ATP modulation of the ligand binding and signal transduction activities of the type C natriuretic peptide receptor guanylate cyclase

The type C natriuretic peptide (CNP)-activated guanylate cyclase (CNP-RGC) is a single-chain transmembrane-spanning protein, containing both CNP binding and catalytic cyclase activities. Upon binding CNP to the extracellular receptor domain, the cytosolic catalytic domain of CNP-RGC is activated, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening signal transduction step which is regulated by ATP binding to the cyclase. This bridges the events of ligand binding and cyclase activation. A defined sequence motif (Gly⁴⁹⁹-Xa-Xa-Xa-Gly⁵⁰³), termed ATP regulatory module (ARM), is critical for this step. The present study shows that ATP not only amplifies the signal transduction step, it also concomitantly reduces the ligand binding activity of CNP-RGC. Reduction in the ligand binding activity is a consequence of the transformation of the high affinity receptor-form to the low affinity receptor-form. A single ARM residue Gly⁴⁹⁹ is critical in the mediation of both ATP effects, signal transduction and ligand binding activity of the receptor. Thus, this residue represents an ATP bimodal switch to turn the CNP signal on and off.     

Six- to five-coordinate heme-nitrosyl conversion in cytochrome c' and its relevance to guanylate cyclase

The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c' reacts with NO to form a 6-coordinate nitrosyl intermediate (lambdaSoret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (lambdaSoret at 395 nm) in a rate-determining step. Stopped-flow measurements at pH 8.9, 25 degrees C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k(on) = (4.4 +/- 0.5) x 10(4) M(-1) x s(-1), which is 3-4 orders of magnitude lower than the values for other pentacoordinate ferrous hemes and is consistent with NO binding within the sterically crowded distal heme pocket. Resonance Raman measurements of the freeze-trapped 6-coordinate nitrosyl intermediate reveal an unusually high Fe-NO stretching frequency of 579 cm(-1), suggesting a distorted Fe-N-O coordination geometry. The rate of 6- to 5-coordinate heme nitrosyl conversion is also dependent upon NO concentration, with a rate constant, k(6-5) = (8.1 +/- 0.7) x 10(3) M(-1) x s(-1), implying that an additional molecule of NO is required to form the 5c-NO adduct. Since crystallographic studies have shown that the 5-coordinate nitrosyl complex of cytochrome c' binds NO to the proximal (rather than distal) face of the heme, the NO dependence of the 6- to 5-coordinate NO conversion supports a mechanism in which the weakened His ligand, as well as the distally bound NO, is displaced by a second NO molecule which attacks and is retained in the proximal coordination position. The fact that a dependent 6- to 5-coordinate nitrosyl conversion has been previously reported for soluble guanylate cyclase suggests that the mechanism of Fe-His bond cleavage may be similar to that of cytochrome c' and strengthens the recent proposal that both proteins exhibit proximal NO binding in their 5-coordinate nitrosyl adducts.     

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.     

In vivo ligand specificity of E-selectin binding to multivalent sialyl Lewisx N-linked oligosaccharides.

The in vivo specificity for E-selectin binding to a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl Lewisx (SLex; NeuAcalpha2-3Gal[Fucalpha1-3]beta1-4GlcNAc) determinants was studied in mice. Following intraperitoneal dosing with lipopolysaccharide, radioiodinated tyrosinamide N-linked oligosaccharides were dosed i.v. and analyzed for their pharmacokinetics and biodistribution. Specific targeting was determined from the degree of SLex oligosaccharide targeting relative to a sialyl oligosaccharide control. Oligosaccharides targeted the kidney with the greatest selectivity after a 4-h induction period following lipopolysaccharide dosing. Unique pharmacokinetic profiles were identified for SLex biantennary and triantennary oligosaccharides but not for monovalent and tetraantennary SLex oligosaccharides or sialyl oligosaccharide controls. Biodistribution studies established that both SLex biantennary and triantennary oligosaccharides distributed to the kidney with 2-3-fold selectivity over sialyl oligosaccharide controls, whereas monovalent and tetraantennary SLex oligosaccharides failed to mediate specific kidney targeting. Simultaneous dosing of SLex biantennary or triantennary oligosaccharide with a mouse anti-E-selectin monoclonal antibody blocked kidney targeting, whereas co-administration with anti-P-selectin monoclonal antibody did not significantly block kidney targeting. The results suggest that SLex biantennary and triantennary are N-linked oligosaccharide ligands for E-selectin and implicate E-selectin as a bivalent receptor in the murine kidney endothelium.     

Oxygen binding and redox properties of the heme in soluble guanylate cyclase: implications for the mechanism of ligand discrimination

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O(2), thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O(2) has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe(2+)-O(2)) when frozen at 77 K in the presence of O(2). The ligation of O(2) was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 °C, with the extremely low affinity for O(2). The low O(2) affinity was not caused by a distal steric protein effect and by rupture of the Fe(2+)-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe(2+)-proximal His bond. Based on our results, we propose that the weak Fe(2+)-proximal His bond is a key determinant for the low O(2) affinity of the heme moiety of soluble guanylate cyclase.     

The effect of ligand heterogeneity on the Scatchard plot. Particular relevance to lipoprotein binding analysis

Computer simulations of equilibrium binding studies of a mixture of two labeled ligands binding competitively to a single class of identical and independent sites (receptors) were performed to investigate how ligand heterogeneity affects the observed data in such studies. The simulated data are presented in Scatchard plots. Ligand heterogeneity was generally found to be indistinguishable from the case of a homogeneous ligand when usual experimental conditions applied (that is, Scatchard plots of the data were straight lines). Some factors that increased the probability of recognizing heterogeneity in the system were identified, however. These are 1) a large difference between the dissociation constants of the two ligands, 2) a high concentration of receptors relative to the dissociation constant of the higher-affinity ligand, 3) a high concentration of the lower-affinity ligand relative to that of the higher-affinity ligand, 4) a high specific activity of the lower-affinity ligand relative to that of the higher-affinity ligand, and 5) lack of experimental error. When ligand heterogeneity (under certain conditions) did cause curvilinearity in the Scatchard plot, the curve formed was always concave-downwards. Thus, ligand heterogeneity may occasionally mimic positive cooperativity, but never mimics negative cooperativity or multiple classes of binding sites. Implications of these findings for equilibrium binding studies involving lipoproteins (which are generally isolated as heterogeneous mixtures of particles) are discussed in detail. These findings are also relevant to equilibrium binding studies using ligands which are mixtures of stereoisomers or which contain chemical or radiochemical impurities.     

Characterization of nitrosoalkane binding and activation of soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is the primary receptor for the signaling agent nitric oxide (NO). Electronic absorption and resonance Raman spectroscopy were used to show that nitrosoalkanes bind to the heme of sGC to form six-coordinate, low-spin complexes. In the sGC-nitrosopentane complex, a band assigned to an Fe-N stretching vibration is observed at 543 cm(-)(1) which is similar to values reported for other six-coordinate NO-bound hemoproteins. Nitrosoalkanes activate sGC 2-6-fold and synergize with YC-1, a synthetic benzylindazole derivative, to activate the enzyme 11-47-fold. In addition, the observed off-rates of nitrosoalkanes from sGC were found to be dependent on the alkyl chain length. A linear correlation was found between the observed off-rates and the alkyl chain length which suggests that the sGC heme has a large hydrophobic distal ligand-binding pocket. Together, these data show that nitrosoalkanes are a novel class of heme-based sGC activators and suggest that heme ligation is a general requirement for YC-1 synergism.     

Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase

Microbial cytochromes c' contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 A resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His-Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species. In contrast, CO binds 6-coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c' are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.     

Covalent coupling of a resact analogue to guanylate cyclase

GGGYG-resact (Gly-Gly-Gly-Tyr-Gly-Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg -Leu-NH2) was synthesized and shown to possess the same respiration-stimulating activity and receptor-binding ability as resact. The incubation of intact sperm cells with radioiodinated peptide, 125I-GGGYG-resact, and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of a single, major radioactive band of apparent molecular weight 160,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The interaction was specific since 150 nM nonradioactive resact but not speract (200 nM) blocked formation of the radioactive band. The radioactive, cross-linked protein co-migrated with 32P-labeled guanylate cyclase and could be immunoprecipitated with a polyclonal antibody raised in rabbits against the sperm guanylate cyclase. The incubation of intact cells with NH4Cl resulted in the partial dephosphorylation of guanylate cyclase and a change in its apparent molecular weight from 160,000 to 150,000; NH4Cl also caused the same conversion in the apparent molecular weight of the cross-linked protein. These data demonstrate that an analogue of resact can be covalently coupled to guanylate cyclase with the specificity predicted for the peptide receptor.     

The receptor guanylate cyclase Gyc76C and a peptide ligand, NPLP1-VQQ, modulate the innate immune IMD pathway in response to salt stress

Receptorguanylate cyclases (rGCs) modulate diverse physiological processes including mammalian cardiovascular function and insect eclosion. The Drosophila genome encodes several receptor and receptor-like GCs, but no ligand for any Drosophila rGC has yet been identified. By screening peptide libraries in Drosophila S2 cells, the Drosophila peptide NPLP1-VQQ (NLGALKSSPVHGVQQ) was shown to be a ligand for the rGC, Gyc76C (CG42636, previously CG8742, l(3)76BDl, DrGC-1). In the adult fly, expression of Gyc76C is highest in immune and stress-sensing epithelial tissues, including Malpighian tubules and midgut; and NPLP1-VQQ stimulates fluid transport and increases cGMP content in tubules. cGMP signaling is known to modulate the activity of the IMD innate immune pathway in tubules via activation and nuclear translocation of the NF-kB orthologue, Relish, resulting in increased anti-microbial peptide (AMP) gene expression; and so NPLP1-VQQ might act in immune/stress responses. Indeed, NPLP1-VQQ induces nuclear translocation of Relish in intact tubules and increases expression of the anti-microbial peptide gene, diptericin. Targeted Gyc76C RNAi to tubule principal cells inhibited both NPLP1-VQQ-induced Relish translocation and diptericin expression. Relish translocation and increased AMP gene expression also occurs in tubules in response to dietary salt stress. Gyc76C also modulates organismal survival to salt stress - ablation of Gyc76C expression in only tubule principal cells prevents Relish translocation, reduces diptericin expression, and reduces organismal survival in response to salt stress. Thus, the principal-cell localized NPLP1-VQQ/Gyc76C cGMP pathway acts to signal environmental (salt) stress to the whole organism.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase.

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.     

From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.     

Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain

The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the beta1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate Fe(II)-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate Fe(II)-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal Fe(II)-His bond and suggests that in both the 5- and 6-coordinate Fe(II)-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes.     

Analysis of binding sites on complement factor I using artificial N-linked glycosylation

Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors, such as factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein. FI is composed of two chains linked by a disulfide bridge; the light chain comprises only the serine protease (SP) domain, whereas the heavy chain contains the FI membrane attack complex domain (FIMAC), CD5 domain, and low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based three-dimensional models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface-exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (K(m)) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished the ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespective of the cofactor used. On the other hand, only a few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI.     

Butadiene diepoxide activation of guanylate cyclase.

A variety of nitroso chemical carcinogens increase the activity of guanylate cyclase (EC 4.6.1.2), the enzyme catalyzing the production of guanosine 3',5'-monophosphate. In the present report, the first non-nitroso chemical carcinogen, butadiene diepoxide, was shown to activate guanylate in a variety of tissues over the concentration range 1-100 mmol/l. At 20 mmol/l concentration, increases were 2- to 17-fold above control. These observations have potential importance since guanosine 3',5'-monophosphate may be involved in cell growth and malignant transformation.