A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.     

Interaction of two classes of ADP-ribose transfer reactions in immune signaling

CD38 is a bifunctional ectoenzyme predominantly expressed on hematopoietic cells where its expression correlates with differentiation and proliferation. The two enzyme activities displayed by CD38 are an ADP-ribosyl cyclase and a cyclic adenosine diphosphate ribose (cADPR) hydrolase that catalyzes the synthesis and hydrolysis of cADPR. T lymphocytes can be induced to express CD38 when activated with antibodies against specific antigen receptors. If the activated T cells are then exposed with NAD, cell death by apoptosis occurs. During the exposure of activated T cells to NAD, the CD38 is modified by ecto-mono-ADP-ribosyltransferases (ecto-mono-ADPRTs) specific for cysteine and arginine residues. Arginine-ADP-ribosylation results in inactivation of both cyclase and hydrolase activities of CD38, whereas cysteine-ADP-ribosylation results only in the inhibition of the hydrolase activity. The arginine-ADP-ribosylation causes a decrease in intracellular cADPR and a subsequent decrease in Ca(2+) influx, resulting in apoptosis of the activated T cells. Our results suggest that the interaction of two classes of ecto-ADP-ribose transfer enzymes plays an important role in immune regulation by the selective induction of apoptosis in activated T cells and that cADPR mediated signaling is essential for the survival of activated T cells.     

Changes in CD38 expression and ADP-ribosyl cyclase activity in rat myometrium during pregnancy: influence of sex steroid hormones

Cyclic ADP-ribose (cADPR), synthesized by CD38, regulates intracellular calcium in uterine smooth muscle. CD38 is a transmembrane protein that has both ADP-ribosyl cyclase and cADPR hydrolase enzyme activities involved in cADPR metabolism. CD38 expression and its enzyme activities in uterine smooth muscle are regulated by estrogen. In the present study, we examined CD38 expression, its enzyme activities, and cADPR levels in myometrium obtained from rats at 14-17 days of gestation (preterm) and at parturition (term). CD38 expression, ADP-ribosyl cyclase activity, and cADPR levels were higher in uterine tissues obtained from term rats compared with that of preterm rats, while activity of cADPR hydrolase did not significantly change. In an effort to address whether changes in estrogen: progesterone ratio that occur during pregnancy account for the observed effects on CD38 expression and function, we determined the effect of different doses of progesterone in the presence of estrogen on CD38 expression and its enzyme activities in uterine smooth muscle obtained from ovariectomized rats. In myometrium obtained from ovariectomized rats, estrogen administration caused increased CD38 protein expression and ADP-ribosyl cyclase activity. The estrogen-induced increases in CD38 expression and ADP-ribosyl cyclase activity were inhibited by simultaneous administration of 10 or 20 mg of progesterone. These results indicate that the estrogen:progesterone ratio determines CD38 expression and ADP-ribosyl cyclase activity. These changes in CD38/cADPR pathway may contribute to increased uterine motility and onset of labor.     

CD38 in Bovine Lung: A Multicatalytic NADase

We report the kinetics and molecular properties of CD38 purified from bovine lung microsomal membranes after its solubilization with Triton X-100. The enzyme was found to be a novel member of a multicatalytic NAD⁺-glycohydrolase (NADase, EC 3.2.2.6). It was able to utilize NAD ⁺ in different ways, producing nicotinamide (Nam) and either adenosine diphosphoribose (ADPR, NADase activity) or cyclic ADPR (cADPR, cyclase activity); it also catalyzed the hydrolysis of cADPR to ADPR (cADPR, hydrolase activity). In addition, the enzyme catalyzed the pyridine base exchange reaction with conversion of NAD ⁺ into NAD analogues. These data are evidence that CD38 is involved in the regulation of both NAD⁺ and calcium-mobilizing agents, the concentration resulting in an essential enzyme that plays a key role in cellular energy and signal-transduction systems.     

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.     

Crystal structure of human CD38 extracellular domain

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.     

Nitric oxide inhibits ADP-ribosyl cyclase through a cGMP-independent pathway in airway smooth muscle

There is evidence for a role of cyclic ADP-ribose (cADPR) in intracellular Ca2+ regulation in smooth muscle. cADPR is synthesized and degraded by ADP-ribosyl cyclase and cADPR hydrolase, respectively, by a bifunctional protein, CD38. Nitric oxide (NO) inhibits intracellular Ca2+ mobilization in airway smooth muscle. The present study was designed to determine whether this inhibition is due to regulation of ADP-ribosyl cyclase and/or cADPR hydrolase activity. Sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine, NO donors, produced a concentration-dependent decrease in ADP-ribosyl cyclase, but not cADPR hydrolase, activity. The NO scavenger carboxy-PTIO prevented and reversed, and reduced glutathione prevented, the inhibition of ADP-ribosyl cyclase by SNP, suggesting S-nitrosylation by NO as a mechanism. N-ethylmaleimide, which covalently modifies protein sulfhydryl groups, making them incapable of nitrosylation, produced a marked inhibition of ADP-ribosyl cyclase, but not cADPR hydrolase, activity. SNP and N-ethylmaleimide significantly inhibited the ADP-ribosyl cyclase activity in recombinant human CD38 without affecting the cADPR hydrolase activity. These results provide a novel mechanism for differential regulation of CD38 by NO through a cGMP-independent pathway involving S-nitrosylation of thiols.     

Crystallographic studies on human BST-1/CD157 with ADP-ribosyl cyclase and NAD glycohydrolase activities

cADPR is the novel second messenger that elicits calcium release from intracellular calcium stores and works independently of IP(3). In mammals, the ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and BST-1/CD157. These enzymes, exposed extracellularly, bear cADPR hydrolase and NAD glycohydrolase activities. In spite of its functional importance, the structural basis of these enzymatic reactions remains elusive. We determined the crystal structures of the extracellular region of human BST-1 at atomic resolution in the free form and in complexes with five substrate analogues: nicotinamide, NMN, ATPgammaS, ethenoNADP, and ethenoNAD. The three-dimensional structural views of the reaction centre with these ligands revealed the mode of substrate binding and the catalytic mechanism of the multifunctional enzymatic reactions. In each catalytic cleft of the dimeric enzyme, substrates are recognized predominantly through van der Waals interactions with two tryptophan residues, and thereby the N-glycosidic bond of NAD is correctly exposed near a catalytic glutamate residue. Its carboxyl side-chain stabilizes the catalytic intermediate of the S(N)-1 type reaction. This conformation of the catalytic cleft also implies the mechanism of cyclization between the adenine base and the ribose. The three key residues are invariant among the sequences of BST-1, CD38, and Aplysia cyclase, and hence this substrate recognition mode and catalytic scheme appear to be common in the cyclase family.     

Structural Basis for Enzymatic Evolution from a Dedicated ADP-ribosyl Cyclase to a Multifunctional NAD Hydrolase

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.Cyclic ADP-ribose (cADPR)³ is a calcium messenger ubiquitous in mammals as well as in invertebrates and plants and is responsible for regulating many physiological processes ranging from the simple function of calcium channel operation to the complex higher level organization of hormone secretion and autism (reviewed in Lee (1), Schuber and Lund (2), and Malavasi et al. (3)). The enzymatic production of cADPR from the substrate nicotinamide adenine dinucleotide (NAD) requires first the removal of the nicotinamide moiety followed by a cyclization reaction in which both ends of the remaining nucleotide are annealed (Fig. 1A). ADP-ribosyl cyclase (ADPRC) from Aplysia california was the first enzyme discovered to possess this function (cyclase) (4). Based on sequence homology (5), two human antigens, CD38 and CD157, were identified to also have the cyclase activity (6–8). However, different from ADPRC, which produces only cADPR from NAD, CD38/CD157 has evolved more like an NADase, producing mainly ADP-ribose (ADPR) from NAD, with cADPR being a minor product. The acquisition of the NADase and the cADPR hydrolysis activities of CD38 make it an important signaling enzyme in regulating NAD and cADPR homeostasis (9–11). Genetic analysis, as well as the conservation of sequence and disulfide bonds among these enzymes, establish that they all evolved from a common ancestor (12). Little is known of why this conserved family of enzymes has evolved divergently in their catalytic metabolism of NAD and cADPR.Open in a separate windowFIGURE 1.Schemes of cADPR formation and mechanistic analogs for substrate and product. A, the cyclization reaction producing cADPR from NAD is catalyzed by both ADPRC and CD38. The structural difference between cADPR and N1-cIDPR lies at the 6-position of purine ring (6-NH for cADPR; 6-O for N1-cIDPR). B, an analog of the substrate NAD, N(2F-A)D, is enzymatically converted to 2F-ADPR by ADPRC instead of cyclized to c(2F-A)DPR. The formation of cADPR from NAD requires the intramolecular attack of the reaction intermediate by the adenine N1 atom. The addition of a fluorine atom on the adjacent C2 atom of adenine prevents the cyclization from occurring. C, ara-2′F-NAD and ribo-2′F-NAD are analogs of NAD that inhibit the cyclization reaction by producing covalent adducts during the catalysis by CD38. Both analogs differ only in the orientation of their fluorine atoms at the 2′-position of the adenine ribose.ADPRC, however, is not solely a cyclase because it can also catalyze the hydrolysis of NMN into ribose-5-phosphate and nicotinamide (13, 14). The catalytic outcome of this novel enzyme is thus determined not by the enzyme alone but also by the specific interactions between the active site and a particular substrate. Consistently, using an NAD analog, N(2F-A)D, as substrate, Zhang et al. (15) showed that the hydrolase activity of ADPRC can be dominantly revealed, whereas its cyclase activity is suppressed beyond detection (Fig. 1B). Likewise, human CD38 can be converted to a ADPRC-like enzyme by mutation of a single residue, Glu-146, at the active site (16). In this study, we determined the structural determinants critical for the catalytic characteristics of ADPRC and CD38 by comparing the crystal structures of the complexes of ADPRC and CD38 bound with various catalytically revealing substrates and products (Fig. 1, A–C). The results identify residues Phe-174 in the cyclase and Thr-221 in CD38 as the main determinants for the cyclase and hydrolysis activities of the enzymes. All together, these structures provide insights into the structural requirements for functional evolution from a cyclase to a hydrolase.     

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in hepatocyte nucleus

CD38 is a type II transmembrane glycoprotein found on both hematopoietic and non-hematopoietic cells. It is known for its involvement in the metabolism of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. It is generally believed that CD38 is an integral protein with ectoenzymatic activities found mainly on the plasma membrane. Here we show that enzymatically active CD38 is present intracellularly on the nuclear envelope of rat hepatocytes. CD38 isolated from rat liver nuclei possessed both ADP-ribosyl cyclase and NADase activity. Immunofluorescence studies on rat liver cryosections and isolated nuclei localized CD38 to the nuclear envelope of hepatocytes. Subcellular localization via immunoelectron microscopy showed that CD38 is located on the inner nuclear envelope. The isolated nuclei sequestered calcium in an ATP-dependent manner. cADPR elicited a rapid calcium release from the loaded nuclei, which was independent of inositol trisphosphate and was inhibited by 8-amino-cADPR, a specific antagonist of cADPR, and ryanodine. However, nicotinic acid adenine dinucleotide phosphate failed to elicit any calcium release from the nuclear calcium stores. The nuclear localization of CD38 shown in this study suggests a novel role of CD38 in intracellular calcium signaling for non-hematopoietic cells.     

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.     

NADP+-Dependent internalization of recombinant CD38 in CHO cells

CD38 is a 46-kDa type II transmembrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose (cADPR) from NAD+. cADPR is a second messenger known to regulate intracellular Ca2+-induced Ca2+-release (CICR). A recent study has revealed that CD38 in Namalwa B cells undergoes internalization upon exposure to external NAD+. In this study, recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells and the possibility of the protein to undergo internalization upon exposure to a substrate analog NADP+ was examined. It was found that such treatment of CHO cells resulted in a decrease of ADP-ribosyl cyclase activity, as well as immunofluorescence of CD38 on the cell surface. The same treatment of CHO cells also resulted in intracellular clustering of CD38 molecules as revealed by confocal microscopic analysis. The internalized CD38 was purified using a streptavidin/biotin-based method and was found to exhibit both ADP-ribosyl cyclase and cADPR hydrolase activities. On immunoblot, the internalized CD38 appeared as a monomer of 46 kDa under reducing condition of SDS-PAGE. Our data demonstrate that NADP+ can efficiently induce internalization of CD38, a process that may be important in the production of cADPR intracellularly to regulate CICR.     

Identification of a major enzyme for the synthesis and hydrolysis of cyclic ADP-ribose in amphibian cells and evolutional conservation of the enzyme from human to invertebrate

Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to function as a second messenger for intracellular Ca(2+) mobilization in various vertebrate and invertebrate tissues. In this study, we isolated two Xenopus laevis cDNAs (frog cd38 and cd157 cDNAs) homologous to the one encoding the human cADPR-metabolizing enzyme CD38. Frog CD38 and CD157 are 298-amino acid proteins with 35.9 and 27.2 % identity to human CD38 and CD157, respectively. Transfection of expression vectors for frog CD38 and CD157 into COS-7 cells revealed that frog CD38 had NAD(+) glycohydrolase, ADP-ribosyl cyclase (ARC), and cADPR hydrolase activities, and that frog CD157 had no enzymatic activity under physiological conditions. In addition, when recombinant CD38 and frog brain homogenate were electrophoresed on an SDS-polyacrylamide gel, ARC of the brain homogenate migrated to the same position in the gel as that of frog CD38, suggesting that frog CD38 is the major enzyme responsible for cADPR metabolism in amphibian cells. The frog cd38 gene consists of eight exons and is ubiquitously expressed in various tissues. These findings provide evidence for the existence of the CD38-cADPR signaling system in frog cells and suggest that the CD38-cADPR signaling system is conserved during vertebrate evolution.     

Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.     

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.     

Regulation of intracellular levels of NAD: a novel role for CD38

Nicotinamide adenine dinucleotide (NAD) plays key roles in many cellular functions. In addition to its well-known role in energy metabolism, NAD also plays a role in signal transduction, ageing, and cellular injury. NAD is also involved in many signal transduction pathways. Therefore, it is imperative to understand the mechanisms that control intracellular NAD levels. However, to date, the mechanisms that regulate intracellular levels of NAD have not been completely elucidated. CD38 is a multifunctional enzyme ubiquitously distributed in mammalian tissues. CD38 has been implicated as the enzyme responsible for the synthesis of the second messengers. However, its major enzymatic activity is the hydrolysis of NAD, in fact, CD38 will generate one molecule of cADPR for every 100 molecules of NAD hydrolyzed. To date, the role of CD38 as a modulator of levels of NAD has not been explored. We postulated that CD38 is the major NADase in mammalian cells and that it regulates intracellular NAD levels. In the current studies we examined the NADase activities and NAD levels in a variety of tissues from both wild-type and CD38 deficient mice. In accordance with our hypothesis, we found that tissue levels of NAD in CD38 deficient mice are 10- to 20-fold higher than in wild-type animals. In addition, NADase activity in the plasma membrane, mitochondria, sarcoplasmic reticulum, and nuclei is essentially absent in most tissues from CD38 deficient mice. These data support the novel concept that CD38 is a major regulator of cellular NAD levels. These findings have implications for understanding the mechanisms that regulate intracellular NAD levels and its role in energy homeostasis, signal transduction, and ageing.     

Catalysis-based inhibitors of the calcium signaling function of CD38

CD38 is a signaling enzyme responsible for catalyzing the synthesis of cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate; both are universal Ca(2+) messenger molecules. Ablation of the CD38 gene in mice causes multiple physiological defects, including impaired oxytocin release, that result in altered social behavior. A series of catalysis-based inhibitors of CD38 were designed and synthesized, starting with arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide. Structure-function relationships were analyzed to assess the structural determinants important for inhibiting the NADase activity of CD38. X-ray crystallography was used to reveal the covalent intermediates that were formed with the catalytic residue, Glu226. Metabolically stable analogues that were resistant to inactivation by phosphatase and esterase were synthesized and shown to be effective in inhibiting intracellular cADPR production in human HL-60 cells during induction of differentiation by retinoic acid. The inhibition was species-independent, and the analogues were similarly effective in blocking the cyclization reaction of CD38 in rat ventricular tissue extracts, as well as inhibiting the α-agonist-induced constriction in rat mesentery arteries. These compounds thus represent the first generally applicable and catalysis-based inhibitors of the Ca(2+) signaling function of CD38.     

Extracellular NAD(+) induces calcium signaling and apoptosis in human osteoblastic cells

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.     

Purification and characterization of ADP-ribosyl cyclase from Euglena gracilis

ADP-ribosyl cyclase, which catalyzes the conversion from NAD+ to cyclic adenosine diphosphoribose (cADPR), is proposed to participate in cell cycle regulation in Euglena gracilis. This enzyme, which was found as a membrane-bound protein, was purified almost the homogeneity after solubilization with deoxycholate, and found to be a monomeric protein with a molecular mass of 40 kDa. Its Km value for NAD+ was estimated to be 0.4 mM, and cADPR, a product of the enzyme, inhibited the enzyme competitively with respect to NAD+ whereas another product, nicotinamide, showed noncompetitive (mixed-type) inhibition. In contrast to mammalian CD38 and BST-1, Euglena ADP-ribosyl cyclase lacked cADPR hydrolase activity.