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.     

Cyclic ADP-ribose as a universal calcium signal molecule in the nervous system

beta-NAD(+) is as abundant as ATP in neuronal cells. beta-NAD(+) functions not only as a coenzyme but also as a substrate. beta-NAD(+)-utilizing enzymes are involved in signal transduction. We focus on ADP-ribosyl cyclase/CD38 which synthesizes cyclic ADP-ribose (cADPR), a universal Ca(2+) mobilizer from intracellular stores, from beta-NAD(+). cADPR acts through activation/modulation of ryanodine receptor Ca(2+) releasing Ca(2+) channels. cADPR synthesis in neuronal cells is stimulated or modulated via different pathways and various factors. Subtype-specific coupling of various neurotransmitter receptors with ADP-ribosyl cyclase confirms the involvement of the enzyme in signal transduction in neurons and glial cells. Moreover, cADPR/CD38 is critical in oxytocin release from the hypothalamic cell dendrites and nerve terminals in the posterior pituitary. Therefore, it is possible that pharmacological manipulation of intracellular cADPR levels through ADP-ribosyl cyclase activity or synthetic cADPR analogues may provide new therapeutic opportunities for treatment of neurodevelopmental disorders.     

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.     

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.     

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.     

The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.     

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme: active site H103W mutation restores ADP-ribosyl cyclase activity

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes that are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change, we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products (i.e., a value larger than observed for other members of this family such as CD38). Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provides new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge, however, in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR.     

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.     

Metabolism of cyclic ADP-ribose: Zinc is an endogenous modulator of the cyclase/NAD glycohydrolase ratio of a CD38-like enzyme from human seminal fluid

CD38, a bifunctional enzyme capable of both synthesis and hydrolysis of the second messenger cyclic ADP-ribose (cADPR). Using the natural substrate of the enzyme, NAD+, the ratio of ADP-ribosyl cyclase/NAD glycohydrolase of CD38 is about 1/100. Here we describe that human seminal fluid contain a soluble CD38 like enzyme with an apparent M.W. of 49 kDa. When purified this enzyme has a cyclase/NAD glycohydrolase ratio of about 1/120. However, the in situ cyclase/NAD glycohydrolase ratio measured in seminal plasma approaches 1/1. We also found that physiological concentrations of zinc present in the seminal fluid, in the range of 0.6 to 4 mM, are responsible for the modulation of the cyclase/NAD glycohydrolase ratio. This new information indicates that the cyclase/NAD glycohydrolase ratio can be modified in vivo.     

Adenoviral mediated anti-sense CD38 attenuates TNF-alpha-induced changes in calcium homeostasis of human airway smooth muscle cells

CD38 is a membrane-bound protein involved in the synthesis and degradation of cyclic-ADP-ribose (cADPR). cADPR mobilizes calcium from intracellular stores in airway smooth muscle cells. To determine the role of CD38/cADPR signaling in calcium regulation in human airway smooth muscle (HASM) cells, we downregulated CD38 expression using a recombinant replication-defective adenovirus with anti-sense human CD38 (Ad-asCD38). CD38 expression was determined by RT-PCR and real-time quantitative PCR, and ADP-ribosyl cyclase (cyclase) activity was determined by competitive binding assay. In HASM cells infected with Ad-asCD38, TNF-alpha-induced, augmented-CD38 expression and cyclase activity were significantly lower than in TNF-alpha-treated cells. The net intracellular calcium responses to 10 nmol/L bradykinin were measured in HASM cells by fluorescence imaging. In cells infected with Ad-asCD38 in the presence of TNF-alpha, the net intracellular Ca2+ responses were significantly lower than in cells treated with TNF-alpha in the presence of the control vector (p < 0.001). These results provide evidence for the feasibility of using adenoviral vectors for gene transfer to down regulate gene expression, and confirm the role of CD38 in calcium homeostatis in ASM cells.     

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway Wei W,Graeff R,Yue J简

Mobilization of intracellular Ca²⁺ stores is involved in many diverse cell functions, including: cell proliferation; differentiation; fertilization; muscle contraction; secretion of neurotransmitters, hormones and enzymes; and lymphocyte activation and proliferation. Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca²⁺ mobilizing nucleotide present in many cell types and species, from plants to animals. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide. The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. It has been shown that many extracellular stimuli can induce cADPR production that leads to calcium release or influx, establishing cADPR as a second messenger. cADPR has been linked to a wide variety of cellular processes, but the molecular mechanisms regarding cADPR signaling remain elusive. The aim of this review is to summarize the CD38/cADPR/Ca²⁺ signaling pathway, focusing on the recent advances involving the mechanism and physiological functions of cADPR-mediated Ca²⁺ mobilization.     

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.     

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.     

Antidiabetic effect of a prodrug of cysteine, L-2-oxothiazolidine-4-carboxylic acid, through CD38 dimerization and internalization.

CD38 is a bifunctional enzyme synthesizing (ADP-ribosyl cyclase) and degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a potent Ca(2+) mobilizer from intracellular pools. CD38 internalization has been proposed as a mechanism by which the ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce the internalization of CD38. Here, we show that the disulfide bond between Cys-119 and Cys-201 in CD38 may be involved in CD38 dimerization and internalization. We tested the effect of a reducing agent, l-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, on CD38 internalization in pancreatic islets. OTC enhanced insulin release from isolated islets as well as CD38 internalization and cytoplasmic Ca(2+) level. Furthermore, islet cells treated with antisense CD38 oligonucleotide showed inhibition of OTC-induced insulin secretion. Intake of OTC in db/db mice ameliorated glucose tolerance, insulin secretion, and morphology of islets when compared with control mice. These data indicate that OTC improves glucose tolerance by enhancing insulin secretion via CD38/cADPR/Ca(2+) signaling machinery. Thus, OTC may represent a novel class of antidiabetic drug.     

Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts

CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.     

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.     

Ectocellular CD38-catalyzed synthesis and intracellular Ca2+-mobilizing activity of cyclic ADP-ribose

CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD⁺ to nicotinamide and cyclic ADP-ribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca²⁺-mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca²⁺ response to KCl-induced depolarization, a process that can be qualified as a Ca²⁺-induced Ca²⁺ release (CICR) mechanism. On the other hand, in the CD38⁺ human Namalwa B lymphoid cells, NAD⁺ (and thiol compounds as well) induced a two-step process of self-aggregation followed by endocytosis of CD38, which resulted in a shift of cADPR metabolism from the cell surface to the cytosol. Both distinctive types of cellular responses to extracellular NAD⁺ seem to be suitable to elicit changes in the intracellular Ca²⁺ homeostasis.     

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.     

Identification of the enzymatic active site of CD38 by site-directed mutagenesis

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.