Thiopurine S-methyltransferase alleles, TPMT(*)2, (*)3B and (*)3C, and genotype frequencies in an Indian population.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds including thiopurine drugs such as 6-mercaptopurine, 6-thioguanine and azathioprine. TPMT activity exhibits genetic variation and shows tri-modal distribution with 89-94% of individuals possessing high activity, 6-11% intermediate activity and approximately 0.3% low activity. Patients with intermediate or deficient TPMT activity exposed to thiopurine drugs show severe hematopoietic toxicity. Three single nucleotide polymorphisms (SNPs) in TPMT (NM_000367.2:c.238G>C, NM_000367.2:c.460G>A and NM_000367.2:c.719A>G) define the most prevalent mutant alleles associated with loss of catalytic activity reported in several populations. The present study investigated, for the first time, the frequency distribution of these three SNPs of TPMT, their alleles and genotypes in a Southern Indian population. Peripheral blood was obtained from 326 individuals of a Southern Indian population, and genomic DNA was isolated from total peripheral white blood cells. The genotypes at the polymorphic loci were determined by allele-specific polymerase chain reaction, restriction fragment length polymorphism and confirmatory DNA sequencing. The estimated genotype frequency for homozygous TPMT(*)1/(*)1 was 97.24%, for heterozygous TPMT(*)1/(*)2 and TPMT(*)1/(*)3B, 0.61% each, and for heterozygous TPMT(*)1/(*)3C, 1.53%. The frequency of heterozygous mutants in the studied Indian population was 2.76%. This study demonstrated significant variations in TPMT gene polymorphisms in an Indian population in relation to other human populations and may help to predict both clinical efficacy and drug toxicity of thiopurine drugs.

Deletion of (54)FLRAPSWF(61) residues decreases the oligomeric size and enhances the chaperone function of alphaB-crystallin.

AlphaB-crystallin is a member of the small heat shock protein family and is known to have chaperone activity. Using a peptide scan approach, we previously determined that regions 42-57, 60-71, and 88-123 in alphaB-crystallin interact with alphaA-crystallin during heterooligomer formation. To further characterize the significance of the N-terminal domain of alphaB-crystallin, we prepared a deletion mutant that lacks residues (54)FLRAPSWF(61) (alphaBDelta54-61) and found that the absence of residues 54-61 in alphaB-crystallin significantly decreased the homooligomeric mass of alphaB-crystallin. The average oligomeric mass of wild-type alphaB-crystallin and of alphaBDelta54-61, calculated using multiangle light scattering, was 624 and 382 kDa, respectively. The mutant subunits aggregate to form smaller, less-compact oligomers with a 4-fold increase in subunit exchange rate. Deletion of the 54-61 region resulted in a 50% decrease in intrinsic tryptophan fluorescence. The alphaBDelta54-61 mutant showed a 2-fold increase in 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding as compared to the wild-type protein, suggesting increased hydrophobicity of the mutant protein. Accompanying the evidence of increased hydrophobicity in the deletion mutant was a 10-fold increase in antiaggregation activity. Homooligomers of 6HalphaA (750 kDa) readily exchanged subunits with alphaBDelta54-61 homooligomers at 37 degrees C, forming heterooligomers with an intermediate mass of 625 kDa. Our data suggest that residues (54)FLRAPSWF(61) contribute to the higher order assembly of alphaB-crystallin oligomers. Residues (54)FLRAPSWF(61) in alphaB-crystallin are not essential for target protein binding during chaperone action, but this region apparently has a role in the chaperone activity of native alphaB-crystallin.

Role of alphaBI5 and alphaBT162 residues in subunit interaction during oligomerization of alphaB-crystallin.

PURPOSE: To determine whether the residues in the NH(2)- and COOH-terminal extensions interact with one another during oligomerization of alphaB-crystallin. METHODS: Site-directed mutagenesis was used to mutate alphaBI5 and alphaBT162 residues to Cys. The recombinant I5C and T162C proteins were expressed in Escherichia coli cells and purified using chromatographic techniques. These proteins were analyzed by SDS-PAGE and mass spectrometry and characterized by multi-angle light scattering and circular dichroism (CD) spectroscopy methods. Fluorescence resonance energy transfer (FRET) assay was used to determine the interaction between the subunits. RESULTS: Dimer formation was observed in both alphaBI5C and alphaBT162C in storage at 4 degrees C. During air oxidation at room temperature, alphaBT162C formed dimers to a greater extent than alphaBI5C. The average molar masses, secondary structures, and chaperone-like activities of the reduced forms of I5C and T162C were comparable to that of wild type alphaB-crystallin. The oligomeric assembly of reduced forms of I5C and T162C appeared homogenous under JEOL 1200EX Electron microscope whereas the oxidized proteins appeared as irregular aggregates. FRET assay demonstrated interactions between alphaBI5C-alphaBI5C and alphaBT162C-alphaBT162C. However, there was no evidence of an interaction between alphaBI5C and alphaBT162C residues during oligomerization. CONCLUSIONS: This study suggests that residues from the NH(2)- and COOH-terminal regions in alphaB-crystallin interact with residues from the corresponding regions of another subunit, but there exists no interaction between the residues at the COOH-terminal extension region and the residues at the NH(2)-terminal region.

Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation.

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, alphaB-(1-18) (MDIAIHHPWIRRPFFPFH) and betaA3/A1-(59-74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, gammaS-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact alpha-, beta-, and gamma-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of alphaB-(1-18) and betaA3/A1-(59-74) peptides increased the scattering of light by beta- and gamma-crystallin and alcohol dehydrogenase. The ability of alpha-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with alphaB-(1-18) and betaA3/A1-(59-74) peptides, whereas gammaS peptide had no effect on chaperone-like activity of alpha-crystallin. The betaA3/A1-(59-74 peptide caused a 5.64-fold increase in alphaB-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in alphaB-(1-18) and betaA3/A1-(59-74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.

Cataract-causing alphaAG98R mutant shows substrate-dependent chaperone activity.

PURPOSE: The G98R mutation in human alphaA-crystallin is associated with autosomal dominant cataract (presenile type). The reasons for cataract development in alphaAG98R individuals are not fully understood. Therefore we undertook this study to analyze the stability, structural changes and chaperone function of alphaAG98R protein. METHODS: Site-directed mutagenesis was employed to generate alphaAG98R mutant protein. Human alphaA-crystallin cDNA cloned into the pET23d vector was used as the template. The recombinant proteins were expressed in E. coli and purified using chromatographic methods. Both the wild-type and mutant proteins were characterized by SDS-PAGE, transmission electron microscopy, static and dynamic light scattering, and spectroscopic analysis. The chaperone-like function of the mutant protein was compared with wild-type protein using different substrates. RESULTS: The G98R mutant protein formed larger oligomers compared to the wild-type alphaA-crystallin. Circular dichroism studies showed altered secondary and tertiary structure whereas bis-ANS binding studies showed a gain of surface hydrophobicity in the alphaAG98R protein. The alphaAG98R protein displayed a substrate-dependent chaperone-like activity. The mutant protein appeared to have diminished chaperone-like activity toward aggregating alpha-lactalbumin, whereas citrate synthase and alcohol dehydrogenase were efficiently protected from aggregation. CONCLUSIONS: The present results reveal that the G98R mutation causes conformational changes in alphaA-crystallin and that with certain substrates the mutant protein forms complexes that are prone to precipitate over time. The accumulation of mutant protein-substrate complexes may be the reason for cataract development in individuals carrying the G98R mutation in alphaA-crystallin.