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E Zocchi

Publications and source records attributed to E Zocchi.

At least 19 recordsLinked to original sources
PubMed1998

Characterization of a CD38-like 78-kilodalton soluble protein released from B cell lines derived from patients with X-linked agammaglobulinemia.

Studies on murine B lymphocytes showed that Bruton's tyrosine kinase mediates signal transduction induced via CD38, a nonlineage-restricted 45-kD ectoenzyme. This signaling is defective in B cells from X-linked immunodeficient mice affected with the analogue of human X-linked agammaglobulinemia (XLA). We performed a structural and functional analysis of CD38 in XLA and other immunodeficiencies, using EBV-immortalized B cells derived from such patients. Membrane CD38 was not significantly different from controls in structure, epitope density, enzymatic activity, and internalization upon binding of agonistic mAbs. Meanwhile, an increased release of soluble CD38 from XLA cells was observed: immunoprecipitation from XLA culture media yielded a protein of approximately 78 kD (p78), reacting also in Western blot and displaying both enzymatic activities and a peptide map similar to membrane CD38. Soluble forms and homotypic aggregations of CD38 were documented in different cell models and by crystallographic analysis of the Aplysia ADP-ribosyl cyclase, the ancestor of human CD38. p78 might represent the product of an altered turn-over of membrane CD38, a starting point for studying its association with Bruton's tyrosine kinase and its role in XLA and other B cell immunodeficiencies.

ADP-ribosyl Cyclase↗
PubMed1998

Expression of CD38 increases intracellular calcium concentration and reduces doubling time in HeLa and 3T3 cells.

CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.

3T3 Cells↗
PubMed1998

Ectocellular CD38-catalyzed synthesis and intracellular Ca(2+)-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 Ca(2+)-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 Ca(2+)-response to KCl-induced depolarization, a process that can be qualified as a Ca(2+)-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.

ADP-ribosyl Cyclase↗
PubMed1997

CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose.

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 microM 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 N1 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 Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.

ADP-ribosyl Cyclase↗
PubMed1997

The CD38/cyclic ADP-ribose system: a topological paradox.

CD38 was first identified as a lymphocyte differentiation antigen that showed typical properties of an orphan receptor involved in many programs of cell proliferation and activation. However, CD38 proved also to be a bifunctional ectoenzyme that catalyzes the transient formation of cyclic ADP-ribose (cADPR) in a variety of cell types. This property raises many intriguing and so far unanswered questions, since cADPR is a new second messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores. The relationship between receptor-like and enzymatic properties of CD38 is still unknown. The apparent topological paradox of ectocellular synthesis and intracellular activity of cADPR might be explained by: (a) influx of cADPR across the plasma membrane to reach its target stores, as suggested by experiments on cerebellar granule cells; and (b) NAD(+)-induced internalization, following membrane oligomerization, of CD38 with consequent partial import of cADPR metabolism to an intracellular compartment, as recently observed in lymphoid B cells. These two distinct mechanisms and other potential ones (e.g. binding of ectocellularly formed cADPR to cell surface receptors and initiation of signal-transducing pathways across the plasmamembrane) seem to be paradigmatic of processes affecting different types of cells. Although in some biological systems, such as Aplysia and sea urchin egg, cADPR metabolism is restricted to the intracellular environment, in mammalian cells the CD38/cADPR system provides new challenges in terms of subcellular compartmentation and qualifies as an unusual example of "ectobiochemistry" with potential, still unrecognized, properties of cellular regulation.

ADP-ribosyl Cyclase↗
PubMed1996

Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum.

CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5 +/- 3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum.

ADP-ribosyl Cyclase↗
PubMed1996

NAD+-dependent internalization of the transmembrane glycoprotein CD38 in human Namalwa B cells.

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD+ (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium-releasing activity of its intermediate product cADPR. Incubation of CD38+ human Namalwa B cells with external NAD+ elicited extensive membrane down-regulation of CD38 and its internalization in non-clathrin-coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD+-dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.

ADP-ribosyl Cyclase↗
PubMed1995

Structural role of disulfide bridges in the cyclic ADP-ribose related bifunctional ectoenzyme CD38.

Human CD38, a type II cell surface glycoprotein, is a bifunctional ectoenzyme catalyzing both ADP-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase reactions. It shares a high degree of sequence homology with the cyclase from Aplysia species and studies of site-directed mutagenesis have recently demonstrated the importance, but not elucidated the role, of several cysteine residues highly conserved between these proteins. N-Ethylmaleimide, iodoacetamide and thiol-oxidizing reagents failed to affect either the cyclase or the weaker hydrolase activity of the Aplysia californica protein. Likewise, these reagents did not impair the two activities of CD38 purified from human erythrocytes. beta-mercaptoethanol had no effect on the Aplysia enzyme activities, while it inactivated both the cyclase and the cADPR hydrolase of CD38 by inducing its extensive oligomerization. In intact erythrocytes the beta-mercaptoethanol-dependent enzyme inactivation was completely prevented by prior cross-linking of the membrane proteins with glutaraldehyde. These data demonstrate that none of the cysteine residues plays any direct catalytic role in CD38 and Aplysia proteins, and that disulfide bridges are essential for maintaining the monomeric, catalytically active structure of CD38.

ADP-ribosyl Cyclase↗
PubMed1995

Self-aggregation of purified and membrane-bound erythrocyte CD38 induces extensive decrease of its ADP-ribosyl cyclase activity.

The transmembrane glycoprotein CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain both the synthesis and the hydrolysis of cyclic ADP-ribose (cADPR). The complete reaction, converting NAD+ to nicotinamide and ADP-ribose, reproduces an NAD+glycohydrolase (NADase) reaction. CD38 purified from human erythrocyte membranes has been recently shown to undergo stable oligomerization induced by either NAD+ or beta-mercaptoethanol. We demonstrate that oligomerization is also triggered by reduced glutathione (GSH) and that the GSH-induced self-aggregation of purified CD38 is accompanied by extensive and comparable decrease of its ADP-ribosyl cyclase and NADase activities. GSH-induced oligomerization of CD38 and strong enzyme inactivation take place also in situ on erythrocyte membranes.

ADP-ribosyl Cyclase↗
PubMed1994

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 beta-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 [32P]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.

ADP-ribosyl Cyclase↗
PubMed1993

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.

ADP-ribosyl Cyclase↗
PubMed1993

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins.

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Adenine Nucleotides↗
PubMed1993

Production and hydrolysis of cyclic ADP-ribose at the outer surface of human erythrocytes.

Hemoglobin-free membranes from human erythrocytes are able to convert beta-NAD+ to cyclic ADP-ribose, a calcium mobilizer as potent as inositol 1,4,5-trisphosphate. Identification of cyclic ADP-ribose was based on HPLC analyses and its Ca(2+)-mobilizing activity on sea urchin egg microsomes. Erythrocyte membranes also hydrolyze cyclic ADP-ribose to ADP-ribose. By comparing the cyclic ADP-ribose-synthesizing and -hydrolyzing activities on unsealed and right-side-out resealed ghosts, it can be concluded that both are localized at the extracellular side of the membrane. This is confirmed by the demonstration of both enzyme activities on the surface of intact human red cells. Identification of the two enzymes involved in cyclic ADP-ribose metabolism might suggest some physiological role of this nucleotide in red cells.

ADP-ribosyl Cyclase↗
PubMed1993

Adenosine diphosphate ribulose in human erythrocytes: a new metabolite with membrane binding properties.

Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [32P]ADPribulose from [32P]NAD+ by the combined activities of commercial NAD+ glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its concentration in human erythrocytes was estimated to be 0.11 +/- 0.1 microM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. The labeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

Adenosine Diphosphate Ribose↗
PubMed1992

Presence and turnover of adenosine diphosphate ribose in human erythrocytes.

ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.

Adenosine Diphosphate↗