Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.
 ...
PMID:Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction. 144 18

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.
 ...
PMID:Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme. 165 Feb 54

The monoclonal antibody termed 1B7 neutralizes pertussis toxin in vivo in cell culture systems and can also passively protect mice from a challenge with live Bordetella pertussis (9). It has been suggested that most other independently derived neutralizing monoclonal antibodies recognizing the S1 subunit apparently recognize the same epitope as 1B7, and that the S1 subunit contains only one immunodominant protective epitope (1). These antibodies have been termed Class A antibodies (8) and inhibit the ADP-ribosyltransferase but not the NAD glycohydrolase activity of the toxin (7). We are testing the hypothesis that immunization with inactivated preparations of pertussis toxin that lead to protection are associated with the production of Class A antibodies. If true, then identification of Class A antibodies in sera might provide a serological correlate of protection. If false, then development of assays designed to detect the important protective antibodies are necessary. Our initial results suggest that Class A antibodies are not the predominant neutralizing antibody in mice immunized with vaccines containing formalin-treated pertussis toxin.
 ...
PMID:Evaluation of antibodies elicited by immunization with pertussis toxin. 177 8

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.
 ...
PMID:Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins. 183 88

Purified recombinant S1 subunit of pertussis toxin (rS1) possessed similar NAD glycohydrolase and ADP-ribosyltransferase activities as S1 subunit purified from pertussis toxin. Purified rS1 and C180 peptide, a deletion peptide which contains amino acids 1-180 of rS1, had Km values for NAD of 24 and 13 microM and kcat values of 22 and 24 h-1, respectively, in the NAD glycohydrolase reaction. In contrast, under linear velocity conditions, the C180 peptide possessed less than 1% of the ADP-ribosyltransferase activity of rS1 using transducin as target. Radiolabeled tryptic peptides of transducin that had been ADP-ribosylated by either rS1 or C180 peptide were identical which suggested that both rS1 and C180 peptide ADP-ribosylated the same amino acid within transducin. To extend the functional primary amino acid map of the S1 subunit, two carboxyl-terminal deletions were constructed. One deletion, C195, removed the 40 carboxyl-terminal amino acids and the other, C219, removed the 16 carboxyl-terminal amino acids of the S1 subunit. Both C195 and C219 migrated in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular masses of 22,000 and 27,500 Da, respectively. Relative to the C180 peptide C195 possessed 10-20-fold increase and C219 possessed 100-150-fold increase in ADP-ribosyltransferase activities. In addition, C219 appeared to have the same ADP-ribosyltransferase activity as rS1. These studies indicate that (i) rS1, purified from Escherichia coli, possesses biochemical properties similar to S1 subunit purified from pertussis toxin, (ii) amino acids 1-180 of the S1 subunit contain residues required for NAD binding, N-glycosidic cleavage, and transfer of ADP-ribose to transducin, and (iii) residues between 181 and 219 of the S1 subunit are required for efficient ADP-ribosyltransferase activity.
 ...
PMID:Localization of a region of the S1 subunit of pertussis toxin required for efficient ADP-ribosyltransferase activity. 199 75

Bordetella pertussis TOX3201 has a 12-base-pair insertion in the S1 subunit gene of pertussis toxin (PTX), which encodes for a 4-amino-acid insertion between residues 107 and 108 of the mature S1 subunit (Black et al., Science 240:656-659, 1988). This mutant strain has been shown to secrete a holotoxin analog of PTX, designated CRM3201, with reduced ADP-ribosyltransferase activity. In the present study, we evaluated the biochemical, biological, and immunoprotective activities of purified CRM3201. Assay of enzymatic activities showed that CRM3201 had 20 to 30% of the ADP-ribosyltransferase activity and 55 to 60% of the NAD glycohydrolase activity of native PTX. CRM3201, however, had only 2 to 6% of the activity of PTX in clustering CHO cells, promoting leukocytosis, inducing histamine sensitization, and potentiating an anaphylactic response to bovine serum albumin. In contrast, activities associated with the B oligomer (binding to fetuin, hemagglutination of goose erythrocytes, and lymphocyte mitogen activity) were comparable to those of native PTX. Injection of BALB/c mice with CRM3201 mixed with Al(OH)3 elicited high titers of antibody to PTX (as measured by enzyme-linked immunosorbent assay), which neutralized a leukocytosis-promoting dose of PTX in these mice and neutralized PTX in a CHO cell assay. Passive transfer of the anti-CRM3201 antibody protected 20-day-old Swiss-Webster mice against a lethal aerosol challenge with B. pertussis 18323. Active immunization with CRM3201 significantly reduced lung colonization in adult BALB/c mice with a B. pertussis respiratory infection. These results demonstrate (i) that the reduced ADP-ribosyltransferase activity of CRM3201 is associated with reductions in certain biological and toxic activities of PTX (the enzymatic and biological activities are not, however, totally concordant); (ii) that CRM3201 possesses a functional B oligomer; and (iii) that CRM3201 can induce toxin-neutralizing antibodies which protect mice against a respiratory challenge with B. pertussis. Our studies with CRM3201 show that recombinant analogs of PTX have the potential to be developed into safe, protective immunogens for use in new acellular pertussis vaccines.
 ...
PMID:Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen. 211 44

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.
 ...
PMID:Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. 247 88

The epitope specificities of 13 hybridomas secreting monoclonal antibodies (MAbs) specific for pertussis toxin (PT) is described. Hybridoma lines were derived by the fusion of spleen cells from mice immunized with native PT, Formalin-detoxified PT, or isolated PT subunits (S1 to S5) with the myeloma line X63-Ag8.653. Five MAbs showed a toxin-neutralizing ability, which was demonstrated by use of a Chinese hamster ovary cell assay system and by a NAD glycohydrolase assay. All five toxin-neutralizing MAbs demonstrated high specificities for and reactivities with native PT but were unable to bind to denatured PT. One MAb was able to neutralize the enzymatic activity of PT. The other four neutralizing MAbs inhibited the binding of PT or PT subunits to the surface of Chinese hamster ovary cells, as shown by an immunofluorescence assay. All neutralizing MAbs reacted with purified S2-S4 or S3-S4 dimers but not with S4 alone. Three MAbs which recognized a common epitope shared by S2 and S3 (which are about 70% homologous at the DNA level) and one MAb which recognized S4 were not neutralizing. Isolated S2-S4 and S3-S4 dimers bound to Chinese hamster ovary cells. These results indicate that the majority of critical epitopes which elicit neutralizing antibody are conformation dependent.
 ...
PMID:Monoclonal antibodies that define neutralizing epitopes of pertussis toxin: conformational dependence and epitope mapping. 247

The 2 cysteine residues present in the A subunit of pertussis toxin form a disulfide bond in the conformation of the toxin secreted from the bacteria. Previous studies have shown that reduction of this bond is necessary for activation of the enzyme. We have found that reduction of this bond also alters the conformation of the A subunit such that it no longer readily associates with the B oligomer of the toxin, a finding which may have implications concerning the form of the toxin found within the eukaryotic cell. In addition, we have demonstrated that reduction of the disulfide bond of the purified A subunit followed by treatment with sulfhydryl-modifying reagents such as N-ethylmaleimide or 5,5'-dithiobis-(2-nitrobenzoic acid) results in inhibition of the NAD glycohydrolase activity of the protein. When a tryptic fragment of the A subunit which contains only 1 of the cysteine residues (Cys-41) of the native protein was reacted with N-ethylmaleimide, the NAD glycohydrolase activity of this fragment was substantially reduced. These data indicate that Cys-41 may be in a region of the molecule which is critical for the enzymatic activity of the toxin.
 ...
PMID:Role of cysteine 41 of the A subunit of pertussis toxin. 253 46

The gene encoding a catalytically active deletion peptide, the C180 peptide, of the S-1 subunit of pertussis toxin was engineered to facilitate mutagenesis at the Trp-26 (wild-type) coding sequence. A synthetic double-stranded oligonucleotide was inserted into the C180 gene such that all possible codons would be introduced into position 26. Seven individual mutants of the C180 peptide which possessed amino acid substitutions at residue 26 (collectively termed C180W26n peptides) were purified from periplasmic extracts of Escherichia coli. Each C180W26n peptide was present as a single major peptide that had an apparent molecular mass of between 20.9 and 24.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and each showed similar immunoreactivity relative to the C180 peptide. The C180W26n peptides demonstrated marked reduction of both ADP-ribosyltransferase and NAD glycohydrolase activities at 25 nM and 10 microM NAD, respectively. Kinetic analysis of the two most active mutants, C180W26F and C180W26Y, revealed that the major perturbation of NAD glycohydrolase activity was due to an increase (approximately 20-fold) in the Km for NAD between these mutants and the C180 peptide.
 ...
PMID:Role of tryptophan 26 in the NAD glycohydrolase reaction of the S-1 subunit of pertussis toxin. 255 99


1 2 3 4 Next >>