Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases kappa and zeta in human cells.

Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5' base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Pol kappa and Pol zeta function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Pol kappa at the insertion step and of Pol zeta at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.

Mutations in the regulatory domain of phenylalanine hydroxylase and response to tetrahydrobiopterin.

Tetrahydrobiopterin (BH4) is a co-factor that enhances the activity of other enzymes, and this co-factor level is found to be affected in phenylketonuria (PKU), an amino acid metabolism disorder. The present study was aimed at understanding the effect of BH4 on mutations in the regulatory domain of phenylalanine hydroxylase (PAH). Among 14 patients, 5 patients were classical PKU, 3 were atypical PKU, and 6 were mild PKU. All of these patients had at least one mutation in the regulatory domain. Patients were given 10 mg/kg BH4, and the response of blood phenylalanine (Phe) levels was monitored following treatment. The level of blood Phe decreased after BH4 treatment in all of the patients. These studies suggest that mutations in the regulatory domain also responded to BH4 even if the patient had classical PKU.

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice.

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögren's syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.

Response of phenylketonuria to tetrahydrobiopterin.

A favorable response, indicated by decline of blood phenylalanine (Phe) in patients with phenylketonuria (PKU), to orally administered 6-R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) has been reported in many countries following the first publication in 1999. In this review, we describe the experience in the United States with PKU patients and their response to BH4. A significant response to BH4 is arbitrarily considered as a decrease of 30% or greater of blood Phe concentration 24 h after administration of BH4. In our studies, 18 of 37 patients with PKU (49%) responded to oral BH4 by >30% decrease in blood Phe concentration. Four PKU patients responded with a decrease of blood Phe concentration between 17.3 and 26.3%. It is suggested that patients with sufficient response to BH4 are candidates who will benefit from BH4 as it becomes available for PKU management. In a separate trial, 20 patients with PKU were screened with ascending doses of BH4: 10, 20, and 40 mg/kg. A favorable response was found in 10 subjects (50%) after 10 mg/kg BH4 and 14 subjects (70%) after 20 mg/kg BH4. There was no additional advantage to 40 mg/kg BH4. A 1-wk trial with 10 and 20 mg/kg BH4 in the same 20 patients showed blood Phe concentrations lowest after 7 d of BH4. The BH4-responsive patients were genotyped and most were compound heterozygotes with 1 mild mutation on 1 allele, responsible for the increase of the residual activity of Phe hydroxylase when BH4 was added. Individuals with the same genotype exhibit different responses upon administration of BH4, attributed to epigenetic factors, such as the metabolic makeup of the individual. Patients with PKU, regardless of their genotype or classification, need to be screened for response to BH4. The majority of patients are identified by 10 mg/kg BH4.

Bimodal occurrence of aspartoacylase in myelin and cytosol of brain.

The growing use of N-acetylaspartate as an indicator of neuronal viability has fostered interest in the biological function(s) of this unusual amino acid derivative. In considering the various physiological roles that have been proposed for this relatively abundant molecule one is obliged to take into account its unusual metabolic compartmentalization, according to which synthesis and storage occur in the neuron and hydrolytic cleavage in the oligodendrocyte. The latter reaction, catalyzed by aspartoacylase (ASPA), produces acetyl groups plus aspartate and has been proposed to occur in both soluble and membranous subfractions of white matter. Our study supports such bimodal occurrence and we now present immunoblot, proteomic, and biochemical evidence that the membrane-bound form of ASPA is intrinsic to purified myelin membranes. This was supported by a novel TLC-based method for the assay of ASPA. That observation, together with previous demonstrations of numerous lipid-synthesizing enzymes in myelin, suggests utilization of acetyl groups liberated by myelin-localized ASPA for lipid synthesis within the myelin sheath. Such synthesis might be selective and could explain the deficit of myelin lipids in animals lacking ASPA.

Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120.

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.

Toward selective covalent inactivation of pathogenic antibodies: a phosphate diester analog of vasoactive intestinal peptide that inactivates catalytic autoantibodies.

We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys(20) residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys(20) enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.

Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity.

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421- 431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421- 436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421- 436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.