Nuclear CD38 in retinoic acid-induced HL-60 cells

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD(+) and hydrolysis of either NAD(+) or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD(+) glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a approximately 43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the approximately 43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.     

Identification of the enzymatic active site of CD38 by site-directed mutagenesis

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.     

CD38 as a therapeutic target

The CD38 molecule is well represented on cell surfaces in many cases of a variety of lymphoid tumors, notably multiple myeloma, AIDS-associated lymphomas, and post-transplant lymphoproliferations. As such, this molecule is a promising target for antibody therapy. After early disappointments, improved anti-CD38 antibodies of strong cytolytic potential have been described by 3 groups. First, a human IgG monoclonal anti-CD38 antibody raised in mice transgenic for human Ig has been found to induce potent complement and cellular cytotoxicities against both myeloma cell lines and fresh harvests from myeloma marrow and leukemic blood. This antibody also exhibits the singular property of inhibiting the CD38 cyclase activity. Second, a series of CD38-specific human antibodies, with high affinities and high ADCC activities against cell lines and primary cultures of myeloma, has been selected from a unique phage-display library. Finally, to enhance specificity for myeloma cells, bispecific domain antibodies targeting both CD38 and CD138 have been developed. As they lack any Fc module, these constructs rely on cytotoxicity for delivering a toxin to tumor cells. The list of candidate CD38-bearing neoplasms as targets for these antibody constructs can now be expanded to include acute promyelocytic leukemia, and possibly other myeloid leukemias, in which surface CD38 can be induced by retinoid treatment. One caveat here is that evidence has been produced to suggest that CD38 promotes pulmonary manifestations of the hazardous retinoic acid syndrome.     

Porcine CD38 exhibits prominent secondary NAD+ cyclase activity

Cyclic ADP‐ribose (cADPR) mobilizes intracellular Ca²⁺ stores and activates Ca²⁺ influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD⁺ by the multifunctional CD38/ADP‐ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD⁺ can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD⁺ to form linear ADPR while Aplysia ADP‐ribosyl cyclase prefers cyclizing NAD⁺ to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD⁺ cyclase activity producing cADPR. We also determined the X‐ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD⁺ reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.     

Changes in CD38 expression and ADP-ribosyl cyclase activity in rat myometrium during pregnancy: influence of sex steroid hormones

Cyclic ADP-ribose (cADPR), synthesized by CD38, regulates intracellular calcium in uterine smooth muscle. CD38 is a transmembrane protein that has both ADP-ribosyl cyclase and cADPR hydrolase enzyme activities involved in cADPR metabolism. CD38 expression and its enzyme activities in uterine smooth muscle are regulated by estrogen. In the present study, we examined CD38 expression, its enzyme activities, and cADPR levels in myometrium obtained from rats at 14-17 days of gestation (preterm) and at parturition (term). CD38 expression, ADP-ribosyl cyclase activity, and cADPR levels were higher in uterine tissues obtained from term rats compared with that of preterm rats, while activity of cADPR hydrolase did not significantly change. In an effort to address whether changes in estrogen: progesterone ratio that occur during pregnancy account for the observed effects on CD38 expression and function, we determined the effect of different doses of progesterone in the presence of estrogen on CD38 expression and its enzyme activities in uterine smooth muscle obtained from ovariectomized rats. In myometrium obtained from ovariectomized rats, estrogen administration caused increased CD38 protein expression and ADP-ribosyl cyclase activity. The estrogen-induced increases in CD38 expression and ADP-ribosyl cyclase activity were inhibited by simultaneous administration of 10 or 20 mg of progesterone. These results indicate that the estrogen:progesterone ratio determines CD38 expression and ADP-ribosyl cyclase activity. These changes in CD38/cADPR pathway may contribute to increased uterine motility and onset of labor.     

Thymidine phosphorylase, thymidine kinase and thymidylate synthetase activities in cerebral hemispheres of developing chick embryos

The changes in activities of thymidine phosphorylase (EC 2.4.2.4), thymidine kinase (EC 2.7.1.75) and thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, EC 2.1.1.-) in the cerebral hemispheres of developing chick embryos were determined and compared with the course of DNA synthesis and of natural cell death in this organ. Thymidine phosphorylase activity reaches a broad maximum at the 12th to 14th day of incubation, followed by a rapid decrease. Thymidine kinase and thymidylate synthetase activities are highest at the earliest time studied (day 10) and decrease until day 14, followed by an increase from day 14 to 16 and a further decrease from day 16 through 1 day post-hatching. The rate of DNA synthesis essentially follows these activities, but the increase at day 16 is not discernible. Our previous study revealed high DNA synthesis at day 10, with natural cell death concurring on days 12-14, followed by another peak after day 16 (glial proliferation) and a decrease after day 16. It appears that thymidine phosphorylase activity reaches a maximum (days 12-14) at the time of maximum cell death, which may be correlated with the degradative function of this enzyme. This was also the time for minimum activities of thymidine kinase and thymidylate synthetase; on the other hand, these activities reach a first (day 10) and second (day 16) maximum at the time of maximum DNA synthesis; this may be correlated with the synthetic functions of these enzymes.     

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.     

Interaction of two classes of ADP-ribose transfer reactions in immune signaling

CD38 is a bifunctional ectoenzyme predominantly expressed on hematopoietic cells where its expression correlates with differentiation and proliferation. The two enzyme activities displayed by CD38 are an ADP-ribosyl cyclase and a cyclic adenosine diphosphate ribose (cADPR) hydrolase that catalyzes the synthesis and hydrolysis of cADPR. T lymphocytes can be induced to express CD38 when activated with antibodies against specific antigen receptors. If the activated T cells are then exposed with NAD, cell death by apoptosis occurs. During the exposure of activated T cells to NAD, the CD38 is modified by ecto-mono-ADP-ribosyltransferases (ecto-mono-ADPRTs) specific for cysteine and arginine residues. Arginine-ADP-ribosylation results in inactivation of both cyclase and hydrolase activities of CD38, whereas cysteine-ADP-ribosylation results only in the inhibition of the hydrolase activity. The arginine-ADP-ribosylation causes a decrease in intracellular cADPR and a subsequent decrease in Ca(2+) influx, resulting in apoptosis of the activated T cells. Our results suggest that the interaction of two classes of ecto-ADP-ribose transfer enzymes plays an important role in immune regulation by the selective induction of apoptosis in activated T cells and that cADPR mediated signaling is essential for the survival of activated T cells.     

Studies on the induction of -aminolevulinic acid synthetase iun mouse liver

Administration of 3,5-diethoxy carbonyl-1,4-dihydrocollidine (DDC) to mice resulted in a striking increase in the level of δ-aminolevulinic acid (ALA) synthetase in liver. Although the enzyme activity was primarily localized in mitochondria and postmicrosomal supernatant fluid, a significant level of activity was also detected in purified nuclei. The time course of induction showed a close parallelism between the bound and free enzyme activities with the former always accounting for a higher percentage of the total activity as compared to the latter. Studies with cycloheximide indicated a half-life of around 3 hr for both the bound and free ALA synthetase. Actinomycin D and hemin prevented enzyme induction when administered along with DDC, but when administered 12 hr after DDC treatment Actinomycin D did not lead to a decay of either the bound or free enzyme activity and hemin inhibited the bound enzyme activity but not the free enzyme level. The molecular sizes of the mitochondrial and cytosolic ALA synthetase(s) were found to be similar on sephadex columns.     

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.     

A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.     

CERTAIN ENZYMATIC ACTIVITIES OF NORMAL AND MOSAIC INFECTED TOBACCO PLANTS

The lower leaves of tobacco plants were inoculated with leaf mosaic virus and the activities of oxygenase, peroxidase, catalase, and invertase were followed in leaves of comparable age at intervals of 2 or 3 days over a period of 21 days. The inoculated leaves exhibited a great decrease relative to normal tissue in the activity of oxygenase and peroxidase on the 6th day. Younger leaves showed this minimum at a progressively later date. A great decrease in the activities of these enzymes was attained by the 14th to the 18th day. This maximum was followed by a decrease. Catalase exhibited an increased activity which reached a maximum at about the 8th day. A second maximum was observed on the 16th to the 18th day. Invertase reached a minimum, relative to normal plants, on about the 8th day. A second minimum was approached on the 16th to the 18th day. These data show that profound disturbances in the physiology of infected plants occur many days before the leaf juice attains an infectious concentration of virus. The observed activities could not be due therefore to metabolic activities of the virus particles themselves. Since infectivity is attained only after a period of profound physiological disturbance, it seems possible that the virus protein develops as a product of abnormal metabolism.     

Potential role of CD34 in cerebral vasospasm after experimental subarachnoid hemorrhage in rats

Inflammatory responses have been implicated in the elaboration of several forms of central nervous system injury, including cerebral vasospasm after subarachnoid hemorrhage (SAH). A critical event participating in such responses is the recruitment of circulating leukocytes into the inflammatory site. CD34 is a key adhesion molecule responsible for recruitment of monocytes/macrophages and the attachment of leukocytes to endothelial cells. However, it has not been investigated whether, and to what degree, CD34 is induced by SAH and also the role of CD34 in the pathogenesis of cerebral vasospasm following SAH remains unknown. Experiment 1 aimed to investigate the timecourse of the CD34 expression in the basilar artery after SAH. In experiment 2, we chose the maximum time point of vasospasm (day 3) and assessed the effect of monoclonal antibody against CD34 on regulation of cerebral vasospasm. As a result, the elevated expression of CD34 was detected in the basilar artery after SAH and peaked on day 3. After intracisternal administration of CD34 monoclonal antibody, the vasospasm was markedly attenuated after blood injection on day 3. Our results suggest that CD34 is increasingly expressed in a parallel time course to the development of cerebral vasospasm in a rat experimental model of SAH and administration of the specific CD34 antibody could prevent or reduce cerebral vasospasm caused by SAH.     

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.     

Evidence for an intracellular ADP-ribosyl cyclase/NAD+-glycohydrolase in brain from CD38-deficient mice

Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.     

Prevention of myosin-induced autoimmune myocarditis in mice by anti-L3T4 monoclonal antibody

This study was aimed at studying the effect of the induction of immune tolerance to swine cardiac myosin from anti-L3T4 monoclonal antibody injection and whether the immune tolerance could protect mice with myosin-induced myocarditis from myocardial injury. Twenty-four Balb/c mice were divided into two groups at random. All of the mice were immunized with swine cardiac myosin on the 1st day, 14th, 28th, 42nd, and 52nd day. Immune tolerance was induced by triplicate injections of 400 microg anti-L3T4 McAb on the 0 day (intravenous), 1st day, and 2nd day (intraperitoneal) in McAb-treated group. In the saline-treated group, saline of the same volume as anti-L3T4 monoclonal antibody was used as a control. The sera and hearts biopsies of all mice were collected on the 58th day. The anti-cardiac myosin antibody was examined with ELISA, and pathological changes of heart were observed by light microscope. It was shown that mice immunized with swine cardiac myosin could produce anti-myosin antibody and the anti-cardiac myosin antibody was positive in most of the saline-treated group but negative in the McAb-treated group. Morphologically, myocardial degeneration, necrosis, and infiltration of inflammatory cells were found in the saline-treated group but not in the McAb-treated group. In conclusion, this study indicated that the immune tolerance to cardiac myosin was induced by the anti-L3T4 monoclonal antibody, and accordingly myocardial injury could be prevented by induction of immune tolerance.     

Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX

Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin. Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.