Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.</p

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides

Inhibition of GC activity by XTP in membrane preparations of HEK293 cells stably overexpressing pGC-A.

<p>Membranes (10 µg protein per tube) was stimulated with 200 µM GTP/Mn²⁺ and 1 µM ANP at 37°C for 5 min and increasing concentrations of XTP/Mn²⁺ (2–1,000 µM). Data were best fitted by using a competitive binding model at one binding site with an IC₅₀ of 145.3 ± 1.2 µM. Values based on the means ± SEM of six independent experiments.</p

Kinetic parameters of membrane preparations of HEK293 stably overexpressing pGC-A for various nucleotides.

<p>NC activities were analyzed as mentioned in Materials and Methods. Membranes from HEK293 cells overexpressing pGC-A (10–80 µg of protein per tube) were incubated with 2–7,500 µM (XTP/Me²⁺: 2–2,000 µM) NTP/Me²⁺ in the presence of 1 µM ANP at 37°C for 5–10 min depending on the analyzed NTP. Apparent s_0.5, V_max, and <i>n</i>_Hill represent the means ± SEM of six independent experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-g002" target="_blank">2</a> and are given in alphabetical order of NTPs. Curves were analyzed by nonlinear regression using Prism version 5.0. nd: not detected, nq: not quantified.</p>*<p>only detectable in the presence of 500 µM ATP/Mg²⁺.</p

Kinetic data of substrate saturation experiments with CyaA-N and EF.

a<p>data-fit ambiguous,</p>*<p>K_i for substrate inhibition; N = 3–6; analysis of the data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone.0070223.s004" target="_blank">Figs. S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone.0070223.s005" target="_blank">S5</a>. AC, adenylyl cyclase; CC, cytidylyl cyclase; GC, guanylyl cyclase; IC, inosityl cyclase; TC, thymidinyl cyclase; UC, uridylyl cyclase; XC, xanthosinyl cyclase.</p

Analysis of NC activity of ROS membrane preparations.

<p>Membranes (81 µg rhodopsin per tube) were incubated for 5 min at 30°C with 3.5 mM MgCl₂ and 1 mM GTP/Mg²⁺, UTP/Mg²⁺ and CTP/Mg²⁺, respectively, and 2 mM EGTA or 2 mM CaCl₂, as indicated. Reactions were stopped by heating at 95°C for 10 min and analyzed as described in Materials and Methods. Values represent the mean with range of 2–3 independent experiments. Please note the different scales of the y-axes of all panels. A, cNMP formation with membranes; B, NMP formation with membranes; C, control experiments showing contamination of NTP solutions with NMPs.</p

Effect of ATP on GC activity in membrane preparations of HEK293 cells stably overexpressing pGC-A.

<p>Membranes (10 µg of protein per tube) were incubated for 25 min at 37°C in the presence of 100 µM GTP/Me²⁺ with or without 500 µM ATP/Me²⁺ as indicated. Values represent the means ± SEM of three independent experiments. Please note the different scales of the y-axes in both panels.</p

cNMP levels in intact HEK293 cells stably overexpressing pGC-A following stimulation with ANP.

<p>HEK293 cells stably overexpressing pGC-A were seeded in 6-well plates for 24 h with 5⋅10⁵ cells per well and stimulated with 1 µM ANP for defined times 10 min after preincubation with IBMX (100 µM). Values of plot A-E are given by means ± SEM of 3–6 independent experiments in µmol/million cells. Please note the different scales of the y-axes in these panels. Plot E: Data points are due to background noise. Dotted lines: lower limit of detection of cIMP, cTMP, and cXMP. ***: p-value ≤0.001.</p

Kinetic analysis of nucleotidyl cyclase activity in membrane preparations of HEK293 cells stably overexpressing pGC-A.

<p>Membranes (10–80 µg protein per tube) were incubated with 2–7,500 µM NTP/Me²⁺ (XTP/Me²⁺: 2–2,000 µM) in the presence of 1 µM ANP at 37°C for 5–10 min. Values represent the mean ± SEM of six independent experiments. Except data of plot D, data were fitted using specific binding with Hill slope. Data of plot D were best fitted by substrate inhibition model. Please note the different scales of the x- and y-axes of all panels. *: only detectable in the presence of 500 µM ATP/Mg²⁺. Kinetic parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-t002" target="_blank">Table 2</a>.</p