Cyrus Munshi

Evidence for a causal role of CD38 expression in granulocytic differentiation of human HL-60 cells

Granulocytic differentiation of human HL-60 cells can be induced by retinoic acid and is accompanied by a massive expression of CD38, a multi-functional enzyme responsible for metabolizing cyclic ADP-ribose (cADPR), a Ca2+messenger. Immunofluorescence staining showed that CD38 was expressed not only on the surface of intact HL-60 cells but also intracellularly, which was revealed after permeabilization with Triton. Concomitant with CD38 expression was the accumulation of cADPR, and both time courses preceded the onset of differentiation, suggesting a causal role for CD38. Consistently, treatment of HL-60 cells with a permeant inhibitor of CD38, nicotinamide, inhibited both the CD38 activity and differentiation. More specific blockage of CD38 expression was achieved by using morpholino antisense oligonucleotides targeting its mRNA, which produced a corresponding inhibition of differentiation as well. Similar inhibitory effects were observed when CD38 expression was reduced by the RNA interference technique targeting two separate regions of the coding sequence of CD38. Further support came from transfecting HL-60 cells with a Tet-On expression vector containing a full-length CD38. Subsequent treatments with doxycycline induced both CD38 expression and differentiation in the absence of retinoic acid. These results provide the first evidence that CD38 expression and the consequential accumulation of cADPR play a causal role in mediating cellular differentiation.

Characterization of the active site of ADP-ribosyl cyclase

ADP-ribosyl cyclase synthesizes two Ca2+ 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. Glu98, Asn107 and Trp140 were within 3.5 Å of the bound nicotinamide and appeared to coordinate it. Substituting Glu98 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 Glu179 with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp77 to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [32P]8-azido-NAD. These results indicate that both Glu179 and Trp77 are crucial for catalysis and that Glu179 may indeed be the catalytic residue.

LARGE-SCALE PRODUCTION OF HUMAN CD38 IN YEAST BY FERMENTATION

Publisher Summary Human CD38 is a lymphocyte antigen that shares considerable sequence homology with Aplysia ADP-ribosylcyclase, an enzyme that cyclizes NAD+ to produce a Ca2+-mobilizing metabolite, cyclic ADP-ribose. CD38 not only can synthesize cyclic ADP-ribose but can also hydrolyze it to ADP-ribose. It is thus a bifunctional enzyme similar to the one purified from canine spleen. To facilitate the understanding of this novel bifunctionality of catalysis, an efficient method for obtaining large amounts of the protein for biochemical and X-ray crystallography analyses, is described. A construct consisting of the extracellular domain of human CD38, with the putative glycosylation sites eliminated by site-directed mutagenesis (CD38S2), was spliced into a yeast expression vector, pHIL-S1. Pichia transfected with the construct secretes soluble CD38 that exhibits bifunctionality of catalysis similar to that of the native protein. To increase the efficiency of protein secretion, procedures are described to subclone CD38S2 into a different vector, pPIC9. This vector is driven by the alcohol oxidase promoter (AOX1) and utilizes the α-factor mating signal sequence (Mfα1). Production of up to 455 mg/liter of soluble CD38 was achieved by fermentation.

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 Ca2+ 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.

STRUCTURES AND ACTIVITIES OF CYCLIC ADP-RIBOSE, NAADP AND THEIR METABOLIC ENZYMES

ADP-ribosyl cyclase and CD38 are multi-functional enzymes involved in calcium signaling. Both can cyclize NAD and its guanine analog, NGD, at two different sites of the purine ring, N1 and N7, respectively, to produce cyclic ADP-ribose (cADPR) and cyclic GDP-ribose, a fluorescent but inactive analog. Both enzymes can also catalyze the exchange of the nicotinamide group of NADP with nicotinic acid, producing yet another potent activator of Ca2+ mobilization, nicotinic acid adenine dinucleotide phosphate (NAADP). The Ca2+ release mechanism activated by NAADP is totally independent of cADPR and inositol trisphosphate indicating it is a novel and hitherto unknown Ca2+ signaling pathway. This article summarizes the current results on the structures and activities of cADPR, NAADP and the enzymes that catalyze their syntheses. A comprehensive model accounting for the novel multi-functionality of ADP-ribosyl cyclase and CD38 is presented.

Structural basis for the mechanistic understanding of human CD38-controlled multiple catalysis.

The enzymatic cleavage of the nicotinamide-glycosidic bond on nicotinamide adenine dinucleotide (NAD+) has been proposed to go through an oxocarbenium ion-like transition state. Because of the instability of the ionic intermediate, there has been no structural report on such a transient reactive species. Human CD38 is an ectoenzyme that can use NAD+ to synthesize two calcium-mobilizing molecules. By using NAD+ and a surrogate substrate, NGD+, we captured and determined crystal structures of the enzyme complexed with an intermediate, a substrate, and a product along the reaction pathway. Our results showed that the intermediate is stabilized by polar interactions with the catalytic residue Glu226 rather than by a covalent linkage. The polar interactions between Glu226 and the substrate 2′,3′-OH groups are essential for initiating catalysis. Ser193 was demonstrated to have a regulative role during catalysis and is likely to be involved in intermediate stabilization. In addition, a product inhibition effect by ADP-ribose (through the reorientation of the product) or GDP-ribose (through the formation of a covalently linked GDP-ribose dimer) was observed. These structural data provide insights into the understanding of multiple catalysis and clues for drug design.

THE HOMO-DIMERIC FORM OF ADP-RIBOSYL CYCLASE IN SOLUTION

ADP-ribosyl cyclase is a multi-functional enzyme that catalyzes the formation of two Ca2+ signaling molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). X-ray crystallography of three different crystal forms shows that it is a non-covalent dimer. Chemical cross-linking and dynamic light scattering were used in this study to determine if the cyclase is also a non-covalent dimer in solution. Treatment of the cyclase in dilute solution (0.05 mg/ml) with dimethylsuberimidate resulted in complete conversion to a species with molecular weight about twice that of the monomeric cyclase. Prolonged cross-linking of the cyclase at four times higher concentration produced also only the covalently linked dimers and no multimer formation was observed. The cross-linked dimer retained full enzymatic activity and readily catalyzed the formation of cADPR from NAD, NAADP from NADP, cyclic ADP-ribose phosphate from NADP, and cyclic GDP-ribose from nicotinamide guanine dinucleotide. Analysis of the autocorrelation functions obtained from dynamic light scattering measurements indicated the cyclase solution (2 mg/ml) was composed of a single molecular species and its diffusion coefficient was measured to be 7. 4x10-7 cm2/s. Computer modeling using the crystallographic dimensions of the non-covalent cyclase dimer, a donut shaped molecule with a central cavity and overall dimensions of 7x6x3 nm, gave a value for the diffusion coefficient essentially the same as that measured. These results indicate the cyclase is a non-covalent dimer in solution.

Large-scale purification of Aplysia ADP-ribosylcyclase and measurement of its activity by fluorimetric assay

Publisher Summary Cyclic ADP-ribose (cADPR) is a Ca 2+ -mobilizing cyclic nucleotide that functions as an endogenous modulator of the Ca 2+ -induced Ca 2+ release mechanism in cells. Its synthesizing enzyme, ADP-ribosylcyclase, is widely distributed among animal tissues and is particularly abundant in Aplysia ovotestis. This chapter describes a procedure for a one-step large-scale purification of the Aplysia cyclase that is useful for biochemical analyses and crystallography. A lymphocyte antigen, CD38, which shares considerable sequence homology with the Aplysia cyclase, is shown to possess not only the cyclase activity but also to catalyze the hydrolysis of cADPR. A fluorimetric assay based on using nicotinamide guanine dinucleotide (NGD + ), a guanine analog of NAD + , is proposed in the chapter. NGD is cyclized by CD38 to produce cyclic GDP-ribose (cGDPR), which is fluorescent. The product is also resistant to hydrolysis and accumulates, making this simple fluorimetric assay ideally suitable for monitoring the cyclize activity of CD38-like bifunctional enzymes in crude tissue extracts and during purification.

ADP-Ribosyl Cyclases - A Family of cADPR and NAADP Metabolizing Enzymes

ADP-ribosyl cyclase cyclizes NAD to produce cyclic ADP-ribose (cADPR) and releases nicotinamide in the process [1]. It is a ubiquitous enzyme widely present in animal cells [1, 2] and has even been identified in Euglena, a protozoan [4]. The first cyclase purified was from Aplysia ovotestis [1, 5]. It is a soluble protein of approximately 30 kDa. Sequence comparison reveals that CD38, a lymphocyte antigen, is a mammalian homolog [6]. CD38 is a transmembrane glycoprotein of approximately 45 kDa [7]. It catalyzes not only the synthesis of cADPR but also its hydrolysis to ADP-ribose [8, 11]. Additionally, both CD38 and the Aplysia cyclase use NADP as a substrate and catalyze the exchange of the nicotinamide group with nicotinic acid, producing nicotinic acid adenine dinucleotide phosphate (NAADP) [12]. The Aplysia cyclase has been crystallized and its structure solved [13, 14]. Current knowledge of the structures of these enzymes and the novel multiplicity of their catalytic activities are described in this chapter. Since both of their products are Ca2+ messengers, this class of enzymes is crucial in mediating Ca2+ signaling functions in cells. Some of their physiological functions have now been revealed by gene ablation and are summarised here.

A Yeast Expression System of High Efficiency for Producing Recombinant Enzymes

Notwithstanding their structural and functional differences, cADPR and NAADP are synthesized by the same homologous enzymes. Three have so far been identified and they are the Aplysia ADP-ribosyl cyclase and the mammalian antigens, CD38 and CD157 (formerly called BST-1) [1–4]. The sequence homology among these three enzymes is shown in Figure 1. All three enzymes are widespread in occurrence and are localized in a variety of tissues [1, 5–8]. The novel multifunctionality of these enzymes calls for detailed structural studies of their catalytic properties by site-directed mutagenesis, enzyme kinetics and X-ray crystallography. Large quantities of proteins are needed for these kinds of studies, necessitating the development of an efficient expression system for producing the necessary recombinant proteins. In addition, the Aplysia cyclase is the most critical component of the recently developed cycling assay for cADPR [9]. CD38, on the other hand, is the only known enzyme that can hydrolyze cADPR and is used in its radioimmunoassay to verify that the signal is indeed due to cADPR [10].