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. 2008 Apr 1;411(1):63-70.
doi: 10.1042/BJ20071616.

Sperm express a Ca2+-regulated NAADP synthase

Sridhar R Vasudevan  1 , Antony Galione, Grant C Churchill
Affiliations

Sperm express a Ca2+-regulated NAADP synthase

Sridhar R Vasudevan et al. Biochem J. .

Abstract

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 (EC(50) of 10 nM and IC(50) of 50 microM) 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.

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Figures

Figure 1
Figure 1
Enzymes in the ADP-ribosyl cyclase family are multifunctional and catalyze 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 2
Figure 2
Sperm exhibit base-exchange activity on their surface. (A) High performance liquid chromatography trace of the substrates (NADP and nicotinic acid) before incubation with sperm. Note that ADPR-phosphate (ADPR-P) is an impurity in commercial NADP. (B) Permeabilized sperm synthesize NAADP by base exchange. High performance liquid chromatography trace taken one hour after incubation of digitonin (50 μM)-permeabllized 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. (E) Intact sperm shows comparable amounts of base-exchange activity to digitonin-permeabilized and freeze-thawed sperm. Data are mean ± standard error of the mean, n=3. (F) Sperm heads and tails can be 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. (G) Both sperm heads and tails exhibit base-exchange activity. Note that the headpiece of the sperm remains with the tail with this procedure. Data are mean ± standard error of the mean, n=3.
Figure 3
Figure 3
The base-exchange enzyme on the surface of sperm shows unique substrate selectivity. All traces show high performance liquid chromatography separation of substrates and products (as labelled). (A, B) Determination of cyclase and glycohydrolase activity using the endogenous substrates NADP and NAD. Inset shows a magnified view of the baseline to demonstrate the absence of cADPR, which elutes between NAD and ADPR.(C, D, E) Determination of hydrolase activity assessed with cADPR, cADPR-P and NAADP. The sharp peak preceding and distorting the cADPR peak is due to a small presence of NAD. (F, G) Determination of base-exchange activity of Neruospora 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 cyclic GDPR, which is fluorescent and hydrolysis resistant. The trace shows a sample incubated in a fluorimeter with excitation of 300 nm and emission of 410 nm, with slit widths of 5 nm. Sperm was added at 10 percent (volume/volume). Aplysia ADP-ribosyl cyclase was added at 100 μg/mL.
Figure 4
Figure 4
Base-exchange activity of the sperm surface enzyme shows Michaelis-Menten kinetics. (A, B) Linear-linear plots of substrate concentration versus initial velocity. (C, D) Double-reciprocal (Lineweaver-Burke) plots of substrate concentration and initial velocity.
Figure 5
Figure 5
Synthesis of NAADP by base exchange is regulated by pH and Ca2+ but not Mg2+. (A) Effect of pH on the initial velocity of the base-exchange reaction. Results shown are mean ± standard error of the mean, n=3. (A, B) Effect of Ca2+ or Mg2+ on the initial velocity of NAADP synthesis. All the reactions contained 1 mM NADP, 10 mM nicotinic acid, pH 7, 21°C and were started with the addition of ∼20 μL sperm (∼1 mg protein/mL). Results shown are mean ± standard error of the mean, n=3-4.
Figure 6
Figure 6
Possible physiological significance of a Ca2+-regulated NAADP synthase. The two inset 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 fund in the lumen, as this would enable regulation.
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