In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides.

Emphasis was placed in this work on the assessment of biological features of 2,2,4-triaminooxazolone, a major one-electron and(. )OH-mediated oxidation product of guanine. For this purpose, two oligonucleotides that contain a unique oxazolone residue were synthesized. Herein we report the mutagenic potential of oxazolone during in vitro DNA synthesis and its behavior towards DNA repair enzymes. Nucleotide insertion opposite oxazolone, catalyzed by Klenow fragment exo(-)and Taq polymerase indicates that the oxazolone lesion induces mainly dAMP insertion. This suggests that the formation of oxazolone in DNA may lead to G-->T transversions. On the other hand, oxazolone represents a blocking lesion when DNA synthesis is performed with DNA polymerase beta. Interestingly, DNA repair experiments carried out with formamidopyrimidine DNA N -glycosylase (Fpg) and endonuclease III (endo III) show that oxazolone is a substrate for both enzymes. Values of k (cat)/ K (m)for the Fpg-mediated removal of oxidative guanine lesions revealed that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than oxazolone. In the case of endo III-mediated cleavage of modified bases, the present results suggest that oxazolone is a better substrate than 5-OHC, an oxidized pyrimidine base. Finally, MALDI-TOF-MS analysis of the DNA fragments released upon digestion of an oxazolone-containing oligonucleotide by Fpg gave insights into the enzymatic mechanism of oligonucleotide cleavage.

Phosphorylation of human sperm protamines HP1 and HP2: identification of phosphorylation sites.

Human sperm is characterized by a high heterogeneity of its basic nuclear protein complement of pro-protamines, protamines and histones. This heterogeneity is increased by the persistence of phosphorylated protamines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to various degrees. Presence of non-phosphorylated and phosphorylated protamines HP1 and HP2 was further demonstrated by electrospray mass spectrometry. Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were identified by automatic Edman degradation of the protein after phosphoserine derivatization to S-ethylcysteine. In both phosphorylated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer electrophoresis.

Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties.

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.

A corrected primary structure for dog-fish Scylliorhinus caniculus protamine Z3.

We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.

Purification and electrospray mass spectrometry of aldose reductase from pig lens.

Aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified from pig lens to homogeneity by a rapid and efficient three-step procedure involving poly(ethylene glycol) fractionation, ion-exchange chromatography and chromatofocusing. The homogeneity of the purified enzyme was examined by polyacrylamide gel electrophoresis under native and denaturing conditions, by isoelectric focusing and by high-performance liquid chromatography on a size-exclusion column. The highly purified enzyme is a monomeric protein with a molecular mass of 35,775 +/- 3 Da as determined by electrospray mass spectrometry (ESMS). This purification procedure is particularly suited for the preparation of triclinic single crystals of pig lens aldose reductase, which are currently used in X-ray studies of this enzyme.

A structural homologue of colipase in black mamba venom revealed by NMR floating disulphide bridge analysis.

The solution structure of mamba intestinal toxin 1 (MIT1), isolated from Dendroaspis polylepis polylepis venom, has been determined. This molecule is a cysteine-rich polypeptide exhibiting no recognised family membership. Resistance to MIT1 to classical specific endoproteases produced contradictory NMR and biochemical information concerning disulphide-bridge topology. We have used distance restraints allowing ambiguous partners between S atoms in combination with NMR-derived structural information, to correctly determine the disulphide-bridge topology. The resultant solution structure of MIT1, determined to a resolution of 0.5 A, reveals an unexpectedly similar global fold with respect to colipase, a protein involved in fatty acid digestion. Colipase exhibits an analogous resistance to endoprotease activity, indicating for the first time the possible topological origins of this biochemical property. The biochemical and structural homology permitted us to propose a mechanically related digestive function for MIT1 and provides novel information concerning snake venom protein evolution.

Electrospray ionization mass spectrometry analysis of the apo- and metal-substituted forms of the Fur protein.

Fur has been purified and reconstituted with Co2+ and Mn2+. The ESI-MS spectra of the apoprotein as well as Mn-Fur and Co-Fur under acidic denaturating conditions showed the existence of two species of molecular mass 16,660 +/- 3 and 16,792 +/- 3 Da, which correspond, respectively, to the N-terminal methionine 'excised' or 'non-excised' forms of the monomer. This result proves the absence of any other post-translational modification or modification due to metal incorporation. On the other hand, under soft conditions, ESI spectra provided for the first time direct evidence for dimeric metal-containing forms in solution.

Stability study of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry.

To estimate the stability of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants, charge state distributions and hydrogen/deuterium exchange rates were monitored by electrospray ionization mass spectrometry. The relative stability of the mutants was observed with the order: V11 insert > Y75F > wild-type = K32E > K12D = K14E > or = K52E > K14E/K32E > W67Y > P35A > I57N > G34S. (A preliminary account has been presented for mutants G34S and P35A [Jaquinod et al. (1995) Rapid Commun. Mass Spectrom. 9, 1135-1140].) This approach is shown to be a useful tool for rapid characterization of mutational effects on protein conformation.

Synthesis and characterization of photoactivatable peptide agonists of the human thrombin receptor.

Chemical synthesis and biochemical analysis of modified agonist peptides of the human thrombin receptor derived from the sequence SFLLRNP containing photoactivatable azido groups and biotin for sensitive detection is described. Substitution of leucine in position three with p-azidophenylalanine and extension of the C-terminus with a KGGK spacer containing biotin covalently linked to the side chain of the C-terminal lysine residue resulted in an active receptor agonist as determined by intracellular Ca2+ mobilization in human erythroleukemia (HEL) cells. In contrast, substitution of phenylalanine in position two with p-azidophenylalanine reduced agonist activity significantly.

New conformational properties induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough ferricytochrome c553 using isotopic exchanges monitored by mass spectrometry.

In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.

MALDI-TOF mass spectrometry as a powerful tool to study enzymatic processing of DNA lesions inserted into oligonucleotides.

MALDI-TOF mass spectrometry measurements, coupled with either exonuclease or DNA N-glycosylases digestions of lesion-containing oligonucleotides, were used to assess biochemical features of several oxidative DNA damage. The latter analytical approach was shown to be an informative and efficient alternative technique to conventional electrophoresis and chromatographic analyses.

Processing in the C-terminal domain of minicollagen XII removes a heparin-binding site.

A minicollagen comprising the two C-terminal domains of collagen XII (COL1 and NC1) has been expressed in insect cells and characterized biochemically. An interaction with heparin is demonstrated, which depends on the correct folding of the molecule. After secretion, minicollagen XII is immediately processed to a form lacking heparin binding ability. Processed and unprocessed trimers differ only at the level of the eight or nine C-terminal residues but they reveal different structures as judged from rotary shadowing images. Similar processing is also observed in the medium of transfected human HeLa cells. These data show that a heparin-binding site is present in the C-terminal end of the chicken collagen XII sequence and strongly suggest that proteolytic processing in the NC1 domain can occur in vivo and regulate the interactive properties of collagen XII.

Characterization of Dac g 4, a major basic allergen from Dactylis glomerata pollen.

Monoclonal antibodies were produced against Dac g 4, a purified major basic allergen from Dactylis glomerata pollen. Their ability to be used for immunopurification of Dac g 4 was studied on a BIAcore apparatus (Pharmacia). The allergen was purified by affinity chromatography with one monoclonal antibody. Its precise molecular mass, 59,185 +/- 30 d, was determined by mass spectrometry. Its isoelectric point is 10.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting showed that Dac g 4-related proteins of similar molecular mass were detected in the majority of allergenic grass pollen species. By double-site ELISAs, we have estimated that Dac g 4 represents about 6% of the total proteins from a water-soluble extract. One monoclonal antibody (mAb H) recognized a 60 kd cross-reactive protein in other grass pollens, though none in any of the tree or weed pollens tested. Inhibition studies of IgE antibody binding to Dac g 4 with pollen extracts confirmed the presence of cross-reactive allergens in Secale cereale, Lolium perenne, Festuca elatior, Holcus lanatus, Bromus arvensis, Poa pratense, Hordeum sativum, and Phleum pratense.

Fatty acid and lipoic acid biosynthesis in higher plant mitochondria.

Fatty acid and lipoic acid biosynthesis were investigated in plant mitochondria. Although the mitochondria lack acetyl-CoA carboxylase, our experiments reveal that they contain the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein (ACP). We demonstrated, by a new method based on a complementary use of high performance liquid chromatography and mass spectrometry, that the soluble mitochondrial fatty-acid synthase produces mainly three predominant acyl-ACPs as follows: octanoyl(C8)-, hexadecanoyl(C16)-, and octadecanoyl(C18)-ACP. Octanoate production is of primary interest since it has been postulated long ago to be a precursor of lipoic acid. By using a recombinant H apoprotein mutant as a potential acceptor for newly synthesized lipoic acid, we were able to detect limited amounts of lipoylated H protein in the presence of malonate, several sulfur donors, and cofactors. Finally, we present a scheme outlining the new biochemical pathway of fatty acid and lipoic acid synthesis in plant mitochondria.

Repair and coding properties of 5-hydroxy-5-methylhydantoin nucleosides inserted into DNA oligomers.

1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) (3) has been shown to be a major oxidation product of thymidine formed upon exposure of DNA to (*)OH-radical and excited photosensitizers. To investigate the biological and structural significance of the 5-OH-5-Me-dHyd residue to DNA, the latter modified 2'-deoxyribonucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides. This was efficiently achieved using the phosphoramidite approach that involved mild deprotection conditions. The purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The piperidine test applied to 5-OH-5-Me-dHyd containing oligonucleotides showed a weak instability of hydantoin nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biochemical features of such a DNA lesion were carried out. Thus, processing of 5-OH-5-Me-dHyd by nuclease P(1), snake venom phosphodiesterase, and calf spleen phosphodiesterase was investigated. The specificity and the mechanism of excision of the lesion by several bacterial and yeast DNA N-glycosylases, namely, endonuclease III (endo III), endonuclease VIII (endo VIII), formamidopyrimidine DNA N-glycosylase (Fpg), Ntg1 protein (Ntg1), Ntg2 protein (Ntg2), and Ogg1 protein (yOgg1), were also determined. These repair studies clearly showed that all these enzymes, with the exception of the yOgg1 protein, are able to recognize and remove 5-hydroxy-5-methylhydantoin from the double-stranded DNA fragment. Finally, a 22-mer DNA oligomer bearing a 5-OH-5-Me-dHyd residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase, and DNA polymerase beta. Thus, it may be concluded that the oxidized thymine residue is a strongly blocking lesion for the three studied DNA polymerases.

The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer.

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which migrates exclusively as a monomer and for the Cys108-->Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67-->Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108-->Ser mutant, which show that they both contain a Cys67-Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356-Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579-585].

Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects.

Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg). Three techniques of analysis were employed. A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage. Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT. This confirms that Tg is an excellent substrate for endonuclease III. Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III. Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine. While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS. From these data it may be suggested that 5-OHC could be modified during its enzymatic excision. Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected.

Synthesis and biochemical properties of cyanuric acid nucleoside-containing DNA oligomers.

1-(2-Deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers. To investigate the biological and structural significance of dY residue in DNA, the latter modified 2'-deoxynucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using the phosphoramidite approach. For this purpose, a convenient glycosylation method involving 3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) was devised. The anomeric mixture of modified 2'-deoxyribonucleosides (1/2 alpha/beta) was resolved by silica gel purification of the 5'-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired beta-phosphoramidite monomer (5). After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double-stranded DNA fragment as inferred from melting temperature measurements. The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease P(1), snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of E. coli polymerase I.

Study of the new stability properties induced by amino acid replacement of tyrosine 64 in cytochrome C553 from Desulfovibrio vulgaris Hildenborough using electrospray ionization mass spectrometry.

Hydrogen/deuterium exchange as well as charge state distribution monitored by electrospray ionization mass spectrometry were demonstrated to be a powerful and effective new tool for probing conformational properties of proteins in solution. In this paper, the influence of single amino acid replacements on the global conformation of cytochrome C553 from Desulfovibrio vulgaris Hildenborough using isotopic exchange monitored by electrospray ionization mass spectrometry is reported. Based on their respective charge state distributions and isotopic exchanges, we have differentiated relative stability of mutants and a ladder classification with the order being wild-type > Y64F = Y64L > Y64V > Y64A, under specific conditions of pH, is proposed.

Mass spectrometric characterisation of post-translational modification and genetic variation in human tetranectin.

Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein primarily involved in tissue remodeling and development, was scanned for covalent modifications and sequence heterogeneity, using a combination of mass spectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of different mass and abundance in plasma tetranectin, all of higher mass than that calculated from the cDNA sequence. To identify and locate residues accounting for the heterogeneity, samples of tetranectin were subjected to proteolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry and, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due to sequence heterogeneity at position 85 and the presence of a partially sialylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is encoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixture of Ser85 and Gly-85 sequence variants. Mass spectrometric analysis of enzymatic and mild acid hydrolysates of an N-terminal glycopeptide showed that the composition and partial covalent structure of the O-linked oligosaccharide prosthetic group is < or =N-acetylhexosamine < or =[hexose, (sialic acid)0-3].