Thymine Residues Research Articles

Pyrimidine oligodeoxyxylonucleotides form triplexes with purine DNA in neutral media

Interactions of oligonucleotides comprising (1-beta-D-2'-deoxy-threo-pentafuranosyl)thymine and (1-beta-D-2'-deoxy-threo-pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine-polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B.

Catechol and hydroquinone have different redox properties responsible for their differential DNA-damaging ability.

We examined the redox properties of the "carcinogenic" catechol and the "noncarcinogenic" hydroquinone in relation to different DNA damaging activities and carcinogenicity using 32P-labeled DNA fragments obtained from the human genes. In the presence of endogenous NADH and Cu2+, catechol induces stronger DNA damage than hydroquinone, although the magnitudes of their DNA damaging activities were reversed in the absence of NADH. In both cases, DNA damage resulted from base modification at guanine and thymine residues in addition to strand breakage induced by Cu+ and H2O2, generated during the oxidation of catechol and hydroquinone into 1,2-benzoquinone and 1,4-benzoquinone, respectively. EPR and 1H NMR studies indicated that 1,2-benzoquinone is converted directly into catechol through a nonenzymatic two-electron reduction by NADH whereas 1,4-benzoquinone is reduced into hydroquinone through a semiquinone radical intermediate through two cycles of one-electron reduction. The reduction of 1,2-benzoquinone by NADH proceeds more rapidly than that of 1,4-benzoquinone. This study demonstrates that the rapid 1,2-benzoquinone two-electron reduction accelerates the redox reaction turnover between catechol and 1,2-benzoquinone, resulting in the enhancement of DNA damage. These results suggest that the differences in NADH-mediated redox properties of catechol and hydroquinone contribute to their different carcinogenicities.

A "master" in base unpairing during isomerization of a promoter upon RNA polymerase binding.

Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the -10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the -10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A small middle dotT base pair at the -11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a "master" role of the adenine base at -11 of the template strand in overall base unpairing. 2-AP at -11 did not inhibit the formation of RNA polymerase small middle dotpromoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, -11.

Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide.

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site‐specific DNA damage induced by hydroxyurea using 32P‐5′‐end‐labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c‐Ha‐ras‐1 protooncogene. Hydroxyurea caused Cu(II)‐mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea‐induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)‐specific chelator, suggesting the involvement of hydrogen peroxide (H2O2) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto‐hydroxyl radicals such as Cu(I)‐hydroperoxo complex (Cu(I)‐OOH) cause DNA damage. Formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) was induced by hydroxyurea in the presence of Cu(H). An electron spin resonance spectroscopic study using N‐(dithiocarboxy)sarcosine as a nitric oxide (NO)‐trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography‐mass spectrometry (GC‐MS) and time‐of‐flight‐mass spectrometry (TOF‐MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H2O2‐independent manner, and endonu‐clease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti‐cancer activity, but also carcinogenesis.

Binding activity of replication protein A to single-stranded DNA containing oxidized pyrimidine base.

To obtain the information for the role of replication protein A (RPA) on the detection of oxidized lesion in the single-stranded DNA, the binding preference of RPA purified from Xenopus egg lysate against the oligonucleotide containing one of three kinds of oxidized thymine residues, 5-formyluracil, 5-hydroxymethyluracil and 5-(1,2-dihydroxyethyl)uracil, was studied by the gel shift assay. Results of competition assay indicate that RPA preferentially binds to the oligonucleotide containing these oxidized thymine residues than the undamaged DNA.

A G577R Mutation in the Human AR P Box Results in Selective Decreases in DNA Binding and in Partial Androgen Insensitivity Syndrome

We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the Cβ of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.

Acrylonitrile enhances H2O2-mediated DNA damage via nitrogen-centered radical formation.

Acrylonitrile (ACN) is widely used as a monomer in the polymer industry. Studies on carcinogenicity in rats exposed to ACN showed increased incidences of tumors including glial cell tumors of central nervous system and increased production of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in glial cells. Using a high performance liquid chromatograph equipped with an electrochemical detector, we revealed that ACN enhanced the formation of 8-oxo-dG induced by H2O2 and Cu(II) whereas ACN itself did not cause DNA damage. The enhancing effect of ACN was much more efficient in the double-stranded DNA than that in the single-stranded DNA. Experiments with 32P-labeled DNA revealed that addition of ACN enhanced the site-specific DNA damage at guanines, particularly at 5'-site of the GG and GGG sequences while H2O2/Cu(II) induced piperidine-labile sites at thymine, cytosine, and guanine residues. An electron spin resonance spectroscopy using alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone showed that a nitrogen-centered radical was generated from ACN in the presence of H2O2 and Cu(II). It is considered that ACN enhances H2O2-mediated DNA damage via nitrogen-centered radical formation. We will discuss the mechanism of the enhancing effect on oxidative DNA damage in relation to expression of ACN carcinogenicity.

Photo-Cross-Linked Oligonucleotide Duplex as a Decoy-DNA for Inhibition of Restriction Endonuclease Activity

As a novel type of regulator molecule for DNA-recognizing proteins, a photo-cross-linked oligonucleotide duplex was designed and synthesized. The molecule regulated the activity of a restriction endonuclease by being recognized as a substrate. This type of regulating molecule is regarded as a decoy-DNA. 4,5′,8-[4-Aminoethylaminomethyl]-trioxalen (aeAMT) was conjugated with an oligodeoxyribonucleotide (ODN) at the 5′-end and the aeAMT was cross-linked with the thymine residue of the complementary oligonucleotide upon UVA irradiation. The terminally cross-linked oligonucleotides, singly clipped (SC) decoy-DNA, acquired thermal stability. An oligonucleoside phosphorothioate (OPT) was also introduced as one or both components, yielding three types of decoy-DNAs, SC-ODN-ODN (SC.DD), SC-OPT-ODN (SC.SD), and SC-OPT-OPT (SC.SS). The SC decoy-DNAs inhibited the function of the restriction endonuclease, AatII, in a sequence-specific and concentration-dependent manner with an appreciable IC 50 value (1.3 μM for SC.DD, 0.016 μM for SC.SD, 0.002 μM for SC.SS). The SC decoy-DNAs were found to be effective for regulating the DNA recognizing proteins.

Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol.

Catechol, a naturally occurring and an important industrial chemical, has been shown to have strong promotion activity and induce glandular stomach tumors in rodents. In addition, catechol is a major metabolite of carcinogenic benzene. To clarify the carcinogenic mechanism of catechol, we investigated DNA damage using human cultured cell lines and 32P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 proto-oncogene. Catechol increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is known to be correlated with the incidence of cancer, in a human leukemia cell line HL-60, whereas the amount of 8-oxodG in its hydrogen peroxide (H2O2)-resistant clone HP100 was not increased. The formation of 8-oxodG in calf thymus DNA was increased by catechol in the presence of Cu(2+). Catechol caused damage to 32P-labeled DNA fragments in the presence of Cu(2+). When NADH was added, DNA damage was markedly enhanced and clearly observed at relatively low concentrations of catechol (<1 microM). DNA cleavage was enhanced by piperidine treatment, suggesting that catechol plus NADH caused not only deoxyribose phosphate backbone breakage but also base modification. Catechol plus NADH frequently modified thymine residues. Bathocuproine, a specific Cu(+) chelator and catalase inhibited the DNA damage, indicating the participation of Cu(+) and H2O2 in DNA damage. Typical hydroxyl radical scavengers did not inhibit catechol plus Cu(2+)-induced DNA damage, whereas methional completely inhibited it. These results suggest that reactive species derived from the reaction of H2O2 with Cu(+) participates in catechol-induced DNA damage. Therefore, we conclude that oxidative DNA damage by catechol through the generation of H2O2 plays an important role in the carcinogenic process of catechol and benzene.

Oxidative DNA Damage Induced by a Metabolite of Carcinogenic o-Anisidine: Enhancement of DNA Damage and Alteration in Its Sequence Specificity by Superoxide Dismutase

The mechanism of DNA damage by a metabolite of the carcinogen o-anisidine in the presence of metals was investigated by the DNA sequencing technique using 32P-labeled human DNA fragments. The o-anisidine metabolite, o-aminophenol, caused DNA damage in the presence of Cu(II). The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine by o-aminophenol increased in the presence of Cu(II). We conclude that Cu(II)-mediated oxidative DNA damage by this o-anisidine metabolite seems to be relevant for the expression of the carcinogenicity of o-anisidine. o-Aminophenol plus Cu(II) caused preferential DNA damage at the 5′-site guanine of GG and GGG sequences. When CuZn-SOD or Mn-SOD was added, the DNA damage was enhanced and its predominant cleavage sites were changed into thymine and cytosine residues. We consider that SOD may increase the frequency of mutations due to DNA damage induced by o-aminophenol and thus increase its carcinogenic potential.

Time-Resolved Resonance Raman Study of the Exciplex Formed between Excited Cu−Porphyrin and DNA

The photoinduced reversible process of exciplex formation and decay between the water-soluble cationic metalloporphyrin 5,10,15,20-tetrakis[4-(N-methylpyridyl)]porphyrin (Cu(T4MPyP)) and calf-thymus DNA has been studied by a picosecond time-resolved resonance Raman (ps-TR3) technique. For a detailed analysis of the exciplex properties, the following model compounds have also been investigated: double-stranded polynucleotides poly(dA-dT)2, poly(dG-dC)2, and poly(dA-dC)·poly(dG-dT), single-stranded poly(dT), and the 32-mer d[(GC)7ATAT(GC)7]2. Additional Raman measurements have also been done in using cw and 20-ns laser sources. It is shown that this reversible exciplex is formed, with a yield depending on the nucleic base sequence, in less than 2 ps after photoexcitation, between photoexcited Cu(T4MPyP) and CO groups of thymine residues in all thymine-containing sequences of nucleic acids. Such a rapid exciplex building process implies that it involves porphyrin molecules initially located, in the steady st...

Superoxide dismutases enhance H 2O 2-induced DNA damage and alter its site specificity

Superoxide dismutases (SODs) are involved in the protection of cells from oxygen toxicity. However, several papers have reported that the overexpression of CuZn-SOD causes oxidative damage to cells. We investigated a mechanism by which an excess of SODs accelerates oxidative stress. The presence of CuZn-SOD, Mn-SOD or Mn(II) enhanced the frequency of DNA damage induced by hydrogen peroxide (H 2O 2) and Cu(II), and altered the site specificity of the latter: H 2O 2 induced Cu(II)-dependent DNA damage with high frequency at the 5′-guanine of poly G sequences; when SODs were added, the frequency of cleavages at thymine and cytosine residues increased. SODs also enhanced the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine by H 2O 2 and Cu(II). We conclude that SODs may increase carcinogenic risks, e.g. of tumors in Down syndrome.

Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.

Strains of methicillin-resistant Staphylococcus aureus (MRSA) have become the most important causative agents of hospital-acquired diseases worldwide. The genetic determinant of resistance, mecA, is not a gene native to S. aureus but was acquired from an extraspecies source by an unknown mechanism. We recently identified a close homologue of this gene in isolates of Staphylococcus sciuri, a taxonomically primitive staphylococcal species recovered most frequently from rodents and primitive mammals. In spite of the close sequence similarity between the mecA homologue of S. sciuri and the antibiotic resistance determinant mecA of S. aureus, S. sciuri strains were found to be uniformly susceptible to beta-lactam antibiotics. In an attempt to activate the apparently "silent" mecA gene of S. sciuri, a methicillin-resistant derivative, K1M200 (for which the MIC of methicillin is 200 microg/ml), was obtained through stepwise exposure of the parental strain S. sciuri K1 (methicillin MIC of 4 microg/ml) to increasing concentrations of methicillin. DNA sequencing of the mecA homologue from K1M200 revealed the introduction of a point mutation into the -10 consensus of the promoter: the replacement of a thymine residue at nucleotide 1577 in the susceptible strain K1 by adenine in the resistant strain K1M200, which was accompanied by a drastic increase in transcription rate and the appearance of a new protein that reacted with monoclonal antibody prepared against the penicillin-binding protein 2A (PBP2A), i.e., the gene product of S. aureus mecA. Transduction of mecA from K1M200 (cloned into a plasmid vector) into a methicillin-susceptible S. aureus mutant resulted in a significant increase of methicillin resistance (from a methicillin MIC of 4 micro/ml to 12 and up to 50 microg/ml), the appearance of a low-affinity PBP detectable by the fluorographic assay, and the production of a protein that reacted in a Western blot with monoclonal antibody to PBP2A. Antibiotic resistance and the protein products disappeared upon removal of the plasmid-borne mecA homologue. The observations support the proposition that the mecA homologue ubiquitous in the antibiotic-susceptible animal species S. sciuri may be an evolutionary precursor of the methicillin resistance gene mecA of the pathogenic strains of MRSA.

Mechanism of oxidative DNA damage induced by carcinogenic 4-aminobiphenyl

DNA adduct formation is thought to be a major cause of DNA damage by carcinogenic aromatic amines. We investigated the ability of an aromatic amine, 4-aminobiphenyl (4-ABP) and its N-hydroxy metabolite (4-ABP(NHOH)) to cause oxidative DNA damage, using 32P-labeled human DNA fragments from the p53 tumor suppressor gene and the c-Ha- ras-1 protooncogene. 4-ABP(NHOH) was found to cause Cu(II)-mediated DNA damage, especially at thymine residues. Addition of the endogenous reductant NADH led to dramatic enhancement of this process. Catalase and bathocuproine, a Cu(I)-specific chelator, reduced the amount of DNA damage, suggesting the involvement of H 2O 2 and Cu(I). 4-ABP(NHOH) dose-dependently induced 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in the presence of Cu(ll) and NADH. 4-ABP(NHOH) conversion to nitrosobiphenyl, as measured by UV-visible spectroscopy, occurred rapidly in the presence of Cu(II), suggesting Cu(II)-mediated autoxidation. Increased amounts of 8-OHdG were found in HL-60 cells compared to the H 2O 2-resistant clone HP100 following 4-ABP(NHOH) treatment, further supporting the involvement of H 2O 2. The present study demonstrates that an N-hydroxy derivative of 4-ABP induces oxidative DNA damage through H 2O 2 in both a cell-free system and in cultured human cells. We conclude that, in addition to DNA adduct formation, oxidative DNA damage may play an important role in the carcinogenic process of 4-ABP.

Haplotype analyses with the human leucocyte antigen and tumour necrosis factor-alpha genes in narcolepsy families.

Our previous study suggested that the tumour necrosis factor-alpha gene with thymine residue at position -857 in its promoter region [TNF-alpha(-857T)] could be associated with human narcolepsy independently of a strong association of the human leucocyte antigen (HLA)-DRB1*1501 with the disorder. To understand the relationship of DRB1*1501 with TNF-alpha(-857T) in narcoleptic patients, we investigated 28 members of four Japanese narcolepsy families and determined the haplotypes with the HLA-B, TNF-alpha(-857C/T) and HLA-DRB1 in the members. The resultant haplotypes indicated that not only the DRB1*1501-TNF-alpha(-857C) haplotype but also the DRB1*1501-TNF-alpha(-857T) haplotype, which is rare in healthy individuals and may have a strong predisposition to the disorder, were present in the affected members. From the chromosomal recombination observed in a few members, it is possible that chromosomal recombination could play a role in the generation of the rare DRB1*1501-TNF-alpha(-857T) haplotype.

Characterization of DNA damage at purine residues in oligonucleotides and calf thymus DNA induced by the mutagen 1-nitrosoindole-3-acetonitrile.

N-Nitrosoindoles can efficiently transfer the nitroso group to nucleophilic targets in isolated purine nucleotides, causing depurination, deamination, and the formation of a novel guanine analogue, oxanine [Lucas, L. T., Gatehouse, D., and Shuker, D. E. G. (1999) J. Biol. Chem. 274, 18319-18326]. To determine the likely biological relevance of these modification pathways, the reactivity of 1-nitrosoindole-3-acetonitrile (NIAN), a model 3-substituted N-nitrosoindole, with oligonucleotides and calf thymus DNA was examined at physiological pH and temperature. Reaction of NIAN with single-stranded oligonucleotides containing various guanine motifs resulted in the production of single-strand break products at guanine sites due to the formation of alkali-labile lesions. The number of lesions increased with NIAN concentration and incubation time. Modification of calf thymus DNA by NIAN resulted in depurination, which gave the corresponding purine bases, deamination coupled with depurination, which gave xanthine, and the formation of oxanine. The former pathway was clearly the most important, and all reaction products exhibited a dose-response relationship. Cytosine and thymine residues were inactive toward NIAN. Further studies revealed an additional product in NIAN-treated duplex DNA containing a CCGG motif that was characterized as an interstrand cross-link, the yield of which increased with increasing NIAN concentration. These results indicate that the transnitrosating ability of NIAN to modify purine residues is preserved at the macromolecular level, with guanine residues appearing to be a primary site of reaction. All of these modification processes are potentially mutagenic events if they occur in vivo.

Conformational polymorphism and extensibility of DNA quadruplexes formed from d(GT)n repeats

We showed earlier that oligonucleotides 3'-d(GT)5-pO(CH2CH2O)3p-d(GT)5-3' form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K = (3.3 +/- 0.1) x 10(4) M-1, cooperativity coefficient omega = 2.5 +/- 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N = 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.

Catechol estrogens induce oxidative DNA damage and estradiol enhances cell proliferation.

Estrogen-induced carcinogenesis involves enhanced cell proliferation (promotion) and genotoxic effects (initiation). To investigate the contribution of estrogens and their metabolites to tumor initiation, we examined DNA damage induced by estradiol and its metabolites, the catechol estrogens 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)). In the presence of Cu(II), catechol estrogens formed piperidine-labile sites at thymine and cytosine residues in (32)P 5'-end-labeled DNA fragments and induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine. NADH markedly enhanced Cu(II)-dependent DNA damage mediated by nanomolar concentrations of catechol estrogens. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that H(2)O(2), generated during Cu(II)-catalyzed autoxidation of catechol estrogens, reacts with Cu(I) to form the Cu(I)-peroxide complex, leading to oxidative DNA damage, and that NADH enhanced DNA damage through the formation of redox cycle. To investigate the role of estrogens and their metabolites in tumor promotion, we examined their effects on proliferation of estrogen-dependent MCF-7 cells. Estradiol enhanced the proliferation of MCF-7 cells at much lower concentrations than catechol estrogens. These findings indicate that catechol estrogens play a role in tumor initiation through oxidative DNA damage, whereas estrogens themselves induce tumor promotion and/or progression by enhancing cell proliferation in estrogen-induced carcinogenesis.

Stereoselective Synthesis of RP- and SP-Dithymidine Phosphorothioates via Chiral Indolooxazaphosphorine Intermediates Derived from Tryptophan

A chiral auxiliary 1 and its enantiomer, which are easily prepared from L- and D-tryptophan, facilitate diastereoselective syntheses of phosphorothioate oligonucleotides. Phosphorothioate 2 was obtained from 1 in five steps with over 95 % de, and the enantiomer of 1 afforded (SP)-2 with an equally good de value. T=thymine residue. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2000/z15468_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A Polymorphic Protease-activated Receptor 2 (PAR2) Displaying Reduced Sensitivity to Trypsin and Differential Responses to PAR Agonists

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR(2)F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe(240)) and 0.084 (Ser(240)) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR(2)F240S displayed a significant reduction in sensitivity toward trypsin ( approximately 3.7-fold) and the PAR2-activating peptides, SLIGKV-NH(2) ( approximately 2.5-fold) and SLIGRL-NH(2) ( approximately 2.8-fold), but an increased sensitivity toward the selective PAR2 agonist, trans-cinnamoyl-LIGRLO-NH(2) ( approximately 4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH(2) ( approximately 7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH(2) and a PAR4-derived peptide, trans-cinnamoyl-YPGKF-NH(2), were selective PAR(2)F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR(2)F240S, suggesting that Phe(240) is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.