The transmembrane enzyme CD38, a multifunctional protein ubiquitously present in cells, is the main enzyme that synthesizes and hydrolyzes cyclic adenosine 5'-diphosphate-ribose (cADPR), an intracellular Ca(2+)-mobilizing messenger. CD38 is thought to be a type II transmembrane protein with its carboxyl-terminal catalytic domain located on the outside of the cell; thus, the mechanism by which CD38 metabolizes intracellular cADPR has been controversial. We developed specific antibodies against the amino-terminal segment of CD38 and showed that two opposing orientations of CD38, type II and type III (which has its catalytic domain inside the cell), were both present on the surface of HL-60 cells during retinoic acid-induced differentiation. When activated by interferon-γ, human primary monocytes and the monocytic U937 cell line exhibited a similar co-distribution pattern. Site-directed mutagenesis experiments showed that the membrane orientation of CD38 could be converted from a mixture of type II and type III orientations to all type III by mutating the cationic amino acid residues in the amino-terminal segment of CD38. Expression of type III CD38 construct in transfected cells led to increased intracellular concentrations of cADPR, indicating the importance of the type III orientation of CD38 to its Ca(2+) signaling function. The identification of these two forms of CD38 suggests that flipping the catalytic domain from the outside to the inside of the cell may be a mechanism regulating its signaling activity.

CD38, as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein, we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved, identifying three separate epitopes on the carboxyl domain. Chromobodies, engineered by tagging the nanobody with fluorescence proteins, provide fast, simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin, PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines, with half maximal effective concentration reaching as low as 10−11 molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma.

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.

Intracellular Ca2؉ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca 2؉ changes. Here we explored the role of one endogenous Ca 2؉ -mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca 2؉ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca 2؉ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells. Mobilization of intracellular Ca2ϩ stores is involved in diverse cell functions, including fertilization, cell proliferation, and differentiation (1-4). At least three endogenous Ca 2ϩ -mobilizing messengers have been identified, including inositol trisphosphate (IP 3 ), 3 nicotinic adenine acid dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Similar to IP 3 , cADPR can mobilize calcium release in a wide variety of cell types and species, from protozoa to animals. The cADPR-mediated Ca 2ϩ signaling has been indicated in a variety of cellular processes (5-7), from abscisic acid signaling and regulation of the circadian clock in plants, to mediating long-term synaptic depression in hippocampus.Ample evidence shows that the ryanodine receptors are the main intracellular targets for cADPR (1,2,8). Ryanodine receptors (RyRs) are intracellular Ca 2ϩ channels widely expressed in various cells and tissues, including muscles and neurons. It is the major cellular mediator of Ca 2ϩ -induced Ca 2ϩ release (CICR) in cells. There are three isoforms of ryanodine receptors: RyR1, RyR2, and RyR3, all of which have been implicated in the cADPR signaling (1, 2, 8). However, evidence regarding cADPR acting directly on the receptors is lacking (9). It has been suggested that accessory proteins, such as calmodulin and FK506-binding protein (FKBP), may be involved instead (10 -15).cADPR is formed from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases. Six ADP-ribosyl cyclases have been identified so far: Aplysia ADP-ribosyl cyclase, three sea urchin homologues (16,17), and two mammalian homologues, CD38 and CD157 (18). CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase. As a novel multifunctional enzyme, CD38 catalyzes the synthesis and hydrolysis of both cADPR and NAADP, two structurally and functionally distinct Ca 2ϩ messengers. Virtually all mammalian tissues ever examined have been shown to express CD38. CD38 knockout mice exhibit mult...

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also an enzyme catalyzing the synthesis of a Ca2+ messenger molecule, cyclic ADP-ribose, from NAD+. It is well established that this novel Ca2+ signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD+ complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1–14). A number of these compounds exhibited moderate inhibition of the NAD+ utilizing activity of CD38, with Compound 4 showing the higher potency. The crystal structure of CD38/ Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations form rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca 2؉ messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca 2؉ signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254 -Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism. Mobilization of Ca2ϩ from intracellular stores is of fundamental importance in virtually all aspects of cellular activity. The major intracellular Ca 2ϩ stores are in the endoplasmic reticulum (ER) 2 and are mobilized by specific messenger molecules, inositol trisphosphate and cyclic ADP-ribose (cADPR). The latter is a novel cyclic nucleotide derived from NAD. It was first described in sea urchin eggs (1, 2) but has since been established as a second messenger molecule responsible for regulating a wide range of physiological functions as diverse as abscisic acid signaling in plants (3) and sponges (4) and social behavior in mice (Ref. 5; reviewed in Refs. 6 and 7). It targets the ryanodine receptor of the endoplasmic Ca 2ϩ stores. The synthesis and hydrolysis of cADPR in mammalian cells are catalyzed by CD38 (8), a transmembrane protein ubiquitously expressed in virtually all tissues (reviewed in Ref. 9). Gene knock-out studies have established that CD38 plays a critical role in a wide range of physiological functions, including insulin secretion (10), susceptibility to bacterial infection (11), and social behavior of mice through modulating neuronal oxytocin secretion (5).CD38 is a membrane protein with a short N-terminal tail, a single transmembrane segment, and a large C-terminal domain containing all the enzymatic activities (8, 12, 13). The crystal structure of the catalytic domain of CD38 has been solved, and the mechanism of how it catalyzes the multiple reactions responsible for metabolizing cADPR has also been elucidated to atomic resolution by x-ray crystallography (14 -16). It is established that the s...

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