Inhibition of NAD+ glycohydrolase and ADP-ribosyl cyclase activities of leukocyte cell surface antigen CD38 by gangliosides.

We have recently reported that gangliosides act as inhibitors of ADP-ribosyltransferases and NAD+ glycohydrolases (NADase) of pertussis toxin and the C3 exoenzyme from Clostridium botulinum (Hara-Yokoyama, M., Hirabayashi, Y., Irie, F., Syuto, B., Moriishi, K., Sugiya, H., and Furuyama, S. (1995) J. Biol. Chem. 270, 8115-8121). Here, we investigated the effect of gangliosides on the enzymatic activity of leukocyte cell surface antigen CD38, which is identified as an ecto-NADase (Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898). Gangliosides GM1a and GQ1balpha inhibited the NADase activity in the immunoprecipitate of anti-CD38 antibody from the membrane extract of retinoic acid-treated human leukemic HL-60 cells. Gangliosides also inhibited the NADase activity of the extracellular domain of CD38 antigen that was deprived of the transmembrane domain and was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP-CD38). The order of the inhibitory effect of purified ganglioside species on the NADase activity on MBP-CD38 was as follows: GQ1balpha > GT1b, GQ1b > GD1a, GD1b, GM1a, GM1b, GD3, GM3. GQ1balpha inhibited the NADase of MBP-CD38 in a noncompetitive manner versus NAD+ with a Ki value of about 0.3 microM. Neither ceramide nor the oligosaccharide moiety of GQ1balpha had an effect on the NADase activity. GQ1balpha, GT1b, and GQ1b also efficiently inhibited the ADP-ribosyl cyclase activity of MBP-CD38. At present, gangliosides are the only endogenous species that can block the enzymatic activity of CD38 antigen. The present results suggest a potential role of gangliosides as inhibitors of the ecto-NADases.

G M3 , 1 and G M1 regulate the tyrosine kinase activities of insulin (1), epidermal growth factor (2), and nerve growth factor (3) receptors, respectively. However, the structural bases for such interactions have not been elucidated.
We have reported for the first time that gangliosides act as an inhibitor of the ADP-ribosylation using pertussis toxin and C3 exoenzyme from Clostridium botulinum (4). The inhibition is not due to the interaction of gangliosides with the carbohydrate recognition domain of the proteins, a general motif for the ganglioside-protein interactions. In the case of pertussis toxin, ganglioside G Q1b␣ is the most potent inhibitor, and its tandem sialic acid residues linked to the internal galactose residue are crucial for inhibition. Because the inhibition by G Q1b␣ is competitive versus NAD ϩ , we have proposed that a negative charge cluster, formed by the two carboxyl groups in the tandem sialic acid residues, mimics the diphosphate moiety of NAD ϩ . Based on our speculation, it is expected that gangliosides with the tandem sialic acid residues also inhibit other enzymes that use NAD ϩ as a substrate.
Leukocyte cell surface antigen CD38 is a type II membrane protein expressed on the surface of multiple cell lineages of hematopoetic origin predominantly in early and activated phenotypes (5). The expression of CD38 antigen mRNA was also reported in the rat brain, duodenum, heart, and pancreatic islets (6). Ligation of CD38 antigen with specific antibodies has a stimulatory effect on cultured lymphocytes (7), prevents apoptosis of human germinal center B cells (8,9), or suppresses stroma cell-supported lymphopoesis of human B-cells (10). These results suggest that CD38 antigen positively or negatively modulates the activation signal for lymphocytes. Recently, CD38 antigen is identified as an ecto-enzyme of NAD ϩ glycohydrolase (NADase) 2 (11). CD38 antigen also catalyzes a conversion of NAD ϩ to cyclic ADP-ribose (cADPR), a Ca 2ϩmobilizing factor from inositol 1,4,5-trisphosphate-insensitive Ca 2ϩ stores (12).
To verify the potential role of ganglioside as an inhibitor of the NAD ϩ metabolizing enzymes, we investigated the effect of gangliosides on the NADase and ADP-ribosyl cyclase activities of CD38 antigen in the present study. The results clearly demonstrated that both activities of MBP-CD38 were inhibited by gangliosides, especially by G T1b , G Q1b , and G Q1b␣ . These data support the idea that gangliosides are physiological regulators of the NAD ϩ metabolizing enzymes on the cell surface.
Purification of the MBP-CD38 Fusion Protein and Preparation of the Immunoprecipitate-The extracellular domain of human CD38 cDNA was obtained by reverse transcriptase-mediated polymerase chain reaction from mRNA of human leukemic HL-60 cells that had been differentiated by retinoic acid. Construction of plasmid that encodes a fusion of maltose-binding protein (MBP) to the extracellular domain of CD38 antigen and subsequent purification of the fusion protein were previously described (13). The purified MBP-CD38 was treated with 8 M guanidine hydrochloride, dialyzed, and stored in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1% Chaps. The immunoprecipitate of anti-CD38 antibody was prepared from the membrane extract of retinoic acidtreated human leukemic HL-60 cells as described previously (11).
Purification of ADP-ribosyl Cyclase from Aplysia Ovotestis-ADPribosyl cyclase was purified from the cytosolic fraction of ovotesits of Aplysia kurodai as described previously (13).
Preparation of Individual Gangliosides-Total bovine brain gangliosides were applied to a Q-Sepharose column and fractionated into 23 fractions as described previously (14). G M3 , G D3 , G M1a , G M1b , G D1a , G D1b , G T1b , and G Q1b were purified from the fractions. G T1a␣ and G Q1b␣ were purified as described previously (15,16).
Assay of Enzyme Activity-To measure the NADase activity of MBP-CD38, the reaction mixture (10 l) containing 50 nM MBP-CD38 in Buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA) was preincubated for 10 min at 37°C. Then the reaction was started by the addition of 10 l of 4.6 M [carbonyl-14 C]NAD ϩ (2.5 nCi/assay) in Buffer A. After 10 min of incubation at 37°C, the reaction mixture was mixed with 5 l of 50 mM NAD ϩ and 10 mM nicotinamide and was spotted onto Whatmann 3MM paper. The paper was developed by 1 M AcONH 4 , pH 5.0, 95% EtOH (3:7, v/v) as described previously (17), and NAD ϩ and nicotinamide were detected under UV light. The radioactivities in these spots were measured. The release of [ 14 C]nicotinamide from [ 14 C]NAD ϩ was linear up to 15 min of incubation. Because the stock solution of MBP-CD38 contained 1% Chaps, the final concentraion of Chaps in the reaction mixture was 18 M. We noticed that the specific activity of NADase and the extent of the inhibition by gangliosides depend on the concentration of Chaps in the reaction mixture. The specific activity was ϳ30 and ϳ60 nmol/min/mg in the presence of 18 and 90 M Chaps, respectively. The apparent IC 50 value of gangliosides in the presence of 90 M Chaps were about 10-fold greater than those in the presence of 18 M Chaps (data not shown). For the assay of the NADase activity in the immunoprecipitates, the immunocomplexes were suspended in Buffer A and preincubated for 10 min. The subsequent procedures is according to that for MBP-CD38.
As for the ADP-ribosyl cyclase activity of MBP-CD38, the reaction mixture (10 l) containing 250 nM MBP-CD38 in Buffer A was preincubated, and the reaction was started by the addition of 10 l of 200 M NAD ϩ in Buffer A. The amount of cyclic ADP-ribose produced after 10 min of incubation at 37°C was measured using radioimmunoassay system as described previously (18).
For the assay of Aplysia ADP-ribosyl cyclase activity, the release of nicotinamide from NAD ϩ was measured, because the release of nicotinamide and production of cyclic ADP-ribose are almost equal (19). The reaction mixture was composed of 20 mM Tris-Hepes, pH 7.4, 1 mM EDTA, 2 mM MgCl 2 , 30 mM KCl, 50 M [carbonyl-14 C]NAD ϩ , and 0.8 nM Aplysia ADP-ribosyl cyclase. The reaction mixture without [ 14 C]NAD ϩ was preincubated for 2 min and further incubated with [ 14 C]NAD ϩ for 2 min. The linearity of the generation of cyclic ADP-ribose or release of nicotinamide versus time was confirmed in the case of ADP-ribosyl cyclase assay of MBP-CD38 or Aplysia enzyme, respectively. In all cases, enzyme was omitted from the reaction mixtures to obtain the background level. The value in the absence of other additions was used as the control. After subtracting the background, the relative values to the control were indicated.
Preparation of the Oligosaccharide Moiety of G Q1b␣ -G Q1b␣ (50 g) was dissolved in buffer (30 l) containing 100 mM sodium acetate, pH 6.0, and 1 mg/ml sodium taurocholate, and endoglycoceramidase (0.75 milliunits). After incubation for 14 h at 37°C, the reaction mixture was applied to a C18 Sep-Pak cartridge (Waters) previously washed with methanol and subsequently with water. The oligosaccharide moiety (6 g) passed through the cartridge as eluted with water, whereas G Q1b␣ remained. The oligosaccharide moiety was completely separated from G Q1b␣ as checked by thin-layer chromatography.

RESULTS
Inhibition of the CD38 Antigen-catalyzed NAD ϩ Glycohydrolysis by Gangliosides-In human leukemic HL-60 cells, the expression of CD38 antigen is induced by retinoic acid (11). The NADase activity in the immunoprecipitate of anti-CD38 antibody from retinoic acid-treated HL-60 cell membranes is attributed to CD38 antigen (11). As shown in Fig. 1, the NADase activity in the immunoprecipitate was inhibited by either G M1a or G Q1b␣ . G Q1b␣ inhibited more effectively than G M1a , suggesting the specificity of the gangliosides in the inhibition.
Inhibitory Effect of Gangliosides on the NAD ϩ Glycohydrolysis Catalyzed by MBP-CD38 -CD38 antigen is type II integral membrane protein composed of the large extracellular domain with the 256 amino acid residues in the C-terminal region, the transmembrane domain with the 23 amino acid residues, and the small cytoplasmic domain with the 21 amino acid residues in the N-terminal region (20). The extracellular domain of CD38 antigen is an NAD ϩ glycohydrolase (11). To investigate whether the transmembrane domain of CD38 antigen is involved in the inhibitory effect of gangliosides, the extracellular domain of CD38 antigen was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP-CD38). As shown in Fig. 2, the MBP-CD38 catalyzed-NAD ϩ glycohydrolysis was inhibited by gangliosides. Thus, the transmembrane domain is not required for the inhibitory effect of gangliosides. The potency of the inhibitory effect was clearly different among the ganglioside species. G Q1b␣ most effectively inhibited the NAD ϩ glycohydrolysis. By contrast, G T1a␣ up to 1 M did not have such an inhibitory effect. G Q1b␣ inhibited the NADase of MBP-CD38 in a noncompetitive manner versus NAD ϩ with a K i value of about 0.3 M (Fig. 3). G Q1b and G T1b were also more effective than the other gangliosides, G D1a , G D1b , G M1a , G M1b , G M3 , and G D3 . G Q1b␣ , G Q1b , and G T1b belong to the b-series ganglioside group containing tandem sialic acid residues linked to the internal galactose residue.
Requirement of Both Oligosaccharide and Ceramide Moiety of G Q1b␣ on the Inhibition of NAD ϩ Glycohydrolysis-The oligosaccharide moiety of G Q1b␣ was prepared by cleaving G Q1b␣ with endoglycoceramidase. As shown in Fig. 4, neither the oligosaccharide moiety nor the ceramide alone was effective in the inhibition. It should be noted that the inhibitory effect of  Inhibitory Effect of Gangliosides on the ADP-ribosyl Cyclase Activity-CD38 antigen is homologous to Aplysia ADP-ribosyl cyclase that efficiently generate cADPR from NAD ϩ (21). Indeed, CD38 antigen has the activity of synthesizing cADPR from NAD ϩ , although the specific activity of ADP-ribosyl cyclase is 50 -100 times lower than that of NADase. As the NAD ϩ glycohodrolysis and cADPR formation occur at the same catalytic site of CD38 antigen, we investigated the effect of gangliosides on the ADP-ribosyl cyclase activity of MBP-CD38.
As shown in Fig. 5A, gangliosides inhibited the ADP-ribosyl cyclase activity of MBP-CD38. The order of the inhibitory effect was G Q1b␣ Ͼ G Q1b Ͼ G T1b Ͼ G M3 , G D3 , G M1a , G D1a , indicating that the specificity of the inhibition of ADP-ribosyl cyclase was similar to that of NADase of MBP-CD38.
In the case of Aplysia ADP-ribosyl cyclase, the activity was enhanced nearly 2-fold by G M3 and G D3 , whereas it was inhibited by G D1b and G T1b (Fig. 5B). In contrast with the effect on the activities of MBP-CD38, G Q1b␣ did not inhibit the Aplysia ADP-ribosyl cyclase activity.

Inhibition of the Enzymatic Activities of CD38 Antigen and
Aplysia ADP-ribosyl Cyclase by Gangliosides-The present study demonstrates that gangliosides inhibit the NADase and ADP-ribosyl cyclase activities of MBP-CD38. G Q1b␣ , G Q1b , and G T1b were more effective in the NADase inhibition than the other gangliosides. Because the IC 50 values of these gangliosides (less than 1 M) were much lower than the critical micelle concentrations (about 100 M according to Ref. 22), the multivalency of ganglioside micelles is not required for the inhibition. G Q1b␣ , G Q1b , and G T1b were also effective in the inhibition of ADP-ribosyl cyclase activity of MBP-CD38. Furthermore, G D1b and G T1b inhibited the activity of Aplysia ADP-ribosyl cyclase. In the previous paper, we showed the inhibitory effect of gangliosides on the ADP-ribosyltransferase and NADase activities of pertussis toxin and C3 exoenzyme (4). Here, we confirmed the inhibitory effect of gangliosides on the endogenous NADases.
The physiological role of the NADase or ADP-ribosyl cyclase activity of CD38 antigen is controversial, probably because antibodies of CD38 antigen that inhibit the enzymatic activity have not been obtained. Gangliosides are the first species that are found to block the enzymatic activity of CD38 antigen. Depletion of gangliosides by inhibitors of glycolipid biosynthesis or overexpression of gangliosides may be helpful to investigate the physiological role of the enzymatic activity of CD38 antigen.
The Importance of the Tandem Sialic Acid Residues-G Q1b␣ and G T1b were more effective in the NADase inhibition of MBP-CD38 than G T1a␣ andG D1a , respectively, indicating that the tandem sialic acid residues linked to the internal galactose residue are important for the inhibition. To investigate whether the negative charges in the carboxyl groups of the sialic acid residues are involved in the inhibition, we blocked the negative charges by the lactonization of gangliosides (23). The lactonization of G T1b greatly diminished the inhibitory effect (data not shown). Thus, the negative charges in the carboxyl groups are probably involved in the inhibition. Mimicking the diphosphate moiety of NAD ϩ by the two carboxyl groups of the tandem sialic acid residues can explain the inhibitory effect of gangliosides, as we have proposed in our previous paper (4).
Information on the recognition of the sialic acid residue by proteins so far elucidated has been restricted to that of one sialic acid residue either ␣233 or ␣236 linked. For example, the ␣233 linked sialic acid residue is recognized by selectins (24), and the ␣236 linked sialic acid residue is recognized by CD22 antigen (25). However, the recognition of tandem sialic acid residues has not been reported. The importance of the tandem sialic acid residues in the inhibition of the NAD ϩinvolving reaction, ADP-ribosylation, NAD ϩ glycohydrolysis, or generation of cyclic ADP-ribose, would be a new aspect to elucidate the physiological role of b-series gangliosides.
Among the b-series gangliosides, G Q1b␣ was the most potent inhibitor in the case of the ADP-ribosyltransferase of pertussis toxin (4) or the NADase of MBP-CD38, whereas the inhibitory effect of G T1b prevailed against that of G Q1b␣ for the NADase of C3 exoenzyme (4) or Aplysia ADP-ribosyl cyclase. Although the presence of the tandem sialic acid residues is a basic motif of the inhibition, the specificity of the inhibition is also determined by the other part of ganglioside structure. The sialic acid residue linked to the GalNAc residue facilitates the inhibition of the NADase of MBP-CD38. By contrast, the absence of the sialic acid residue linked to the GalNAc residue is necessary for the inhibition of C3 exoenzyme or Aplysia ADP-ribosyl cyclase. The difference of the specificity in the inhibition suggests the variety of the interaction between b-series gangliosides and the enzymes.
Possible Interaction of Gangliosides with CD38 Antigen-The tandem sialic acid residues of gangliosides are important for the inhibition of the NADase activity of MBP-CD38. However, the oligosaccharide moiety of G Q1b␣ did not have the inhibitory effect. Thus, the remaining part of G Q1b␣ , the ceramide moiety, is also required for the inhibition. The decrease of the inhibitory effect of G Q1b␣ in the presence of excess amount of ceramide suggests that the ceramide moiety is involved in the binding of G Q1b␣ to MBP-CD38. The binding of ceramide moiety is considered to be a prerequisite for the inhibition. Such binding of the ceramide moiety probably do not compete with that of NAD ϩ , causing the noncompetitive type inhibition of G Q1b␣ versus NAD ϩ .
MBP-CD38 does not contain the hydrophobic transmembrane region of CD38 antigen. Although the hydrophobic region is not predicted in the primary structure (20), the binding of 8-anilino-1-naphthalenesulfonate to MBP-CD38 suggests the presence of hydrophobic area on the surface of MBP-CD38. 3 Such hydrophobic area on MBP-CD38 probably interacts with the ceramide moiety. If a similar interaction occurs between native CD38 antigen and gangliosides, gangliosides may induce the internalization of the extracellular domain of CD38 antigen, because the ceramide moiety is usually embedded in the lipid bilayer.
Biological Implication-CD38 antigen is supposed to modulate the onset of signals for lymphocyte activation (5,(7)(8)(9)(10). So far, the natural ligands of CD38 antigen have not been definitely identified. The present result raised a possibility that gangliosides interact with CD38 antigen in vivo.
In lymphocytes, the association of glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor type tyrosine kinases has been reported (26 -28). Although a GPI-anchored protein Thy-1 is present in detergent-insoluble glycolipid microdomains, neither caveolin nor the caveolae-like structure is present in lymphocytes (29). There may be another type of core structure consisting of glycolipids and membrane-spanning proteins to connect the GPI-anchored proteins to the tyrosine kinases in lymphocytes. CD38 antigen is an integral membrane protein and probably linked to the tyrosine kinases, because ligation of CD38 antigen induces the tyrosine phosphorylation of proteins (30). Recently, we identified one of the tyrosinephosphorylated proteins stimulated by anti-CD38 monoclonal antibody as c-cbl proto-oncogene product (p120 c-cbl )(31). Accordingly, the association of b-series gangliosides with CD38 antigen, if occurring in vivo, is a candidate for the transmembrane core. It is speculated that diverse effects of CD38 ligation (5,(7)(8)(9)(10) are due to the stabilization or disruption of the CD38mediated membrane machinery.
Several ecto-NADase or ecto-ADP-ribosyltransferases have been identified in addition to CD38 antigen. BST-1 is a stromal cell-derived GPI-anchored protein that facilitates pre-B-cell growth (32). BST-1 is homologous to CD38 antigen and has an activity of NADase and ADP-ribosyl cyclase (33). The rat alloantigen RT6 is a GPI-anchored ecto-ADP-ribosyltransferase expressed on lymphocytes (34,35). RT6 may regulate the activity of cytotoxic T cells (34). The present study provides an idea that b-series gangliosides interact with the ecto-NADase molecules. Further study should be required as to verify the in vivo interactions between b-series gangliosides and ecto-NADase molecules.