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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.

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.

Mice lacking CD38, an ectoenzyme that generates the calcium-mobilizing metabolite cADPR, make reduced T cell-dependent antibody responses. Despite the predicted role for CD38 in B cell activation, we find that CD38 regulates the migration of dendritic cell (DC) precursors from the blood to peripheral sites and controls the migration of mature DCs from sites of inflammation to lymph nodes. Thus, T cells are inefficiently primed in Cd38(-/-) mice, leading to poor humoral immune responses. We also show that CD38 and cADPR modulate calcium mobilization in chemokine-stimulated DCs and are required for the chemotaxis of immature and mature DCs to CCL2, CCL19, CCL21, and CXCL12. Therefore, CD38 regulates adaptive immunity by controlling chemokine receptor signaling in DCs.

CD38 is a transmembrane glycoprotein that functions as an ectoenzyme and as a receptor. Based on the structural similarity between CD38 and ADP-ribosyl cyclase from Aplysia californica, it was hypothesized that CD38 is expressed as a homodimer on the surface of cells. Indeed, CD38 dimers have been reported, however, the structural requirements for their stabilization on the plasma membrane are unknown. We demonstrate that the majority of CD38 is assembled as noncovalently associated homodimers on the surface of B cells. Analysis of CD38 mutants, expressed in Ba/F3 cells, revealed that truncation of the cytoplasmic region or mutation of a single amino acid within the alpha1-helix of CD38 decreased the stability of the CD38 homodimers when solubilized in detergent. Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were significantly reduced. Together, these results show that CD38 is expressed as noncovalently associated homodimers on the surface of murine B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells.

The lymphoid surface antigen CD38 is a NAD(+)-glycohydrolase that also catalyzes the transformation of NAD(+) into cyclic ADP-ribose, a calcium mobilizing second messenger. In addition, ligation of CD38 by antibodies triggers signaling in lymphoid cells. Since the cytoplasmic tail of CD38 is dispensable for this latter property, we have previously proposed that CD38-mediated receptor signal transduction might be regulated by its conformational state. We have now examined the molecular changes of this protein during its interaction with NAD(+) by measuring the intrinsic fluorescence of CD38. We have shown that addition of the substrate produced a dramatic decrease in the fluorescence of the catalytically active recombinant soluble ectodomain of murine CD38. Analysis of this event revealed that the catalytic cycle involves a state of the enzyme that is characterized by a low fluorescence which, upon substrate turnover, reverts to the initial high intrinsic fluorescence level. In contrast, non-hydrolyzable substrates trap CD38 in its altered low fluorescence state. Studies with the hydrophilic quencher potassium iodide revealed that the tryptophan residues that are mainly involved in the observed changes in fluorescence, are remote from the active site. Similar data were also obtained with human CD38, indicating that studies of intrinsic fluorescence will be useful in monitoring the transconformation of CD38 from different species. Together, these data demonstrate that CD38 undergoes a reversible conformational change after substrate binding, and suggest a mechanism by which this change could alter interactions with different cell-surface partners.