ADP-ribosyl Cyclase Research Articles

Self-Aggregation of the Transmembrane Glycoprotein CD38 Purified from Human Erythrocytes

The 46 kDa human transmembrane glycoprotein CD38 is a multicatalytic enzyme exhibiting ADPribosyl cyclase, cyclic ADPribose hydrolase and NAD+-glycohydrolase activities at its extracellular domain. When CD38, purified to homogeneity from human erythrocyte membranes, was incubated with NAD+ or β-mercaptoethanol, extensive aggregation took place. Addition of both compounds to CD38 led to the formation of still larger aggregates (over 300 nm), which were resistant to TCA precipitation. Extensive and stable CD38 self-aggregation was shown by, i) SDS-PAGE and autoradiography of the [38P]NAD+-incubated CD38, ii) SDS-PAGE followed by immunochemical detection of CD38 on the transblots, iii) direct electron microscopy on negatively stained CD38 samples. Self-aggregation of CD38 might be correlated with its putative function as a transducer of activation and proliferation signals in a number of hematopoietic cells.

Cyclic ADP-Ribose: Metabolism and Calcium Mobilizing Function

Publisher Summary This chapter describes the discovery of cyclic adenosine diphosphate–ribose (cADPR) as a novel endogenous Ca 2+ -mobilizing agent, and the way by which it fulfills most of the criteria necessary for it to be considered a second messenger. The enzymatic pathways for the synthesis and degradation of the metabolite are also summarized. The metabolic pathway of cADPR consists of synthesis from nicotinamide adenine dinucleotide (NAD + ) by ADP–ribosyl cyclase and degradation by the cADPR hydrolase to ADP-ribose. CD38-like bifunctional enzymes are responsible for regulating the cellular concentration of cADPR. CD38 is an ecto-enzyme catalyzing the synthesis and the degradation of cADPR raises the possibility that cADPR may have extracellular functions. The properties of its intracellular receptor and the mechanism of its Ca 2+ -mobilizing activity are discussed. The physiological roles of cADPR in two specific cellular systems are reviewed in the chapter: the sea urchin egg, an invertebrate cell; and the pancreatic β cell, a mammalian system.

Human lymphocyte antigen CD38 catalyzes the production of cyclic ADP-ribose

The human lymphocyte antigen CD38 has been shown to share sequence homology with ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of NAD + to cyclic ADP-ribose (cADPR), a potent Ca 2+-mobilizing agent. In this study COS1 cells from African Green Monkey kidney were transiently transfected with CD38 cDNA, inducing expression of authentic CD38 on the cell surface. We demonstrate that CD38 expressed in this manner can convert NAD + to cADPR in the extracellular medium as assessed by Ca 2+ release from sea-urchin egg microsomes.

A Single Protein Immunologically Identified as CD38 Displays NAD + Glycohydrolase, ADP-Ribosyl Cyclase and Cyclic ADP-Ribose Hydrolase Activities at the Outer Surface of Human Erythrocytes

The three ectoenzyme activities, NAD + glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase were purified to homogeneity from solubilized human erythrocyte membranes. The purification procedure involved three sequential chromatography steps on hydroxylapatite, immobilized Cu ++ and immobilized anti-CD38 monoclonal antibody resins. The final step yielded a single 46 kDa protein displaying all three enzymatic activities. Since the protein bound specifically to the anti-CD38 resin, it was immunologically identified as CD38, a 46 kDa surface antigen involved in activation and proliferation of lymphocyte populations.

CGMP mobilizes intracellular Ca2+ in sea urchin eggs by stimulating cyclic ADP-ribose synthesis.

Many hormones or neurotransmitters act at cell surface receptors to increase the intracellular free calcium concentration, triggering a wide range of cellular responses. As the source of this Ca2+ is often internal stores, additional messengers are required to convey the hormonal message from the plasma membrane. Cyclic ADP-ribose (cADPR) has been proposed as the endogenous activator of Ca(2+)-induced Ca2+ release by the ryanodine receptor in sea urchin eggs and in several mammalian cell types. A second messenger role for cADPR requires that its intracellular levels be under the control of extracellular stimuli. Here we demonstrate a novel action of 3',5'-cyclic guanosine monophosphate (cGMP) in stimulating the synthesis of cADPR from beta-NAD+ by activating its synthetic enzyme ADP-ribosyl cyclase in sea urchin eggs and egg homogenates. We suggest that cADPR may transduce signals generated by cell surface receptors or gaseous transmitters linked to cGMP production.

Wide distribution of an enzyme that catalyzes the hydrolysis of cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a metabolite of NAD + that is as effective as inositol trisphosphate in mobilizing intracellular Ca 2+ stores. Its synthesizing enzyme, ADP-ribosyl cyclase, has been shown to be present in mammalian and invertebrate tissues. In this study we identify another widely-distributed enzyme that can hydrolyze cADPR to ADP-ribose. Incubation of cADPR with brain extracts resulted in progressive decrease in its Ca 2+ mobilizing activity. The degradation of cADPR was catalyzed by a heat-labile protein factor in the brain extracts. Analysis by HPLC indicated a single degradation product was produced in equal molar quantity and that it has identical elution time as ADP-ribose. Proton NMR confirmed that the product was ADP-ribose. The degradation enzyme had a Michaelis constant of 0.16 mM and a broad pH maximum around neutrality. Centrifugation studies of the total brain extracts showed that the degradation activity was membrane-bound. Survey of tissues from various animals established that both the degradation and the synthesizing enzyme of cADPR were widely distributed from mammals to invertebrates. Since the degradation enzyme hydrolyzes an unique linkage between the adenine group and the terminal ribosyl moiety of cADPR, we propose to call it cyclic ADP-ribose hydrolase.

Similarities in amino acid sequences of Aplysia ADP-ribosyl cyclase and human lymphocyte antigen CD38.

Similarities in amino acid sequences of Aplysia ADP-ribosyl cyclase and human lymphocyte antigen CD38.

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.