Purification and Characterization of NAD Glycohydrolase from Rabbit Erythrocytes

doi:10.1006/abbi.1993.1404

Archives of Biochemistry and Biophysics

Volume 305, Issue 1, 15 August 1993, Pages 147-152

https://doi.org/10.1006/abbi.1993.1404 Get rights and content

Abstract

The NAD glycohydrolase (NADase) was solubilized from intact erythrocytes with bacterial phosphatidylinositol-specific phospholipase C and purified to homogeneity by affinity chromatography on Cibacron blue-agarose. This purification procedure resulted in an ∼85-fold purification with an overall recovery of 75%. The purified NADase has a molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 63,000 as determined by gel permeation column chromatography at pH 7.0. Two hybridoma cell lines secreting antibodies against NADase were established and the antibodies recognized the purified enzyme as well as a 65-kDa band from the extracts of rabbit erythrocyte ghost. The enzyme displayed a K_m of 43 μM for β-NAD, a V_max of 23 μmol/min/mg, a broad pH optimum around pH 7.0, and pI of 5.0. Nicotinamide and isoniazid are inhibitors (K_i values, 2.5 and 3.5 mM, respectively) of the noncompetitive type. Adenosine diphosphoribose acts as a competitive inhibitor (K_i = 2.0 mM). Cibacron blue 3GA is a potent competitive inhibitor of NADase (K_i = 96 nM). The purified enzyme splits β-NAD, NADP, and nicotinamide hypoxanthine dinucleotide among the compounds tested and does not exhibit transglycosidase activity. Amino acid composition of the rabbit erythrocyte enzyme differed from that of NADases of other species, and the purified NADase contains 8% carbohydrate and a stoichiometric amount of inositol.

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Citation Excerpt :
Another enzyme from the Gram-positive pathogen Streptococcus pyogenes was reported to be an NADase with no apparent ART, ADP-ribosyl cyclase, or cyclic ADPR hydrolase activities (2). We identified and characterized a rabbit erythrocyte enzyme with pure NADase activity (3) that was anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) linkage and could be solubilized by incubation with Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) (4, 5). Of the NAD-degrading enzymes, which have the potential to control of NAD(P) levels, CD38, a mammalian ADP-ribosyl cyclase that exhibits significant NADase activity in addition to its intrinsic ADP-ribosyl cyclase activity, has been the most extensively studied (6).
NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.
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ADP ribosylation has been recently recognised as an important posttranslational modification regulating numerous cellular processes. This enzymatic activity is shared by two major families of enzymes, the extracellular ADP-ribosyl-transferases, or ecto-ARTS and the poly-ADP-ribosyltranferases, whose denomination derives from the capacity of its founding member, PARP1, to synthesise large linear or branched polymers of ADP-ribose on target proteins. This latter post-translational modification has recently attracted much interest based on its role in the cellular response to genotoxic and oxidative stress. Accordingly, a series of PARP-specific pharmacological inhibitors have demonstrated cell survival and anti-inflammatory properties in vivo, promoting a renewed interest in the potential immunoregulatory role of this gene family. More recently, the role of ADP-ribosylation in regulating several aspects of intracellular signalling and gene transcription has been uncovered, in particular within cells of the immune system, revealing the potential immunomodulatory role of several members of this family in addition to PARP1. We review herein the experimental evidence illustrating the complex role played by this gene family in regulating multiple aspects of the immune response, including cell survival, cytokine gene transcription and antiviral innate defences. In particular, the unexpected potential anti-inflammatory role of members of this family (including in particular PARP5a, 5b and PARP14) will be briefly discussed, raising some concern on the use of pan-specific PARP inhibitors to treat chronic inflammatory diseases.
Extracellular NAD+ induces a rise in [Ca2+]<inf>i</inf> in activated human monocytes via engagement of P2Y<inf>1</inf> and P2Y<inf>11</inf> receptors
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Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.
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NAD is available in the extracellular environment and elicits immune modulation such as T cell apoptosis by being used as the substrate of cell surface ADP-ribosyl transferase. However, it is unclear whether extracellular NAD affects function of macrophages expressing cell surface ADP-ribosyl transferase. Here we show that extracellular NAD enhances Fcγ receptor (FcγR)-mediated phagocytosis in J774A.1 macrophages via the conversion into cyclic ADP-ribose (cADPR), a potent calcium mobilizer, by CD38, an ADP-ribosyl cyclase. Extracellular NAD increased the phagocytosis of IgG-coated sheep red blood cells (IgG-SRBC) in J774A.1 macrophages, which was completely abolished by pretreatment of 8-bromo-cADPR, an antagonist of cADPR, or CD38 knockdown. Extracellular NAD increased basal intracellular Ca²⁺ concentration, which also was abolished by pretreatment of 8-bromo-cADPR or CD38 knockdown. Moreover, the chelation of intracellular calcium abolished NAD-induced enhancement of phagocytosis of IgG-SRBC. Our results suggest that extracellular NAD act as a regulator for FcγR-mediated phagocytosis in macrophages.
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The ADP-ribose (ADPR) pyrophosphatase (ADPRase) NUDT5, a member of a superfamily of Nudix hydrolases, hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5′-phosphate. Nitric oxide (NO) enhances nonenzymatic ADP-ribosylation of proteins such as β-actin and glyceraldehydes 3-phosphate dehydrogenase in the presence of free ADPR, suggesting a possibility that NUDT5 could also be ADP-ribosylated by its substrate, ADPR. Here, we show that NO stimulates nonenzymatic ADP-ribosylation of NUDT5 using ADP-ribose and consequently activates its ADPRase activity. We found that ADPRase activity in J774 macrophage cells is increased by the treatment with SNP, an exogenous NO generator or TNF-α/IFN-γ, endogenous NO inducers. Anti-NUDT5 antibody pulled down most of the ADPRase activity increased by NO, indicating that the ADPRase regulated by NO is NUDT5. Using recombinant human NUDT5, we also demonstrated that the increase of ADPRase activity is mediated via ADP-ribosylation at cysteine residue(s) in the presence of reductant. This result suggests that NO activates NUDT5 through ADP-ribosylation at cysteine residues of the enzyme in macrophages.

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Abstract

References (0)

Cited by (58)

Location, Location, Location: Compartmentalization of NAD<sup>+</sup> Synthesis and Functions in Mammalian Cells

Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content

Complex roles of members of the ADP-ribosyl transferase super family in immune defences: Looking beyond PARP1

Extracellular NAD<sup>+</sup> induces a rise in [Ca<sup>2+</sup>]<inf>i</inf> in activated human monocytes via engagement of P2Y<inf>1</inf> and P2Y<inf>11</inf> receptors

Extracellular NAD is a regulator for FcγR-mediated phagocytosis in murine macrophages

Activation of NUDT5, an ADP-ribose pyrophosphatase, by nitric oxide-mediated ADP-ribosylation

Regular ArticlePurification and Characterization of NAD Glycohydrolase from Rabbit Erythrocytes

Abstract

Regular Article
Purification and Characterization of NAD Glycohydrolase from Rabbit Erythrocytes