NAADP (nicotinic acid–adenine dinucleotide phosphate), the most potent Ca2+-mobilizing second messenger, is active in a wide range of organisms and cell types. Until now, all NAADP-producing enzymes have been thought to be members of the ADP-ribosyl cyclase family. ADP-ribosyl cyclases exhibit promiscuous substrate selectivity, synthesize a variety of products and are regulated in a limited manner, which may be non-physiological. In the present paper, we report the presence of an enzyme on the surface of sea urchin sperm that exhibits bell-shaped regulation by Ca2+ over a range (EC50 of 10 nM and IC50 of 50 μM) that is physiologically relevant. Uniquely, this surface enzyme possesses complete selectivity for nucleotides with a 2′-phosphate group and exhibits only base-exchange activity without any detectable cyclase activity. Taken together, these findings indicate that this novel enzyme should be considered as the first true NAADP synthase.

INTRODUCTION

Currently, there are four generally accepted Ca2+-releasing second messengers: IP3 (inositol 1,4,5-trisphosphate) [1], cADPR (cADP-ribose) [2], sphingosine 1-phosphate [3] and NAADP (nicotinic acid–adenine dinucleotide phosphate) [4]. Compared with IP3 and cADPR, NAADP is unique in many respects, including releasing Ca2+ from lysosome-related organelles [5], exhibiting potent self-desensitization [6,7], which may relate to pseudo-irreversible binding [6], and the ability to initiate Ca2+ increases via either Ca2+-induced Ca2+ release [8] or store overloading [911]. Nevertheless, many features of NAADP behaviour are contested or remain unknown. For example, the Ca2+ store targeted by NAADP has been suggested to be the ER (endoplasmic reticulum) [12], and the channel on which NAADP acts directly has been suggested to be the ryanodine receptor rather than a novel channel [1315]. There is also a large gap in our understanding of the signalling steps involved in NAADP synthesis. Indeed, if NAADP is synthesized by base exchange, it is not entirely clear how this synthesis is regulated endogenously by enzymes categorized as ADP-ribosyl cyclases [16].

To date, all known ADP-ribosyl cyclases exhibit both cyclase (cADPR synthesis) and base-exchange (NAADP synthesis) activity, with the relative ratio mainly regulated by pH and substrate availability [16] and moderately regulated by Zn2+ [17], phosphorylation and cAMP and cGMP [18,19]. That is, ADP-ribosyl cyclases can perform a multitude of reactions dictated entirely by substrate availability (Figure 1): NAD is converted into cADPR, and nicotinic acid and NADP are converted into NAADP [20,21]. One concern with all mechanisms of regulation described thus far is that the reaction conditions are not physiologically relevant (e.g. pH of 4 or millimolar concentrations of Zn2+ and Cu2+) [16,20,21].

Enzymes in the ADP-ribosyl cyclase family are multifunctional and catalyse four classes of reaction: base-exchange, cyclase, glycohydrolase and hydrolase

Figure 1
Enzymes in the ADP-ribosyl cyclase family are multifunctional and catalyse four classes of reaction: base-exchange, cyclase, glycohydrolase and hydrolase

Although several exogenous nucleotides and pyridine bases can serve as substrates, NADP is used to illustrate each reaction.

Figure 1
Enzymes in the ADP-ribosyl cyclase family are multifunctional and catalyse four classes of reaction: base-exchange, cyclase, glycohydrolase and hydrolase

Although several exogenous nucleotides and pyridine bases can serve as substrates, NADP is used to illustrate each reaction.

Previously, we demonstrated that NAADP fulfilled all the criteria necessary to be a bona fide second messenger during sea urchin sperm activation [22]. We reasoned that, given the capacity of sea urchin sperm to provide rapid and large synthesis of NAADP, sperm would be a good model system to study the regulation of NAADP synthesis. In the present paper, we report that sea urchin sperm possess an enzyme capable of base exchange, with selectivity for nucleotides with a 2′-phosphate, that is regulated by Ca2+ in a bell-shaped manner. Moreover, within our limits of detection, this enzyme can only perform base exchange and not cyclization. Therefore we propose that this enzyme is most appropriately termed NAADP synthase.

EXPERIMENTAL

Materials

Lytechinus pictus sea urchins were obtained from Marinus Scientific. All chemicals were from Sigma, except where indicated.

Sperm collection

Sperm were shed from Lytechinus pictus sea urchins by intracoelomic injection of approx. 1 ml of 0.5 M KCl. Sperm were collected on a ‘dry’ Petri dish and were transferred to a microcentrifuge tube and stored at 4 °C until required.

Permeabilization and homogenization of sperm

Before permeabilization and homogenization, the viability of the sperm was tested visually under a microscope as determined by the induction of swimming on dilution into artificial seawater, to ensure they were healthy. Sperm were permeabilized using 50 μM digitonin. Homogenization was carried out using Jencons VC50 sonicator. Three pulses of 50 W and 50 Hz for 3 s were delivered to the samples, followed by freeze–thawing [−80 °C for 10 min and thawing at room temperature (23 °C)].

HPLC

Nucleotides were separated on a column (3 mm×150 mm) packed with AGMP-1 resin (Bio-Rad Laboratories). Samples (200 μl) were injected on to a column equilibrated with water. The bound material was eluted with a concave-up gradient of trifluoroacetic acid, which increased linearly to 2% at 1.5 min, and to 4, 8, 16, 32 and 100% (150 mM trifluoroacetic acid) at 3, 4.5, 6.0, 7.5 and 7.51 min respectively. The flow rate was 4 ml/min. The nucleotides were detected by their absorbance at 254 nm.

ADP-ribosyl cyclase activity

The cyclase activity was determined by an assay that relies on the fluorescence increase caused by the formation of cGDPR (cGDP-ribose) from NGD (nicotinamide guanine dinucleotide) [23]. The reaction was carried out in the presence of 500 mM KCl, 4 mM NGD, 20 mM Hepes (pH 7) and intact sperm (final concentration of 20%, v/v). The fluorescence intensity was monitored using a PerkinElmer fluorescence spectrometer at an excitation wavelength of 300 nm and an emission wavelength of 410 nm.

Ca2+ and Mg2+ buffer preparation

Solutions containing various concentrations of free Ca2+ were prepared using the pH-metric method described previously [24]. Absolute Ca2+ concentrations were later verified with a Ca2+-selective electrode. The computer program WinMax chelator (version 2.05) was used to prepare Mg2+ solutions of different buffered concentrations [25].

Enzyme kinetic studies

Intact sperm were incubated (final concentration 20%, v/v) with various concentrations of NADP and 50 mM nicotinic acid at 23 °C in the presence of 500 mM KCl and 20 mM Hepes for 1 h. Similarly, to find the Km for nicotinic acid, the nicotinic acid concentration was varied in the presence of 5 mM NADP. The reaction was stopped after 1 h by addition of ice-cold ethanol and centrifugation at 16100 g for 2 min. The protein pellet was stored at −20 °C for protein analysis by bicinchoninic acid. The production of NAADP was determined using the HPLC method described above.

Analysis

Results are means±S.E.M. Statistical comparisons were made using a non-paired Student's t test. Enzyme kinetic analysis was performed using GraphPad Prism.

RESULTS AND DISCUSSION

We explored NAADP synthesis via base exchange (Figure 1) in sea urchin sperm by adding exogenous NADP and nicotinic acid to sperm permeabilized with digitonin to facilitate the entry of substrates (Figure 2A and 2B). Permeabilized sperm underwent the base-exchange reaction (Figure 2B). Surprisingly, the non-permeabilized sperm, which served as our negative control, synthesized NAADP at levels comparable with those of the permeabilized sperm (Figure 2C), suggesting the presence of an enzyme on the surface of the sperm. We verified that sperm were permeabilized by digitonin by observing Trypan Blue uptake (Figure 2D). Also, there was no additional enzymatic activity in freeze–thawed and sonicated sperm (Figure 2E), providing further evidence that all of the enzymatic activity was on the surface. To localize the enzymatic activity on the sperm, we decapitated sperm with shear (passing them through a 25-gauge needle seven times) and then separated the heads and tails by density-gradient centrifugation [26,27]. We verified that the resulting fractions were highly enriched in heads or tails by visible light microscopy (Figure 2F). NAADP production per mg of protein was approximately four times higher in the isolated tails than in the isolated heads (Figure 2G). Taken together, these data demonstrate that sperm possess base-exchange activity on their surface.

Sperm exhibit base-exchange activity on their surface

Figure 2
Sperm exhibit base-exchange activity on their surface

(A) HPLC trace of the substrates (NADP and nicotinic acid) before incubation with sperm. Note that ADPR-P is an impurity in commercial NADP. (B) Permeabilized sperm synthesize NAADP by base exchange. HPLC trace taken 1 h after incubation of digitonin (50 μM)-permeabilized sperm incubated with NADP (1 mM) and nicotinic acid (10 mM) in artificial seawater (20 mM Hepes, pH 7.0). (C) Intact sperm synthesize NAADP by base exchange. Reaction conditions were as in (B), except without digitonin. (D) Effect of digitonin concentration on sperm permeabilization. Permeabilization was assessed by incubating sperm with Trypan Blue for 5 min and then scoring the number of blue sperm heads in a sample of 100 viewed with bright-field microscopy. Results are means±S.E.M. (n=3). (E) Intact sperm show comparable amounts of base-exchange activity to digitonin-permeabilized and freeze–thawed sperm. Results are means±S.E.M. (n=3). (F) Sperm heads and tails were separated and purified. Visible light images of bands formed in a sucrose gradient after centrifugation of sperm beheaded by the shear force when passed through a needle. Magnification, ×630. (G) Both sperm heads and tails exhibit base-exchange activity. Note that the headpiece of the sperm remains with the tail with this procedure. Results are means±S.E.M. (n=3).

Figure 2
Sperm exhibit base-exchange activity on their surface

(A) HPLC trace of the substrates (NADP and nicotinic acid) before incubation with sperm. Note that ADPR-P is an impurity in commercial NADP. (B) Permeabilized sperm synthesize NAADP by base exchange. HPLC trace taken 1 h after incubation of digitonin (50 μM)-permeabilized sperm incubated with NADP (1 mM) and nicotinic acid (10 mM) in artificial seawater (20 mM Hepes, pH 7.0). (C) Intact sperm synthesize NAADP by base exchange. Reaction conditions were as in (B), except without digitonin. (D) Effect of digitonin concentration on sperm permeabilization. Permeabilization was assessed by incubating sperm with Trypan Blue for 5 min and then scoring the number of blue sperm heads in a sample of 100 viewed with bright-field microscopy. Results are means±S.E.M. (n=3). (E) Intact sperm show comparable amounts of base-exchange activity to digitonin-permeabilized and freeze–thawed sperm. Results are means±S.E.M. (n=3). (F) Sperm heads and tails were separated and purified. Visible light images of bands formed in a sucrose gradient after centrifugation of sperm beheaded by the shear force when passed through a needle. Magnification, ×630. (G) Both sperm heads and tails exhibit base-exchange activity. Note that the headpiece of the sperm remains with the tail with this procedure. Results are means±S.E.M. (n=3).

Having found an enzyme on the surface of the sperm that is capable of producing NAADP (Figure 2), we investigated whether this enzyme was functionally similar to any of the cyclases already characterized in regard to substrate selectivity, enzyme kinetics and regulation (Table 1) [20,21,28]. All of the NAADP-synthesizing ADP-ribosyl cyclases reported so far that are capable of producing NAADP are multifunctional in nature, and exhibit four activities: base-exchange, glycohydrolase, hydrolase and cyclase (Figure 1) [20,21]. All of these reactions can be explained by a unifying enzymatic mechanism involving a covalent intermediate [20,29,30]. Variations in the relative amounts of each of these enzymatic activities are characteristic of different ADP-ribosyl cyclase family members (Table 1). The extremes of the variations are illustrated by the contrast between Aplysia cyclase, which is entirely a cyclase, and CD38, which is mostly a hydrolase [31].

Table 1
Comparison of enzyme kinetics, regulation and enzymatic activities of known ADP-ribosyl cyclases with sperm surface NAADP synthase

GDPR, GDP-ribose; na, not applicable; nr, not reported.

 Enzyme or enzymatic activity 
Parameter CD38 (external) [16,47CD157 (external) [37Aplysia cyclase (luminal) [16Human sperm [17,48Brain cyclase [49Vascular smooth muscle [50NACE, Schistosoma mansoni [32Sea urchin egg, soluble (internal) [18,19Sea urchin egg, membrane-bound (internal) [18,19Sea urchin, soluble (ER, lumen) [33Sea urchin sperm Sea urchin sperm surface NAADP synthase (external) 
Zn2+ Activates [51]; inhibits hydrolase [52Activates [37Activates [50Switches cyclase/ glycohydrolase activity [17Inhibits [49Inhibits [50nr nr Increase [50nr nr nr 
Cu2+ Activates [50,51Inhibits mM [37nr No effect [17Complete inhibition, 10 μM [49Inhibits [50nr nr nr nr nr nr 
Ca2+ No effect [51No effect No effect No effect [17,48No effect [49nr nr nr nr nr nr Low, activates; high, inactivates 
Km NADP 1 mM [53nr nr nr nr 14 μM [32200 μM [18,190.2 mM [19]; 0.4 mM [18nr nr 110 μM  
Km NA 5 mM [16nr ∼10 mM [16nr nr nr nr 10 mM [18,1910 mM [19]; 9 mM [18nr nr 4 mM 
Km NAD 15 μM [23,29]; 200 μM [17nr 39 μM [16150 μM [17]; 8 μM [4821 μM [4937 μM [5039 μM [32660 μM (−cGMP) [19]; 500 μM (+cGMP) [19nr nr 90 μM [27Not a substrate 
pH, cyclase 6–8 [164–6.5 [376–9 [166.8 [17]; 7 [486–7 [49nr na 7–8, [197–8 [18,19nr nr na 
pH, base-exchange 4, optimal; >7, nil [6,29 4–5, optimal; >7, active [16nr nr nr nr 5, optimal; >7, active [195, optimal; >7, active [18,194, active [33nr 5, optimal; >7, active 
Km NGD 1 μM [23]; 2 μM [29 2.3 μM [23nr Not a substrate [49nr 23 μM [32  nr nr Not a substrate 
cAMP nr nr nr nr nr nr nr No effect [18No effect [19]; activates base exchange [18nr nr nr 
cGMP nr nr nr nr nr nr nr Activates cyclase [18,19]; no effect on base exchange [18,19No effect [18 nr nr 
Nitric oxide S-nitrosylation [54nr nr nr nr nr nr nr nr nr nr nr 
Cyclase             
 NAD to cADPR Yes [16Yes [37Yes [16Yes [17,48Yes [49Yes [50Minimal [32Yes [18,19Yes [18,19Yes [33Yes [27No 
 NADP tocADPR-P No [16,34nr Yes [16No [17nr nr nr nr nr nr nr No 
 NGD to cGDPR Yes [23nr Yes [23No [17No [49Yes [50Yes [32Yes [18,19Yes [18,19Yes [33nr No 
Glycohydrolase             
 NAD to ADPR Yes [16nr Yes [16Yes [17Yes [49nr Yes [32nr nr nr Yes [27Minimal 
 NADP to ADPR-P Yes [16,34nr Yes [16nr nr nr Yes [32nr nr nr nr Yes 
 NGD to GDPR Weak [23nr No [23nr nr Yes [50Yes [32nr nr nr nr No 
Base exchange             
 NADP+nicotinicacid to NAADP Yes [16nr Yes [16nr nr nr Yes [32Yes [18,19Yes [18,19Yes [33nr Yes 
Hydrolase             
 cADPR to ADPR Yes [16]; yes [34Yes [37No [23Yes [17,48nr nr nr Yes [18,19Yes [18,19nr Yes [27No 
 cADPR-P to ADPR-P No [16]; no [34nr nr nr nr nr nr nr nr nr nr No 
 cGDPR to GDPR No [23nr No [23nr nr Yes [50nr nr nr nr nr No 
 Enzyme or enzymatic activity 
Parameter CD38 (external) [16,47CD157 (external) [37Aplysia cyclase (luminal) [16Human sperm [17,48Brain cyclase [49Vascular smooth muscle [50NACE, Schistosoma mansoni [32Sea urchin egg, soluble (internal) [18,19Sea urchin egg, membrane-bound (internal) [18,19Sea urchin, soluble (ER, lumen) [33Sea urchin sperm Sea urchin sperm surface NAADP synthase (external) 
Zn2+ Activates [51]; inhibits hydrolase [52Activates [37Activates [50Switches cyclase/ glycohydrolase activity [17Inhibits [49Inhibits [50nr nr Increase [50nr nr nr 
Cu2+ Activates [50,51Inhibits mM [37nr No effect [17Complete inhibition, 10 μM [49Inhibits [50nr nr nr nr nr nr 
Ca2+ No effect [51No effect No effect No effect [17,48No effect [49nr nr nr nr nr nr Low, activates; high, inactivates 
Km NADP 1 mM [53nr nr nr nr 14 μM [32200 μM [18,190.2 mM [19]; 0.4 mM [18nr nr 110 μM  
Km NA 5 mM [16nr ∼10 mM [16nr nr nr nr 10 mM [18,1910 mM [19]; 9 mM [18nr nr 4 mM 
Km NAD 15 μM [23,29]; 200 μM [17nr 39 μM [16150 μM [17]; 8 μM [4821 μM [4937 μM [5039 μM [32660 μM (−cGMP) [19]; 500 μM (+cGMP) [19nr nr 90 μM [27Not a substrate 
pH, cyclase 6–8 [164–6.5 [376–9 [166.8 [17]; 7 [486–7 [49nr na 7–8, [197–8 [18,19nr nr na 
pH, base-exchange 4, optimal; >7, nil [6,29 4–5, optimal; >7, active [16nr nr nr nr 5, optimal; >7, active [195, optimal; >7, active [18,194, active [33nr 5, optimal; >7, active 
Km NGD 1 μM [23]; 2 μM [29 2.3 μM [23nr Not a substrate [49nr 23 μM [32  nr nr Not a substrate 
cAMP nr nr nr nr nr nr nr No effect [18No effect [19]; activates base exchange [18nr nr nr 
cGMP nr nr nr nr nr nr nr Activates cyclase [18,19]; no effect on base exchange [18,19No effect [18 nr nr 
Nitric oxide S-nitrosylation [54nr nr nr nr nr nr nr nr nr nr nr 
Cyclase             
 NAD to cADPR Yes [16Yes [37Yes [16Yes [17,48Yes [49Yes [50Minimal [32Yes [18,19Yes [18,19Yes [33Yes [27No 
 NADP tocADPR-P No [16,34nr Yes [16No [17nr nr nr nr nr nr nr No 
 NGD to cGDPR Yes [23nr Yes [23No [17No [49Yes [50Yes [32Yes [18,19Yes [18,19Yes [33nr No 
Glycohydrolase             
 NAD to ADPR Yes [16nr Yes [16Yes [17Yes [49nr Yes [32nr nr nr Yes [27Minimal 
 NADP to ADPR-P Yes [16,34nr Yes [16nr nr nr Yes [32nr nr nr nr Yes 
 NGD to GDPR Weak [23nr No [23nr nr Yes [50Yes [32nr nr nr nr No 
Base exchange             
 NADP+nicotinicacid to NAADP Yes [16nr Yes [16nr nr nr Yes [32Yes [18,19Yes [18,19Yes [33nr Yes 
Hydrolase             
 cADPR to ADPR Yes [16]; yes [34Yes [37No [23Yes [17,48nr nr nr Yes [18,19Yes [18,19nr Yes [27No 
 cADPR-P to ADPR-P No [16]; no [34nr nr nr nr nr nr nr nr nr nr No 
 cGDPR to GDPR No [23nr No [23nr nr Yes [50nr nr nr nr nr No 

We examined both substrate selectivity and the range of many enzymatic activities possible with cyclases, specific to our enzyme present on the surface of sperm. Sperm readily produced NAADP by base exchange (Figure 3G; 1.72±0.42 pmol/min per mg of protein), but failed to produce detectable cADPR-P (cADPR phosphate) (Figure 3A) or cADPR (Figure 3B). A predominance of base exchange over cyclase activity is unusual and limited to NACE [NAD(P)+-catabolizing enzyme], a cyclase present in Schistosoma mansoni [32]. NACE itself does cyclize the NAD analogue NGD to cGDPR, which is hydrolysis-resistant and fluorescent (300 nm excitation and 410 nm emission) [23]. In contrast, incubation of sperm with NGD did not produce detectable cGDPR (Figure 3H), whereas spiking the same sample with authentic ADP-ribosyl cyclase from Aplysia showed production of cGDPR. The inability of the sea urchin enzyme to use NGD as a substrate proves that it is distinct from NACE. That NGD is not a substrate also suggests that the enzyme activity that we have observed is not that of the sea urchin ADP-ribosyl cyclase isoform 1 [SpARC1 (Strongylocentrotus purpuratus ADP-ribosyl cyclase 1)], which was characterized recently [33]. Sperm exhibited pronounced glycohydrolase activity when incubated with NADP (Figure 3A). However, this activity was minimal when incubated with NAD (Figure 3B; 1.2%). With regard to hydrolase activity, the sperm surface enzyme did not convert cADPR into ADPR (ADP-ribose) (Figure 3C), cADPR-P into ADPR-P (ADPR phosphate) (Figure 3D) or NAADP into ADPR-P (Figure 3E). That the sperm enzyme could convert a small amount of NAD into ADPR, but could not produce any cADPR (Figure 3B), or hydrolyse cADPR to ADPR (Figure 3C), indicates that the surface enzyme does not exhibit any cyclase activity. These results also demonstrate that both the base-exchange and glycohydrolase reactions exhibit selectivity for NADP, probably reflecting a requirement for both nicotinamide base and a 2′-phosphate group. Taken together, these data demonstrate that the sperm surface enzyme is unlike any previously characterized ADP-ribosyl cyclase (Table 1) [32]. It is important to note that classical NADase enzymes, such as the NADase from Neurospora, do not catalyse base exchange (Figure 3F). Similarly, CD38 only hydrolyses NADP, it does not cyclize it to cADPR-P [16,34]. As sperm did not show detectable cyclase activity (Figures 3A and 3B), enzymatically this enzyme is not a cyclase; therefore we suggest that it is more correctly termed an NAADP synthase.

The base-exchange enzyme on the surface of sperm shows unique substrate selectivity

Figure 3
The base-exchange enzyme on the surface of sperm shows unique substrate selectivity

All traces show HPLC separation of substrates and products (as labelled). (A, B) Determination of cyclase and glycohydrolase activity using the endogenous substrates NADP and NAD. The inset in (B) shows a magnified view of the baseline to demonstrate the absence of cADPR, which is eluted between NAD and ADPR. (CE) Determination of hydrolase activity assessed with cADPR, cADPR-P and NAADP. The sharp peak preceding and distorting the cADPR peak is due to the presence of a small amount of NAD. (F, G) Determination of base-exchange activity of Neurospora NADase and sea urchin sperm. (H) Determination of cyclase activity of sperm and Aplysia ADP-ribosyl cyclase using NGD. The product of NGD cyclization is cGDPR, which is fluorescent and hydrolysis-resistant. The trace shows a sample incubated in a fluorimeter with excitation at 300 nm and emission at 410 nm, with slit widths of 5 nm. Sperm was added at 10% (v/v). Aplysia ADP-ribosyl cyclase was added at 100 μg/ml. Abs, absorbance; NiAM, nicotinamide.

Figure 3
The base-exchange enzyme on the surface of sperm shows unique substrate selectivity

All traces show HPLC separation of substrates and products (as labelled). (A, B) Determination of cyclase and glycohydrolase activity using the endogenous substrates NADP and NAD. The inset in (B) shows a magnified view of the baseline to demonstrate the absence of cADPR, which is eluted between NAD and ADPR. (CE) Determination of hydrolase activity assessed with cADPR, cADPR-P and NAADP. The sharp peak preceding and distorting the cADPR peak is due to the presence of a small amount of NAD. (F, G) Determination of base-exchange activity of Neurospora NADase and sea urchin sperm. (H) Determination of cyclase activity of sperm and Aplysia ADP-ribosyl cyclase using NGD. The product of NGD cyclization is cGDPR, which is fluorescent and hydrolysis-resistant. The trace shows a sample incubated in a fluorimeter with excitation at 300 nm and emission at 410 nm, with slit widths of 5 nm. Sperm was added at 10% (v/v). Aplysia ADP-ribosyl cyclase was added at 100 μg/ml. Abs, absorbance; NiAM, nicotinamide.

We next established the Km and the Vmax of NAADP synthase. Plots of initial velocity against nicotinic acid concentration (with 10 mM NADP) (Figure 4A) and initial velocity against NADP concentration (with 100 mM nicotinic acid) (Figure 4B) revealed a rectangular hyperbola that fit the Michaelis–Menten equation by non-linear regression. Such a plot is consistent with a single binding site and no co-operativity. For ease of presentation of these data, double-reciprocal plots are also given for nicotinic acid (Figure 4C) and NADP (Figure 4D). For nicotinic acid, the Km was 2.8 mM and the Vmax was 5.3 pmol/min per mg of protein (Figure 4A), whereas, for NADP, the Km was 111 μM and the Vmax was 5.6 pmol/min per mg of protein (Figure 4B). These values are different from those of all other characterized ADP-ribosyl cyclases which exhibit Km values in the region of 200 μM for NADP and 10 mM for nicotinic acid [19] (Table 1). Note that our enzyme kinetics measurements were not confounded by degradation of NAADP, as the sperm did not significantly metabolize NAADP over the time course of the assays (Figure 3E). Overall, the enzyme kinetic parameters provide additional support for the novelty of this enzyme.

Base-exchange activity of the sperm surface enzyme shows Michaelis–Menten kinetics

Figure 4
Base-exchange activity of the sperm surface enzyme shows Michaelis–Menten kinetics

(A, B) Linear–linear plots of concentration of nicotinic acid (A) or NADP (B) substrate against initial velocity. (C, D) Double-reciprocal (Lineweaver–Burk) plots of concentration of nicotinic acid (C) or NADP (D) substrate and initial velocity.

Figure 4
Base-exchange activity of the sperm surface enzyme shows Michaelis–Menten kinetics

(A, B) Linear–linear plots of concentration of nicotinic acid (A) or NADP (B) substrate against initial velocity. (C, D) Double-reciprocal (Lineweaver–Burk) plots of concentration of nicotinic acid (C) or NADP (D) substrate and initial velocity.

ADP-ribosyl cyclase activity is highly sensitive to pH, with acidic pH favouring base exchange due to the protonation state of a critical aspartate residue at the active site [35]. The base-exchange activity of the sperm surface enzyme was optimal at pH 5 (Figure 5A), as is the case with other members of the cyclase family [16] (Table 1). Other ADP-ribosyl cyclases, such as CD38, have almost no base-exchange activity at pH 7 and can only catalyse the hydrolysis of NADP to ADPR-P [16]. The only exception is Aplysia cyclase, which shows a small amount of base-exchange activity [16]. Surprisingly, at pH 7, the sperm surface enzyme was capable of producing NAADP by base exchange at 25% of its pH optimum (1.8±0.23 pmol/min per mg of protein at pH 7 compared with 7.1±0.34 pmol/min per mg of protein at pH 5). With regard to pH regulation, with the exception of Aplysia cyclase, the sperm base-exchange enzyme differs from all other members of the ADP-ribosyl cyclase family.

Synthesis of NAADP by base exchange is regulated by pH and Ca2+, but not by Mg2+

Figure 5
Synthesis of NAADP by base exchange is regulated by pH and Ca2+, but not by Mg2+

(A) Effect of pH on the initial velocity of the base-exchange reaction. Results are means±S.E.M. (n=3). (B, C) Effect of Ca2+ (B) or Mg2+ (C) on the initial velocity of NAADP synthesis. All the reaction mixtures contained 1 mM NADP and 10 mM nicotinic acid (pH 7) at 21 °C and were started with the addition of ∼20 μl of sperm (∼1 mg of protein/ml). Results are means±S.E.M. (n=3 or 4).

Figure 5
Synthesis of NAADP by base exchange is regulated by pH and Ca2+, but not by Mg2+

(A) Effect of pH on the initial velocity of the base-exchange reaction. Results are means±S.E.M. (n=3). (B, C) Effect of Ca2+ (B) or Mg2+ (C) on the initial velocity of NAADP synthesis. All the reaction mixtures contained 1 mM NADP and 10 mM nicotinic acid (pH 7) at 21 °C and were started with the addition of ∼20 μl of sperm (∼1 mg of protein/ml). Results are means±S.E.M. (n=3 or 4).

Previously, it has been shown that ADP-ribosyl cyclase enzymes are modulated by cations (Table 1). We studied the possible effect of Ca2+ on the base-exchange enzyme. Sperm were incubated in solutions containing different amounts of free Ca2+ buffered with EGTA [24]. The response to Ca2+ concentration was bell-shaped: as the concentration of Ca2+ was increased, NAADP production was stimulated with an EC50 of 10 nM (Figure 5B) and then inhibited with an IC50 of 50 μM (Figure 5B). This regulation is selective for Ca2+, because Mg2+ did not alter NAADP production (Figure 5C). Regulation by Ca2+ is unique to the sperm surface enzyme, as all other ADP-ribosyl cyclases have been shown to be insensitive to Ca2+ [31,36,37] (Table 1). Remarkably, the Ca2+ stimulation occurred at pH 7 and elevated the Vmax to about half that (Figure 5B) obtained by lowering the pH to 5 (Figure 5C). Taken together, the data suggest that either Ca2+ or pH can serve as an endogenous regulator of NAADP synthesis.

Other Ca2+-releasing pathways have several steps that respond to positive feedback by Ca2+; the NAADP-mediated pathway also appears to have positive feedback, but at different stages of the signalling cascade. For example, both ryanodine receptors and IP3 receptors have feedback at the level of the channel enabling Ca2+-induced Ca2+ release [38], and the IP3 pathway is stimulated by Ca2+ acting on phospholipase Cδ1 [39]. As NAADP-mediated Ca2+ release is insensitive to Ca2+ [40], Ca2+ feedback appears to occur exclusively at the enzymatic steps for synthesis (Figure 4) and metabolism [41], which may underlie NAADP-mediated Ca2+ oscillations [11,41].

Considering that many of the other enzymes responsible for producing NAADP and cADPR are surface-bound or in the lumen of organelles [33,42,43], the location of this enzyme may not be a great surprise. Indeed, a recent report of the molecular identity of the urchin ADP-ribosyl cyclases suggests that the enzyme is functional within the lumen of the ER [33]. Combining the conclusions from the study by Churamani et al. [33] with our present results leads us to propose a mechanism for the regulation of NAADP synthase by Ca2+ and pH over concentration ranges that are physiologically relevant when put into the context of the events occurring during fertilization (Figure 6). At fertilization, the sperm and egg fuse and their plasma membranes become contiguous [44]. Subsequently, the sperm contents are engulfed by the egg, and the plasma membrane of the sperm is endocytosed (Figure 6) [44]. It would be particularly informative to evaluate the possible locations of the NAADP synthase within the fertilized egg with regard to the physiological pH and Ca2+ concentrations. In terms of regulatory control, the most effective location for NAADP synthase would be in the lumen of vesicles (Figure 6). A luminal location is consistent with location of the sea urchin egg ADP-ribosyl cyclase 1C, which is present and active in the lumen of the ER when expressed in Xenopus eggs [33].

Possible physiological significance of a Ca2+-regulated NAADP synthase

Figure 6
Possible physiological significance of a Ca2+-regulated NAADP synthase

The two graphs show the dose–response curves for modulation of the NAADP synthase by Ca2+ and pH. The horizontal bars show the environmental and physiological concentrations of Ca2+ and protons. The cartoon highlights sperm–egg fusion and the possible subsequent events that would modulate NAADP synthase activity. 1. NAADP synthase enzyme (E) on the surface of the sperm would be inactive in sea water. 2. Sperm–egg fusion and mixing of their plasma membranes would not activate the enzyme. 3. Endocytosis of the NAADP synthase enzyme would make it luminal and expose it to Ca2+ concentrations in the range capable of regulating its activity. 5. Loss of endosome membrane integrity would expose the enzyme to cytosolic Ca2+ fluctuations. Taken together, it is most reasonable that the NAADP synthase would be found in the lumen, as this would enable regulation.

Figure 6
Possible physiological significance of a Ca2+-regulated NAADP synthase

The two graphs show the dose–response curves for modulation of the NAADP synthase by Ca2+ and pH. The horizontal bars show the environmental and physiological concentrations of Ca2+ and protons. The cartoon highlights sperm–egg fusion and the possible subsequent events that would modulate NAADP synthase activity. 1. NAADP synthase enzyme (E) on the surface of the sperm would be inactive in sea water. 2. Sperm–egg fusion and mixing of their plasma membranes would not activate the enzyme. 3. Endocytosis of the NAADP synthase enzyme would make it luminal and expose it to Ca2+ concentrations in the range capable of regulating its activity. 5. Loss of endosome membrane integrity would expose the enzyme to cytosolic Ca2+ fluctuations. Taken together, it is most reasonable that the NAADP synthase would be found in the lumen, as this would enable regulation.

If NAADP synthase is widely distributed in many tissues and species, the results from the present study will have far-reaching importance, as this synthase may be the physiologically relevant enzyme that is responsible for agonist-induced NAADP increases. Currently, although all ADP-ribosyl cyclases can make NAADP by base exchange, there is no direct evidence that this is the physiologically relevant pathway [45,46]. Such base-exchange activity may be nothing more than a consequence of a covalent intermediate production in the cyclization reaction mechanism [20,29]. Indeed, on the basis of experiments with CD38-knockout mice, agonist-induced NAADP increases are unaffected, demonstrating that CD38 is of questionable importance for NAADP synthesis [45,46].

In summary, we have uncovered an enzymatic activity that not only can synthesize NAADP by base exchange, but also is unique in its regulation by Ca2+ and selectivity for NADP. Unlike all previous enzymes that can synthesize NAADP, which are all members of the family of enzymes termed ADP-ribosyl cyclases, this new enzyme is regulated by physiologically relevant factors and shows no detectable cyclase activity, making it more appropriately termed an NAADP synthase.

This work was supported by grants from The Royal Society and the Wellcome Trust. S. R. V. was supported by a studentship from the Department of Pharmacology, University of Oxford.

Abbreviations

     
  • ADPR

    ADP-ribose

  •  
  • ADPR-P

    ADPR phosphate

  •  
  • cADPR

    cADP-ribose

  •  
  • cADPR-P

    cADPR phosphate

  •  
  • cGDPR

    cGDP-ribose

  •  
  • ER

    endoplasmic reticulum

  •  
  • IP3

    inositol 1,4,5-trisphosphate

  •  
  • NAADP

    nicotinic acid–adenine dinucleotide phosphate

  •  
  • NACE

    NAD(P)+-catabolizing enzyme

  •  
  • NGD

    nicotinamide guanine dinucleotide

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