Ectocellular CD38-catalyzed synthesis and intracellular Ca2+-mobilizing activity of cyclic ADP-ribose (original) (raw)
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FEBS Letters, 1998
Cyclic ADP-ribose (cADPR) is a natural metabolite of L L-NAD + with a potent Ca 2+ -mobilizing activity in different cell types, including T-lymphocytes. We investigated (i) whether stimulation of T-lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP-ribosyl cyclase and cADPR-hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca 2+ -mobilizing effects of this nucleotide in Jurkat and HPB.ALL T-lymphocytes. The anti-CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ). In contrast, activation of an ectocellular ADP-ribosyl cyclase by preincubation of cells with L L-NAD + led to a dose-dependent increase in cADPR, but no changes in [Ca 2+ ] i were observed. However, extensive washing of the cells following preincubation with NAD + demonstrated that the increases in cADPR were not intracellular but due to cell surface-associated nucleotide. Accordingly, measurements of ADP-ribosyl cyclase activity in intact T-cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP-ribosyl cyclase activity of CD38 on the regulation of the cADPR-mediated intracellular Ca 2+ -signalling in T-lymphocytes.
Journal of Biological Chemistry, 1997
CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD ؉ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD ؉ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD ؉ leading to the formation of a dimeric ADPR ((ADPR) 2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38 ؉ Namalwa cells from as low as 10 M NAD ؉ , was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR) 2 was identified as that between the N 1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me 2 SO:tert-butoxide. Although (ADPR) 2 itself did not release Ca 2؉ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca 2؉-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR) 2 is a new product of CD38 that amplifies the Ca 2؉-mobilizing activity of cADPR.
The FASEB Journal
CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38 ؉ cells with either NAD ؉ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR-generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD ؉ , or GSH, or N-acetylcysteine. This effect of CD38-internalizing ligands on intracellular calcium levels was found to involve a two-step mechanism: 1) influx of cytosolic NAD ؉ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8-N 3 -NAD ؉ , and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD ؉ to cADPR, followed by outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigarginsensitive stores. This unknown intracellular trafficking of NAD ؉ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38 ؉ cells.-Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., Passalacqua, M., De Flora, A. Ligand-induced internalization of CD38 results in intracellular Ca 2؉ mobilization: role of NAD ؉ transport across cell membranes. FASEB J. 13, 273-283 (1999)
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999
CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38(+) cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that t...
Journal of Biological Chemistry, 2004
Native human HL-60 cells do not express CD38, a multifunctional ectoenzyme, which generates cyclic ADP-ribose (cADPR), a potent calcium mobilizer. However, when HL-60 cells are induced to differentiate to granulocytes by treatment with retinoic acid (RA), they express CD38 and accumulate cADPR. Both processes play a causal role in RA-induced differentiation. Other granulocyte differentiation-inducers, including dimethyl sulfoxide (Me 2 SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me 2 SO involves any changes in the cADPR/intracellular calcium ([Ca 2؉ ] i) signaling system and, specifically, whether Me 2 SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR. Semiquantitative polymerase chain reaction analysis of transcripts, binding of [ 3 H]nitrobenzylthioinosine (NBMPR) to intact cells, and influx experiments of extracellular cADPR (with selective inhibitors of NT as NBMPR or in specific conditions) were performed in native and Me 2 SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me 2 SOdifferentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me 2 SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca 2؉ ] i levels when grown in a transwell co-culture setting over CD38-transfected 3T3 fibroblasts where nanomolar cADPR concentrations are present in the medium. NBMPR inhibited both responses of Me 2 SO-induced cells. Thus, concentrative influx of extracellular cADPR across CNT3 and cs-csg NT could substitute in the absence of CD38 in eliciting cADPR-dependent [Ca 2؉ ] i increases in granulocyte-differentiated HL-60 cells, as well as in other CD38 ؊ cells. ADP-ribosyl cyclases play a major role in regulating intracellular calcium levels ([Ca 2ϩ ] i) 1 by converting NAD ϩ to nicotina
The FASEB Journal, 2002
CD38 is an ectocyclase that converts NAD ؉ to the Ca 2؉-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD ؉ 3cADPr conversion to cause cytosolic Ca 2؉ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD ؉ surrogate, NGD ؉ , to its fluorescent product, cGDPr (K m ϳ5.13 M). NAD ؉ also triggered a cytosolic Ca 2؉ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The ⌬ ؊49-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD ؉-induced cytosolic Ca 2؉ signal was preserved. Likewise, Ca 2؉ release persisted in cells transfected with the Myr-⌬ ؊49-CD38-EGFP or ⌬ ؊49-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca 2؉ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD ؉ production to cytosolic Ca 2؉ signaling.
Decreased cADPR and increased NAD+ in the Cd38−/− mouse
Biochemical and Biophysical Research Communications, 2006
CD38 is a type II glycoprotein that catalyzes the formation of cyclic ADP-ribose (cADPR), an intracellular calcium signalling molecule, from nicotinamide adenine dinucleotide (NAD + ). Using a modified version of the fluorimetric cycling assay for cADPR which reduces between-subject variability, we report significant decreases in brain and lung cADPR, which although similar to previously published values, showed much less individual variation. The reduced variation within each group suggests that the range of cADPR is narrower than previously thought, and that the regulatory mechanisms controlling these levels are more finely tuned. We also report significant increases in brain, lung, and kidney NAD + in the Cd38 À/À mouse, and provide the first experimental demonstration of the proximate relationship between CD38 and NAD + .