ADP-ribosylation Site Research Articles

ADP-ribosylation of transducin by pertussis toxin.

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [32P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma. The amino terminus of T alpha, while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation.

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.

Primary structure of elongation factor 2 around the site of ADP-ribosylation is highly conserved from archaebacteria to eukaryotes

Elongation factor 2 (EF-2) from eukaryotes and archaebacteria can be ADP-ribosylated by diphtheria toxin (DT) [(1977) Annu. Rev. Biochem. 46, 69-94; (1980) Nature 287, 250-251]. The primary structure of the ADP-ribose accepting region in EFs from the archaebacteria Thermoplasma acidophilum, Halobacterium cutirubrum and Methanococcus vannielli was determined in order to elucidate the degree of conservation compared with 4 previously established eukaryotic sequences [(1971) FEBS Lett. 103, 253-255]. Within a 9-residue sequence including the site of ADP-ribosylation 5 positions were found to be occupied by the same amino acid in all the archaebacterial and eukaryotic factors studied. There were more differences among the 3 archaebacterial sequences than among the 4 eukaryotic ones.

Antibodies against the carboxyl-terminal 5-kDa peptide of the alpha subunit of transducin crossreact with the 40-kDa but not the 39-kDa guanine nucleotide binding protein from brain.

We tested 18 antisera showing reactivity against the alpha subunit of transducin, the guanine nucleotide binding protein from rod outer segment, for crossreactivity against the 40- and 39-kDa guanine nucleotide binding proteins purified from bovine brain. A single antiserum, CW6, showed crossreactivity, and this was predominantly against the 40-kDa protein. Immunoblots of the tryptic fragments of transducin alpha subunit with multiple antisera raised against that subunit showed that only CW6 recognizes a COOH-terminal 5-kDa peptide that includes the site of pertussis toxin ADP-ribosylation. Antibodies against the 5-kDa peptide, affinity-purified from CW6, specifically react with the 40-kDa brain protein on immunoblots. The results show that the 39- and 40-kDa guanine nucleotide binding proteins from brain differ immunochemically and that the COOH-terminal 5-kDa peptide of transducin alpha subunit is homologous to a region in the 40-kDa brain protein. We speculate that this homologous region may be in a domain that confers specificity for receptor interactions of guanine nucleotide binding proteins.

Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2.

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.

The primary structure of the COOH-terminal half of cholera toxin subunit A 1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A 1, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A 1 ( M r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A 1 polypeptide. The site of self ADP-ribosylation in the A 1 subunit [ C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun. 116, 341–348 ] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A 2 subunit in the holotoxin is at position 91.

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

G proteins and dual control of adenylate cyclase

G proteins and dual control of adenylate cyclase

ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation.

Islet-activating protein catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein that mediates activation of a retinal cyclic GMP-selective phosphodiesterase. Radiolabel from [adenylate-32P]NAD+ was incorporated specifically into the alpha subunit of purified transducin. Maximal levels of incorporation approximated 0.8 mol of ADP-ribose/mol of transducin. A peptide containing the ADP-ribosyl moiety was purified from a tryptic digest of radiolabeled transducin. This peptide was characterized by chemical and enzymatic procedures and by fast atom bombardment mass spectrometry. The primary structure of this peptide was Glu-Asn-Leu-Lys-Asn(ADP-ribose)-Gly-Leu-Phe. It is probable that the peptide originated from the carboxyl terminus of the alpha subunit and that the ADP-ribosyl moiety is attached by an N-glycosidic linkage to the asparagine residue. Transducin associated with retinal disc membranes is also ADP-ribosylated by cholera toxin. Cholera toxin and islet-activating protein sequentially catalyze the incorporation of 1.9 mol of ADP-ribose/mol of transducin, indicating two distinct sites of ADP-ribosylation within transducin.

Location and amino acid sequence around the ADP-ribosylation site in the cholera toxin active subunit A 1

Renatured, S-carboxymethylated subunit A 1 of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem. Biophys. Res. Commun. 1981, 102 , 1021). In the absence of acceptor self ADP-ribosylation of A 1 subunit was observed. Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 – 0.2M PO 4(Na) buffer, pH 6.6. On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min. The ADP-ribosylated A 1 was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A 1 subunit. On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg ∗-Asp-Arg-Tyr. The Arg ∗ in the peptide was indicated to be the site of ADP-ribosylation.

Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2.

Diphtheria toxin inactivates protein synthesis elongation factor 2 by catalyzing the ADP-ribosylation of a novel derivative of histidine, diphthamide, in the protein (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). In this report, we describe experiments involving nuclear Overhauser enhancement NMR spectroscopy which were undertaken to elucidate the site of ADP-ribosylation of diphthamide and the configuration of the glycosidic bond formed by the toxin. The essential result of these experiments is that, in ribosyl-diphthamide obtained by enzymatic digestion of ADP-ribosyl-elongation factor-2, the H-5 imidazole proton is near the R-4 proton of ribose. This result and others are consistent with the interpretation that diphtheria toxin covalently attaches ADP-ribose to the imidazole N-1 of diphthamide via an alpha-glycosidic linkage.

ADP ribosylation of rat liver lysine-rich histone in vitro.

Purified rat liver nuclei were incubated with [14C]-NAD+ and the various nuclear protein fractions were separated. Forty per cent of the total radioactivity incorporated was associated with the histone fraction. Of this, about 50% was extracted with H1, in 0.5 N perchloric acid. When crude H1 was purified and fractionated into five different subfractions by chromatography on Bio-Rex 70, it was found that all the H1 subfractions contained radioactivity. This radioactive material was identified as oligomers of adenosine diphosphate ribose (ADP-Rib) with an average chain length which corresponded to trimers. The extent of the modification was dependent on the concentration of NAD+. About 60% of the H1 molecules were modified with a concentration of 1 mM NAD+. The presence of these oligomers of ADP-Rib introduced a large degree of microheterogeneity to H1 as detected by electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid. Bands of H1 with 10 to 20% less mobility than the unmodified H1 were present. Also, as a consequence of large content of ADP-Rib, the absorption maximum shifted from 275 to 259 nm. The half-life of the bond between the oligomers of ADP-Rib and H1 was about 3 min at 37 degrees C in the presence of 0.1 N NaOH, and 10 m1 were modified. The site of ADP ribosylation in the NH2-terminal half was localized in the tryptic peptide extending from the NH2-terminal end to lysine 15. The site of modification of the COOH-erminal half was localized in the tryptic peptide which contained the only glutamic acid residue in this fragment of H1...

Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2.

We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.