The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner.     

Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris

A 371 bp full-length cDNA (GenBank Accession No. DQ232774) was obtained from housefly Musca domestica by using degenerate primers and subsequent amplification by 5'- and 3'-RACE. The cecropin gene, Mdcec and Mdcec/6His, was cloned into expression pPICZalpha-A vector and was expressed in the methylotrophic yeast, Pichia pastoris. The recombinant Mdcec was purified using cationic exchange chromatography and 1.2mg pure active Mdcec was obtained from 100ml culture broth supernatant. To facilitate purification of Mdcec, the C-terminal 6His-tagged Mdcec was also expressed in P. pastoris. The recombinant Mdcec/6His was purified to homogeneity by a nickel chelating sepharose column and 2.0mg pure active Mdcec/6His was obtained from 100ml culture broth supernatant. Anti-microbial assays demonstrated that Mdcec had broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. Mdcec/6His showed a similar activity to Mdcec against bacteria, but a slight higher activity against fungi. These results indicate that the 6His-tag can enhance the cationic nature and stability of Mdcec. This is the first report on the heterologous expression of a cecropin and cecropin with a 6His tag in P. pastoris. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional M. domestica cecropin for both research and industrial purpose.     

A new form of guanylyl cyclase is preferentially expressed in rat kidney

On the basis of the conserved amino acid sequences of the catalytic domain of both soluble and plasma membrane forms of guanylyl cyclase, we have used the polymerase chain reaction to identify a new form of guanylyl cyclase that is expressed principally in kidney. The cDNA for this new form (GC-S beta 2) codes for a 76.3-kDa protein, which most closely resembles a 70-kDa subunit (GC-S beta 1) of the lung soluble guanylyl cyclase. The mRNA for GC-S beta 1 is preferentially expressed in lung and brain, whereas GC-S beta 2 mRNA is more abundant in kidney and liver. An 86 amino acid carboxyl-terminal region extends beyond the C-terminus of GC-S beta 1 and contains a consensus sequence (-C-V-V-L) for isoprenylation/carboxymethylation. This is the first demonstration of heterogeneity among the heterodimeric forms of guanylyl cyclase and suggests differential regulation.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires both 70 and 73 kDa subunits.     

Cyclic GMP controls Rhodospirillum centenum cyst development

Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α-proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homologue of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homologue preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.     

Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells

Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.     

BAY 41-2272 activates two isoforms of nitric oxide-sensitive guanylyl cyclase

Soluble guanylyl cyclase is an important target for endogenous nitric oxide and the guanylyl cyclase modulator, YC-1. Recently BAY 41-2272 was identified as a similar but more potent and more specific substance. While YC-1 also acts as non-specific phosphodiesterase inhibitor, BAY 41-2272 is devoid of an effect on phosphodiesterases. BAY 41-2272 has so far only been tested on the alpha(1)/beta(1) heterodimeric isoform of soluble guanylyl cyclase and its binding site has been mapped to a region in the alpha(1) subunit amino-terminal sequence. Although this region is poorly conserved in the alpha(2) subunit, we show in the current study that the alpha(2)/beta(1) heterodimeric enzyme isoform is activated by BAY 41-2272. Deletion analysis of the alpha(2) subunit and co-expression with the beta(1) subunit in the baculovirus/Sf9 system is consistent with the amino-terminal amino acids 104 to 401 of the alpha(2) subunit as binding site for BAY 41-2272.     

Synthesis of biologically active human interferon α-2b in Nicotiana benthamiana

A method was developed for the production and purification of biologically active recombinant human interferon α-2b (rhIFN α-2b) synthesized by expression in Nicotiana benthamiana plants. A gene construct containing a modified hIFN α-2b gene was cloned in two vectors based on tobacco mosaic virus driven by an actin promoter from Arabidopsis thaliana (pA-IFN-A) and cauliflower mosaic virus driven by a 35S promoter (pA-IFN-S). The expression vectors were introduced into the plant cells by agroinfiltration. The maximum rates of synthesis achieved in the case of pA-IFN-A and pA-IFN-S 5 days after agroinfiltration were determined to be 200 and 20 mg per 1 kg of fresh leaves, respectively. The recombinant hIFN α-2b synthesized in the plant showed high antiviral and antitumor activity comparable with that of commercial drug.     

A role for GCAP2 in regulating the photoresponse. Guanylyl cyclase activation and rod electrophysiology in GUCA1B knock-out mice

Cyclic GMP serves as the second messenger in visual transduction, linking photon absorption by rhodopsin to the activity of ion channels. Synthesis of cGMP in photoreceptors is supported by a pair of retina-specific guanylyl cyclases, retGC1 and -2. Two neuronal calcium sensors, GCAP1 and GCAP2, confer Ca(2+) sensitivity to guanylyl cyclase activity, but the importance and the contribution of each GCAP is controversial. To explore this issue, the gene GUCA1B, coding for GCAP2, was disrupted in mice, and the capacity for knock-out rods to regulate retGC and generate photoresponses was tested. The knock-out did not compromise rod viability or alter outer segment ultrastructure. Levels of retGC1, retGC2, and GCAP-1 expression did not undergo compensatory changes, but the absence of GCAP2 affected guanylyl cyclase activity in two ways; (a) the maximal rate of cGMP synthesis at low [Ca(2+)] dropped 2-fold and (b) the half-maximal rate of cGMP synthesis was attained at a higher than normal [Ca(2+)]. The addition of an antibody raised against mouse GCAP2 produced similar effects on the guanylyl cyclase activity in wild type retinas. Flash responses of GCAP2 knock-out rods recovered more slowly than normal. Knock-out rods became more sensitive to flashes and to steps of illumination but tended to saturate at lower intensities, as compared with wild type rods. Therefore, GCAP2 regulation of guanylyl cyclase activity quickens the recovery of flash and step responses and adjusts the operating range of rods to higher intensities of ambient illumination.     

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.