©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Gangliosides as Inhibitors of ADP-ribosyltransferases of Pertussis Toxin and Exoenzyme C3 from Clostridium botulinum(*)

(Received for publication, October 25, 1994; and in revised form, December 28, 1994)

Miki Hara-Yokoyama (1)(§) Yoshio Hirabayashi (2) Fumitoshi Irie (2) Bunei Syuto (3) Kohji Moriishi (3) Hiroshi Sugiya (1) Shunsuke Furuyama (1)

From the  (1)Department of Physiology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi, Matsudo, Chiba 271, Japan, the (2)Laboratory for Glyco-Cell Biology, Frontier Research Program, The Institute of Physical and Chemical Research (Riken), 2-1 Hirosawa, Wako, Saitama 351-01, Japan, and the (3)Department of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously reported the presence of an endogenous inhibitory activity in bovine brain for the ADP-ribosylation of GTP-binding proteins catalyzed by pertussis toxin (PT) (Hara-Yokoyama, M., and Furuyama, S.(1989) Biochem. Biophys. Res. Commun. 160, 67-71). In the present study, we identified the inhibitor as a ganglioside. The screening of various gangliosides revealed that G most effectively inhibited the ADP-ribosyltransferase activities of both the holoenzyme and the catalytic subunit of PT. G is a ganglioside newly identified as one of the antigens recognized by the cholinergic neuron-specific antibody, anti-Chol-1alpha (Hirabayashi, Y., Nakao, T., Irie, F., Whittaker, V.P., Kon, K., and Ando, S.(1992) J. Biol. Chem. 267, 12973-12978). G also inhibited the PT-catalyzed NAD glycohydrolysis. Unlike PT activity, the ADP-ribosylation and the NAD glycohydrolysis catalyzed by the C3 exoenzyme from Clostridium botulinum type C were inhibited by G and G. The ADP-ribosylation catalyzed by either PT or the C3 exoenzyme was not inhibited by ceramide, galactocerebroside, or sialic acid. In addition to the inhibitory action of gangliosides on ADP-ribosylation, the importance of gangliosides as regulators of NAD metabolism is discussed.


INTRODUCTION

ADP-ribosylation is a post-translational modification of proteins whereby the ADP-ribose moiety of NAD is transferred to proteins as a monomer, oligomer, or polymer. Poly(ADP-ribosyl)ation occurs mostly in nuclei, and accumulating evidence indicates a close relationship between poly(ADP-ribosyl)ation and the regulation of chromatin activities(1, 2, 3) . While the physiological roles of oligo- or mono(ADP-ribosyl)ation in the cytoplasm, organella, or membranes in eukaryotic cells have not been established, the involvement of mono(ADP-ribosyl)ation in the regulation of signaling pathways or membrane traffic has been suggested(4, 5, 6, 7, 8) .

Several mono(ADP-ribosyl)transferases produced by bacteria specifically modify target proteins in eukaryotic cells. Pertussis toxin (PT), (^1)an exotoxin produced by Bordetella pertussis(9, 10) , ADP-ribosylates the alpha subunit of several GTP-binding proteins (G-proteins), which act as central signal transducers(11, 12) , and causes the functional uncoupling of the G-proteins from the receptors(13, 14) . The C3 exoenzyme from Clostridium botulinum types C and D(15, 16, 17, 18) ADP-ribosylates the Rho family of low molecular weight GTP-binding proteins in eukaryotic cells(19) , which are required for the organization of the microfilament network(20, 21) . ADP-ribosylation by the C3 exoenzyme interrupts the interaction of the Rho proteins with the downstream effector molecules(22) . Interestingly, ADP-ribosyltransferases comparable to PT and the C3 exoenzyme have been found in human erythrocytes (23) and in bovine brain(24) , respectively. These endogenous ADP-ribosyltransferases may be involved in the essential cellular functions(25) .

To elucidate how ADP-ribosylation is regulated, we aimed to characterize the endogenous modulator molecules. PT- and the C3 exoenzyme-catalyzed ADP-ribosylation was used as an assay system to detect modulatory activity for ADP-ribosylation. Previously, we reported the presence of an endogenous inhibitory activity of PT-catalyzed ADP-ribosylation in rat liver (26) and in bovine brain (27) . In the present study, we found that the inhibitory activity was present in the crude ganglioside fraction of bovine brain. Consequently, various species gangliosides, including newly identified species(28, 29, 30) , were examined for their effects on the ADP-ribosylation. We found that G, a minor ganglioside species recognized by a cholinergic neuron-specific antibody(30) , has a strong inhibitory activity for PT. On the other hand, gangliosides G and G had inhibitory activity for the C3 exoenzyme. This is the first report that demonstrates the inhibitory effect of gangliosides on ADP-ribosylation. We propose that gangliosides are involved in the cellular NAD metabolism.


EXPERIMENTAL PROCEDURES

Materials

[adenylate-P]NAD (800 Ci/mmol) and [carbonyl-^14C]NAD (25-40 Ci/mmol) were purchased from DuPont NEN. Neuraminidases from Arthrobacter ureafaciens and C. perfringens were from Boehringer Mannheim GmbH (Germany). Ceramide from bovine brain sphingomyelin (type III), galactocerebroside from bovine brain (type II), N-acetylneuraminic acid, G, and G were purchased from Sigma. Oligomers of N-acetylneuraminic acid were from Nakarai (Japan). PT was from Seikagaku Kogyo (Japan). To activate, 500 nM PT was preincubated for 15 min at 30 °C with 50 mM dithiothreitol (31) and 1 mM ATP(32) . G-proteins were partially purified from bovine brain membranes as described previously (33) by successive chromatographies on columns of DEAE-Sephacel, ULTROGEL AcA34 (IBF Biotechnics, France), and heptylamine-Sepharose.

C3 Exoenzyme

The C3 exoenzyme was purified from a culture supernatant of C. botulinum type C, strain 6813, as described previously(18) .

Preparation of the Bovine Brain Membranes and the Cholate Extract

Bovine brain was homogenized in 3 volumes of Buffer A (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 100,000 times g for 60 min. The pellet was washed twice and suspended in the same buffer. This suspension was used as the membrane fraction. To make cholate extract, the membrane fraction (70 mg of protein) was suspended in Buffer A (10 ml) containing 1% sodium cholate, stirred on ice for 60 min, and centrifuged at 100,000 times g for 60 min.

The cholate extract was heated in boiling water for 5 min, dialyzed overnight against water, and concentrated using an Amicon YM5 membrane (extract A). Extract A was mixed with 40 ml of chloroform/methanol (2:1, v/v) and centrifuged at 2000 times g for 15 min. The chloroform/methanol layer (lower), water/methanol layer (upper), and the interface were separated. After evaporation, each fraction was suspended in 10 ml of chloroform/methanol (2:1, v/v).

Preparation of the Ganglioside Fraction from Bovine Brain Membranes

The ganglioside fraction was prepared essentially according to Folch et al.(34) . The membrane fraction (300 mg of protein in 5 ml) was suspended in 100 ml of chloroform/methanol (2:1, v/v) and was centrifuged at 2,000 times g for 15 min. The pellet was resuspended in 100 ml of chloroform/methanol (1:2, v/v) and was centrifuged. The first and the second extracts were combined, evaporated, and suspended in chloroform/methanol (2:1, v/v). After filtration, one-fourth volume of water was added and the suspension was centrifuged. The upper layer (6 ml) was used as the crude ganglioside fraction.

Preparation of Individual Gangliosides

Total bovine brain gangliosides were applied to a Q-Sepharose column and fractionated into 23 fractions as described previously(28) . G and G were purified as described previously(29, 30) .

PT-catalyzed ADP-ribosylation

The typical reaction mixture (50 µl) contained 50 nM activated PT (5 µl), 2.5 µl of partially purified G-protein (0.1 mg/ml), and 1 µM [adenylate-P]NAD (1 µCi/assay) in Buffer B (50 mM Tris-HCl, pH 8.0, 20 mM dithiothreitol, 1 mML-alpha-dimyristoylphosphatidylcholine). The reaction was started by the addition of PT. After a 30-min incubation at 30 °C, the reaction was stopped by the addition of 4% SDS and 16% trichloroacetic acid. Precipitated proteins were collected on a nitrocellulose filter (Schleicher & Schuell BA85) and were washed with 20 ml of 6% trichloroacetic acid. The filters were dried and the retained radioactivity was measured. The incorporation of the radioactivity into the protein was linear versus time within the 30-min incubation. To obtain the background level, PT was omitted from the reaction mixture. The incorporation in the absence of other additions was used as the control. After subtraction of the background value due to nonspecific binding, each value was expressed as a percentage of the control value. For the kinetic measurement, the reaction mixture (50 µl) contained 74 nM G-protein, 0.25-4.0 µM [adenylate-P]NAD (50 Ci/mmol), and 10 nM activated PT in Buffer B. After the reaction was started by adding PT, aliquots (10 µl) were withdrawn at 20, 40, and 60 s.

Preparation of the S1 Subunit of PT and Measurement of ADP-ribosylation Catalyzed by the S1 Subunit

The S1 subunit of PT was prepared using a haptoglobin-Sepharose column(35, 36) . The flow-through fraction contained only the S1 subunit. The fractions containing the S1 subunit were combined and dialyzed against 10 mM Tris-HCl, pH 8.0. The S1 subunit (10 µl) was mixed with 10 µl of 25 mM dithiothreitol and 0.5 mM ATP and was incubated for 15 min at 30 °C for activation. The activated S1 subunit solution (20 µl) was used for the ADP-ribosyltransferase activity as described above. The final concentration of the S1 subunit in the reaction mixture was about 30 nM.

C3 Exoenzyme-catalyzed ADP-ribosylation

The reaction mixture (50 µl) contained 10 nM C3 exoenzyme, 0.5 µl of the cholate extract of bovine brain membranes (3.0 mg/ml), and 0.06 µM [adenylate-P]NAD (0.5 µCi/assay) in Buffer C (10 mM Tris-HCl, pH 7.0, 10 mM dithiothreitol, 1 mM MgCl(2)). After incubation for 15 min at 20 °C, the incorporation of the radioactivity into the protein was measured. The linearity of the incorporation versus time was confirmed.

NAD Glycohydrolysis

The reaction mixture (20 µl) contained 46 µM [carbonyl-^14C]NAD (0.1 µCi/assay) and 50 nM activated PT or C3 exoenzyme in Buffer B or Buffer C, respectively. After a 2.5 h-incubation at 30 °C, the reaction mixture was mixed with 5 µl of 50 mM NAD and 10 mM nicotinamide and was spotted onto Whatman No. 3MM paper. The paper was developed by 1 M AcONH(4) (pH 5.0), 95% EtOH (3:7, v/v) as described previously(37) , and NAD and nicotinamide were detected under UV light. The radioactivities in these spots were measured. The release of nicotinamide from NAD was linear up to 2.5 h of incubation.

Neuraminidase Treatment

The ganglioside fraction from bovine brain (120 µl) was dried under vacuum and redissolved in 50 µl of 0.1 M acetate buffer, pH 5.0, and 10 µl of A. ureafaciens neuraminidase (0.1 unit) or H(2)O. Alternatively, the ganglioside fraction from bovine brain (30 µl) was dried up and redissolved in 6 µl of 1 M acetate buffer, pH 5.0, and either 54 µl of C. perfringens neuraminidase (0.8 unit) in 10 mM EDTA or 54 µl of 10 mM EDTA. After incubation for 24 h at 40 °C, the fraction was boiled for 5 min, and their aliquots (10 µl) were used to examine the inhibitory activity.


RESULTS

Inhibitory Activity of PT-catalyzed ADP-ribosylation Found in the Cholate Extract of Bovine Brain Membranes

We have previously reported the presence of a heat-stable inhibitory activity of PT-catalyzed ADP-ribosylation in the cholate extract of bovine brain membranes(27) . As the inhibitory activity was also stable after treatment with chloroform (data not shown), lipids were extracted and fractionated from the cholate extract by the method of Folch et al.(34) . The inhibitory activity in the upper layer (gangliosides) was higher than that in either the lower layer (phospholipids) or the interface (proteins) fraction (data not shown).

The upper layer fraction was fractionated by gel permeation column chromatography using 45% acetonitrile, 0.1% trifluoroacetic acid. The activity emerged from the column just after 2,4-dinitrophenol-alanine (molecular mass of 260 Da), suggesting that the inhibitor is not a high molecular mass species (data not shown). No proteins or peptides were detected in the peak fraction by silver staining. On the other hand, the inhibitory activity in the cholate extract was concentrated after ultrafiltration using an Amicon YM5 unit (molecular mass cutoff about 5,000 Da). Similar results were obtained when the bovine brain membranes were extracted by the nonionic detergent nonanoyl-N-methylglucamide (MEGA9) or the ampholytic detergent CHAPS (data not shown). The inhibitor behaves as either a low or high molecular mass species depending on the conditions. These results suggest that the inhibitor is a ganglioside. The change in the apparent molecular mass may be explained by micelle formation, which is a characteristic property of gangliosides.

The Inhibitory Activity of PT in the Crude Ganglioside Fraction of Bovine Brain Membranes

To examine the involvement of a ganglioside, a crude ganglioside fraction was prepared from bovine brain membranes, according to Folch et al.(34) . Addition of the crude gangliosides inhibited the PT-catalyzed ADP-ribosylation in a dose-dependent manner (Fig. 1A). The inhibitory activity of the ganglioside fraction was about one-fifth that of the cholate extract obtained from the same amount of membranes. About 50% of the inhibitory activity was relieved by treatment with neuraminidase from A. ureafaciens (Fig. 1B). The treatment with neuraminidase from C. perfringens led to a 30% attenuation of the inhibitory activity (data not shown). Therefore, ganglioside species are probably involved in the inhibition.


Figure 1: Effect of the ganglioside fraction from bovine brain membranes on the PT and C3 exoenzyme-catalyzed ADP-ribosylation. A, The PT and C3 exoenzyme-catalyzed ADP-ribosylation was measured as described under ``Experimental Procedures'' in the presence of the indicated amount of the ganglioside fraction. Various volumes of the ganglioside fractions were separately placed in glass tubes and evaporated, and the reaction mixtures were added. The concentrations of G and G in the fraction were about 0.08 and 0.13 mg/ml, respectively. Values are means ± S.D. from duplicate assays. B and C, the ganglioside fraction was treated with neuraminidase as described under ``Experimental Procedures.'' The ADP-ribosylation was measured in the presence of ganglioside without (1) or with (2) neuraminidase treatment. Values are means ± S.D. from duplicate assays.



Effect of Individual Gangliosides on the PT-catalyzed ADP-ribosylation

For the screening of ganglioside species, crude ganglioside fraction from bovine brain was fractionated by Q-Sepharose column chromatography(28) . All fractions except for the flow-through fraction inhibited the PT-catalyzed ADP-ribosylation (data not shown). The inhibitory activity was highest in the final fraction containing G and G (Fraction 23). A similar result was obtained when the gangliosides were treated in alkaline conditions prior to the separation by Q-Sepharose column chromatography (data not shown). As shown in Fig. 2A, PT-catalyzed ADP-ribosylation was inhibited by purified G with an IC value of about 0.1 mg/ml (40 mM), but not by G. Dose-dependent inhibition by various purified gangliosides was shown in Fig. 2B. The inhibition was 95% when G was added at 0.25 mg/ml, whereas the inhibition did not exceed 50 to 80% when either G, G, G, or G was added up to 0.9 mg/ml. The inhibition depends on the concentration of NAD or L-alpha-dimyristoylphosphatidylcholine. The kinetic analysis suggests that G inhibits the PT-catalyzed ADP-ribosylation in a competitive manner versus NAD (Fig. 3). The apparent inhibitory activity of G in the absence of L-alpha-dimyristoylphosphatidylcholine was higher than that in the presence of L-alpha-dimyristoylphosphatidylcholine (data not shown). G was detected, by monoclonal antibody GGR-41, in the upper layer of the cholate extract as well as in the crude ganglioside fraction from bovine brain membranes (Fig. 4). The amount of G in the cholate extract was larger than that in the crude ganglioside fraction.


Figure 2: Effect of individual gangliosides on the PT-catalyzed ADP-ribosylation. The method for the assay is described under ``Experimental Procedures.'' A, the reaction mixture contained either G () or G (up triangle). B, the reaction mixture contained G (circle), G (bullet), G (box), G (), or G (up triangle). Values are means from duplicate assays. C, structures of ganglioside species.




Figure 3: Effect of NAD concentration on the inhibition of PT-catalyzed ADP-ribosylation by G. The PT-catalyzed ADP-ribosylation was measured in the absence (circle) or presence (bullet) of 0.1 mg/ml G as described under ``Experimental Procedures.'' Data are presented as Lineweaver-Burk plots. Essentially the same result was obtained in a separate experiment.




Figure 4: Thin layer chromatograms of the water/methanol layer of the cholate extract and the crude ganglioside fraction from bovine brain membranes. The upper layer of the cholate extract and the crude ganglioside fraction were prepared as described under ``Experimental Procedures.'' After dialysis, the samples were spotted on precoated high performance thin layer chromatography plates (Silica Gel 60; E. Merck, Darmstadt, Federal Republic of Germany) and were developed with chloroform/methanol/water (5:5:1, v/v/v). Lane 1, standard ganglioside mixture (G, G, G, G, G, G, and G in panel A and G and G in panel B); lane 2, the water/methanol layer of the cholate extract (100 µl); lane 3, the crude ganglioside fraction (40 µl). The gangliosides were visualized by the resorcinol/HCl reagent (A) or by immunostaining with the monoclonal antibody GGR-41 (B).



Effect of Gangliosides on the ADP-ribosylation Catalyzed by the S1 Subunit of PT

PT is a hexameric protein with an A-B architecture (38) . The A protomer is composed of a single S1 subunit containing the catalytic site of ADP-ribosyltransferase. The B oligomer is made up of five subunits (S2, S3, S4, and S5 in an 1:1:2:1 ratio) and is required for the binding of this toxin to the membranes of target cells(35, 38) . The S2 and S3 subunits of the B oligomer contain carbohydrate recognition domains (39) and bind to glycoproteins(40, 41) . Even in the presence of ATP, which promotes dissociation of the PT subunits (42) , more than 80% of the S1 subunit is associated with the B oligomer(31) . To examine whether the inhibition by gangliosides is due to the direct interaction with the S1 subunit or with the carbohydrate recognition domains of the B oligomer, the S1 subunit of PT was isolated by a haptoglobin-Sepharose column chromatography. As shown in Fig. 5, gangliosides inhibited the S1 subunit-catalyzed ADP-ribosylation. G was more effective than either G, G, G, G, or G was. Therefore, the site of ganglioside action is identified as the S1 subunit.


Figure 5: Effect of gangliosides on the ADP-ribosylation catalyzed by the S1 subunit of PT. The ADP-ribosylation catalyzed by the S1 subunit of PT was assayed as described under ``Experimental Procedures'' in the presence of 0.1 mg/ml G (1), G (2), G (3), G (4), G (5), and G (6). Values are means ± S.D. from duplicate assays.



Inhibitory Activity of C3 Exoenzyme-catalyzed ADP-ribosylation in the Bovine Brain Membranes

As shown in Fig. 1A, the ganglioside fraction from the bovine brain membranes inhibited the C3 exoenzyme-catalyzed ADP-ribosylation. The inhibitory activity was completely relieved by treatment with neuraminidase from C. perfringens (Fig. 1C).

Effects of Individual Gangliosides on C3 Exoenzyme-catalyzed ADP-ribosylation

The effect of gangliosides separated by Q-Sepharose column chromatography on the C3 exoenzyme-catalyzed ADP-ribosylation was studied. In contrast to the effect on the PT-catalyzed ADP-ribosylation, the C3-exoenzyme catalyzed ADP-ribosylation was inhibited by the fraction containing G, G, G, or G, but not by G (Fraction 23, data not shown). As shown in Fig. 6, G, G, G, and G inhibited the ADP-ribosylation in a dose-dependent manner. The effects of G and G were slightly greater than those of G and G. The amount of inhibitory activity of G (Fraction 23) was smaller than that of G, G, G, and G. The inhibition approached 80% by the addition of up to 0.9 mg/ml of the gangliosides.


Figure 6: Effect of gangliosides on the C3 exoenzyme-catalyzed ADP-ribosylation. The method is described under ``Experimental Procedures.'' The reaction mixture contained G (circle), G (bullet), G (box), G (), and Fraction 23 from the Q-Sepharose column chromatography (). Values are means from duplicate assays.



Effects of Gangliosides on NAD Glycohydrolysis

The inhibition of ADP-ribosylation can be explained by the interaction of gangliosides with ADP-ribosyltransferase or by the interaction with the substrate proteins. As the heterotrimeric form of G-protein is the preferred substrate of PT(43) , the ADP-ribosylation can be inhibited by the dissociation of heterotrimeric G-proteins. The C3 exoenzyme-catalyzed ADP-ribosylation is inhibited by the formation of the GTP-bound form of the Rho/Rac proteins with guanine nucleotides(44) .

To investigate whether the gangliosides directly interact on the C3 exoenzyme, we examined the effect of gangliosides on the NAD glycohydrolysis activity of the C3 exoenzyme. As shown in Fig. 7, G and G inhibited the C3 exoenzyme-catalyzed NAD glycohydrolysis, indicating that these species act directly on the C3 exoenzyme. On the other hand, the addition of either G, G, or G (Fraction 23) had no effect. The lack of inhibition of NAD glycohydrolysis with G is consistent with its effect on the ADP-ribosylation. As for G and G, our data could not discriminate whether they act directly on the C3 exoenzyme. The crude ganglioside fraction also inhibited the C3 exoenzyme-catalyzed NAD glycohydrolysis. PT-catalyzed NAD hydrolysis was inhibited by the crude ganglioside fraction as well as by G. Accordingly, G/G and G inhibit the ADP-ribosylation by interacting with the C3 exoenzyme and with PT, respectively.


Figure 7: Effect of gangliosides on the NAD glycohydrolysis. A, the C3 exoenzyme-catalyzed NAD glycohydrolysis was measured as described under ``Experimental Procedures'' in the presence of G (1), G (2), G (3), G (4), or the Q-Sepharose Fraction 23(5) , at final concentrations of 0.5 mg/ml, and in the presence of 4 µl of the crude ganglioside fraction(6) . B, the PT-catalyzed NAD glycohydrolysis was measured as described under ``Experimental Procedures'' in the presence of G at a final concentration of 0.1 mg/ml (1) and 4 µl of the crude ganglioside fraction (2). To obtain the background level, the enzymes were omitted from the reaction mixture. The hydrolysis in the absence of other additions was used as the control. After subtraction of the background, each value was expressed as a percentage of the control value. Values are means ± S.D. from duplicate assays.



Effects of Ceramide, Galactocerebroside, and Sialic Acid

To investigate the structural requirement for the inhibitory effect of gangliosides, the effects of ceramide, galactocerebroside, and sialic acid were studied. Ceramide and galactocerebroside at 0.1-0.5 mg/ml did not inhibit the PT and C3 exoenzyme-catalyzed ADP-ribosylation (data not shown). Sialic acid species (monomer, dimer, trimer, tetramer, pentamer, and hexamer at 0.1-0.7 mg/ml) also did not inhibit either ADP-ribosylation (data not shown). Thus, both the lipid components and the sialic acid residues in the gangliosides are required for their inhibitory effect.


DISCUSSION

Inhibition of the PT- and C3 Exoenzyme-catalyzed ADP-ribosylation by Gangliosides

In the present study, we demonstrated that gangliosides inhibit the ADP-ribosyltransferases of PT and the C3 exoenzyme. PT and the C3 exoenzyme modify different target proteins with distinct amino acid specificities, a cysteine residue (11, 12) and an asparagine residue(22) , respectively. The C3 exoenzyme is a basic protein(18) , whereas the catalytic S1 subunit of PT is an acidic protein(35) , and no significant homology was found between PT (45) and the C3 exoenzyme(46) . Therefore, the inhibition of both ADP-ribosyltransferases by gangliosides suggests that gangliosides interact with an essential structure of the enzymes that is required in common for both types of ADP-ribosylation.

G as an Inhibitor of PT

PT has been used to demonstrate the involvement of PT-sensitive G-proteins upstream of the inhibition of adenylate cyclase, the activation of phospholipase A(2), phospholipase C, or ion channels(47, 48) . Sensitivity to PT has also been used to P-label the G-proteins with [adenylate-P]NAD. PT is part of the standard criteria for the characterization of the signaling processes. However, there is an ambiguity in the interpretation of PT-insensitive results. The negative results can be caused either by the presence of PT-insensitive G-proteins or the signaling pathway, or by the presence of endogenous inhibitors. Although the presence of an endogenous inhibitor has been previously suggested(26, 27, 49, 50) , the chemical nature of the inhibitor has never been characterized. Since we found in this investigation that G inhibits PT, some of previous PT-insensitive results may be due to endogenous G. For example, the decrease in the endogenous inhibitory activity of PT after K-ras transformation in thyroid cells (50) is possibly related to the change in the amount of G.

G is a minor ganglioside, identified as the antigen of the cholinergic neuron-specific antibody, anti-Chol-1alpha, and represents about 0.03% of total gangliosides(30) . G was found to exist in the central nervous system tissues as well as in rat liver(51) . Information on ganglioside metabolism in these tissues will be helpful to evaluate the PT-insensitive results.

Mechanism of Inhibition

Gangliosides modulate the activities of various enzymes(52) . G partly mimics the effect of Ca/calmodulin and modulates the Ca/calmodulin-dependent kinase activity(53) . G, G, and G bind to calmodulin or a calmodulin-like binding site of enzymes and affect the activity of the calmodulin-dependent nucleotide phosphodiesterase(54, 55) . G inhibits the kinase activity associated with the epidermal growth factor and platelet-derived growth factor receptors by preventing receptor-receptor aggregation, which is required for the subsequent activation of the kinase domain(56, 57) . However, these mechanisms do not explain the inhibitory effect on the ADP-ribosylation catalyzed by PT or the C3 exoenzyme.

The inhibitory effect of gangliosides on the activities of PT and the C3 exoenzyme is probably not due to their interaction with the carbohydrate recognition domain of the proteins, based on the following reasons. (i) While the specific binding of G to the B oligomer of PT was reported(58) , the inhibitory effect was not specific for G. (ii) The ADP-ribosyltransferase activity of the catalytic S1 subunit of PT, which does not have the carbohydrate recognition domain, was inhibited by gangliosides, particularly by G. (iii) Inherently, the C3 exoenzyme has no carbohydrate recognition domain. The interaction of gangliosides with either PT or the C3 exoenzyme may occur at different sites other than carbohydrate binding domains.

PT-catalyzed ADP-ribosylation was inhibited by G, but not by G. This observation suggests that tandem sialic acid residues linked to the internal galactose residue are involved in the inhibition. Tandem sialic acid residues are also present in the molecules of G and G, which inhibit the C3 exoenzyme. As G acts as a competitive inhibitor versus NAD, a structural similarity may exist between the molecules of G and NAD. It is possible to locate one carboxyl group of one sialic acid close to the other by rotating the C-C or C-O bonds of the glycerol group connecting the two sialic acids (Fig. 8). The distance between the two carboxyl groups is similar to that between the two phosphate groups of NAD. A negative charge cluster, formed by the two carboxyl groups in the tandem sialic acid residues of the b-series gangliosides, can mimic the diphosphate moiety of NAD. As the dimer of sialic acid did not inhibit the ADP-ribosyltransferase activity of PT and the C3 exoenzyme, another component of the ganglioside molecule, such as the gangliotetraose core structure, may also be required. A structural study of the conformation of NAD bound to PT or the C3 exoenzyme will be necessary to elucidate how gangliosides inhibit the activity of these ADP-ribosyltransferases.


Figure 8: Space-filling model of NAD (A) and a sialic acid dimer (B). The conformation of the nicotinamide-ribose bond and the adenine-ribose bond are assumed to be anti, according to the results of NAD bound to L-lactate dehydrogenase(80) . The conformation of the pyranose ring of sialic acid residue is ^1C(4)(81) . Phosphorus, carbon, oxygen, and hydrogen atoms were colored in yellow, blue, red, and white, respectively.



Involvement of Gangliosides in the Inhibition of ADP-ribosylation

Benzamide and its derivatives are commonly used as inhibitors of ADP-ribosyltransferases. Recently, hydrophobic molecules, such as long-chain fatty acids and vitamin K(1), were also reported to be inhibitors(59, 60) . As the intracellular localization of gangliosides was reported(61, 62, 63, 64) , they can be potential members of the endogenous inhibitors of ADP-ribosyltransferases in cells.

Gangliosides reside in the cells as a component of plasma membranes, organella(65) , or in association with cytoskeleton(64, 66, 67) . Gangliosides may be in the sites where the ADP-ribosylation should be regulated. Indeed, several substrate proteins of the endogenous ADP-ribosylation are the membrane proteins (4, 6, 7, 23, 25) and a cytoskeletal protein(68) . Involvement of brefeldin A-sensitive ADP-ribosylation in membrane transport was also suggested(8) . The inhibitory action of gangliosides implies the importance of membrane or cytoskeleton-targeted ADP-ribosylation in the cells.

Possible Action of Gangliosides as Regulators of NAD Metabolism in Cells

In this investigation, we found that gangliosides inhibit the NAD glycohydrolase activities of PT and the C3 exoenzyme. As PT and the C3 exoenzyme are entirely different types of ADP-ribosyltransferases, the crucial target of ganglioside for the inhibition may be the NAD glycohydrolase. The presence of an ectoenzyme of NAD glycohydrolase (69) suggests that gangliosides regulate the NAD metabolism on the surface of the cells. An ectoenzyme of NAD glycohydrolase is induced during retinoic acid-induced differentiation in human leukemic HL-60 cells(70) , and this enzyme was identified as the human leukocyte cell surface antigen CD38(71) . CD38 has a hyaluronic acid binding motif and probably interacts with the extracellular matrix molecules(72) . Also, the rat T-cell alloantigen RT6.2 was found to be a glycosylphosphatidylinositol-anchored NAD glycohydrolase (73) and is homologous to a glycosylphosphatidylinositol-anchored mono(ADP-ribosyl)transferase from rabbit skeletal muscle(74) . Although the physiological significance of the NAD metabolism outside the cells is elusive at present, we speculate that gangliosides mediate signals from the cell surface to the cell interior by regulating the NAD metabolism. An implicated result is that CD38 also has an activity to generate cyclic ADP-ribose(75, 76) , a newly identified second messenger for Ca mobilization(77) . The concept of the regulation of NAD metabolism by gangliosides should be tested in future studies.


FOOTNOTES

*
This work was supported in part by Grants-in-Aid for Scientific Research 02857267 (to M. Y.) and 05274106 (to Y. H.) from the Ministry of Education, Science and Culture of Japan and by a Nihon University Research Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 81-473-68-6111; Fax: 81-473-64-6295.

(^1)
The abbreviations used are: PT, pertussis toxin; G-protein, guanine nucleotide-binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate. The abbreviations for gangliosides are according to Svennerholm nomenclature (78) and our previous publications(29, 79) .


ACKNOWLEDGEMENTS

We thank Prof. Yasuo Kishimoto, at the University of California, for the critical reading of the manuscript and helpful discussions. We also thank Dr. Yutaka Ito, at the Institute of Physical and Chemical Research (Riken), and Tohru Terada, at the University of Tokyo, for the graphic drawings, Dr. Katsumi Nogimori, at the Kaken Pharmaceutical Co., for valuable advice for the isolation of the S1 subunit of PT, and Dr. Zhang Gu, at the Institute of Physical and Chemical Research (Riken), for the preparation of G.


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