Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Moss, Joel; Manganiello, Vincent C.; Vaughan, Martha

doi:10.1073/pnas.73.12.4424

Cited by 207 publications

(101 citation statements)

References 21 publications

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“…An enzymatic assay based on nicotinamide release was employed to measure the NAD-glycohydrolase activity of monomeric CRM197 and DT (41). Full details are outlined in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

Malito

Bursulaya

Chen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

125

107

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CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.

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“…An enzymatic assay based on nicotinamide release was employed to measure the NAD-glycohydrolase activity of monomeric CRM197 and DT (41). Full details are outlined in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

Malito

Bursulaya

Chen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

125

107

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“…The [32P]ADP-ribosylated proteins were also electrophoresed through SDS-polyacrylamide gels (12.5%) as described in [3,16], and an autoradiogram was made with a Kodak X-Omat AR film using an intensifying screen at -85°C. NAD glycohydrolase activity was assayed by a method described in [17,18]. The reaction mixture contained 20 mM sodium Hepes (pH 7.4), 1 mM MgCI2, 20,aM [4)H-nicotinamide]NAD (approximately 1000 cpm/pmol) and other additions as indicated in Table I.…”

Section: Assays Of C3-catalyzed Adp-ribosylation and Nad Glyco-mentioning

confidence: 99%

Botulinum ADP‐ribosyltransferase activity as affected by detergents and phospholipids

et al. 1990

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GTP-binding proteins with Mr values of 22 000 and 25 000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADPribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.

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“…In addition, both choleragen and LT require NAD for activation of adenylate cyclase in disrupted cells (9,11,12) and possess NAD glycohydrolase and ADPribosyltransferase activities (13)(14)(15). It has been proposed that both toxins exert their effects through the NAD-dependent ADP-ribosylation of either adenylate cyclase itself or of a protein critical to cyclase activation (9,11,(13)(14)(15)(16)(17). LT cross-reacts immunologically with antisera directed primarily against the B-subunits of choleragen (9,18,19).…”

Section: Introductionmentioning

confidence: 99%

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Moss¹,

Garrison²,

Fishman³

et al. 1979

J. Clin. Invest.

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A B S T R A C T Chemically transformed mouse fibroblasts did not raise their cyclic AMP level in response to Escherichia coli heat-labile enterotoxin. These fibroblasts did, however, incorporate exogenous mono-, di-, and trisialogangliosides. After the uptake of monosialoganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraininyl]-galactosylglucosylceramide (GM,), the cells responded to E. coli heat-labile enterotoxin. The di-and trisialogangliosides were considerably less effective. GMI, the putative cholera toxin (choleragen) receptor, has been implicated previously as the receptor for E. coli heat-labile enterotoxin based on the ability of the free ganglioside to inhibit the effects of toxin. This investigation establishes that the ganglioside, when incorporated into fibroblasts, serves a functional role in mediating the responsiveness to the toxin.

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Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Cited by 207 publications

References 21 publications

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

Botulinum ADP‐ribosyltransferase activity as affected by detergents and phospholipids

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

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