Functional study of the P32T ITPA variant associated with drug sensitivity in humans

Abstract

Sanitization of the cellular nucleotide pools from mutagenic base analogues is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic, and toxic incorporation of purine base analogues [i.e., ITP, dITP, XTP, dXTP, or 6-hydroxylaminopurine (HAP) deoxynucleoside triphosphate] into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele, 94C-->A, encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analogue drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by Western blot is severely diminished in a human fibroblast cell line with the 94C-->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability, and availability.

Fig. 1. Human ITPA and its variants

A. The location of P32T and E44L mutations on the crystal structure of human ITPase. For the dimer structure, P32 and E44 residues are in red and marked with arrows, ITP is in green. The secondary structure of ITPase is shown with a ribbon diagram colored with β-strands yellow and all other elements blue. The location of N- and C-termini are labeled. The dimer interface is located in the center of the figure. B. Purified proteins separated on SDS-PAGE gel and visualized with Coomassie stain. Lane 1 – MultiMark (Invitrogen) molecular weight marker, lane 2 – wild-type ITPA, lane 3 – ITPA-P32T, lane 4 – ITPA-E44L

Fig. 2. The dependence of ITPase activity on the concentrations of substrates, ITP and dITP

Activity assay was performed as described in Materials and Methods. High R² values close to 1 confirm the validity of approximation. Filled diamonds – wild-type ITPA, open squares – ITPA-P32T

Fig. 3. UV thermal denaturation curves of purified wild-type ITPA and ITPA-P32T

Solutions of wild-type ITPA (A) or ITPA-P32T (B), at the indicated concentrations were heated while monitoring the absorbance at 280 nm. Plots are truncated at the peaks, after which precipitation of the protein led to a decline in absorbance. Melting temperatures, which occur at the inflection point of the plots, were determined by identifying the temperature at the peaks in first derivative plots.

Fig 4. ITPA P32T is functional in bacterium E. coli

A. Sensitivity to high temperature of double mutant ΔrdgB recA200(Ts) strain (EK5) transformed with vector expressing the human wild-type ITPA gene and its mutant variants ITPA-P32T and ITPA-E44L . Five μl of the serial dilutions of the overnight cultures grown at 30°C were spotted on a minimal medium plates containing 50 μM IPTG. The plates were incubated overnight at 30°C or at 42°C. B. The ability of ITPA-P32T to compensate for the HAP-sensitivity of the Rosetta strain. Cells were spotted on a minimal medium plate containing 50 μM IPTG using a multi-prong replicator device, and 50 μg of HAP was spotted onto the filter paper at the center of each plate. The plates were incubated overnight at 37°C. C. Protection of the wild type strain EK1 from HAP-induced mutagenesis by human ITPA and its variants. Spot-tests were performed as in Fig. 4B on a minimal medium with or without ITPG. The cells were then replica-plated onto LB plates containing rifampicin and incubated overnight at 37°C.

Fig 4. ITPA P32T is functional in bacterium E. coli

A. Sensitivity to high temperature of double mutant ΔrdgB recA200(Ts) strain (EK5) transformed with vector expressing the human wild-type ITPA gene and its mutant variants ITPA-P32T and ITPA-E44L . Five μl of the serial dilutions of the overnight cultures grown at 30°C were spotted on a minimal medium plates containing 50 μM IPTG. The plates were incubated overnight at 30°C or at 42°C. B. The ability of ITPA-P32T to compensate for the HAP-sensitivity of the Rosetta strain. Cells were spotted on a minimal medium plate containing 50 μM IPTG using a multi-prong replicator device, and 50 μg of HAP was spotted onto the filter paper at the center of each plate. The plates were incubated overnight at 37°C. C. Protection of the wild type strain EK1 from HAP-induced mutagenesis by human ITPA and its variants. Spot-tests were performed as in Fig. 4B on a minimal medium with or without ITPG. The cells were then replica-plated onto LB plates containing rifampicin and incubated overnight at 37°C.

Fig 4. ITPA P32T is functional in bacterium E. coli

A. Sensitivity to high temperature of double mutant ΔrdgB recA200(Ts) strain (EK5) transformed with vector expressing the human wild-type ITPA gene and its mutant variants ITPA-P32T and ITPA-E44L . Five μl of the serial dilutions of the overnight cultures grown at 30°C were spotted on a minimal medium plates containing 50 μM IPTG. The plates were incubated overnight at 30°C or at 42°C. B. The ability of ITPA-P32T to compensate for the HAP-sensitivity of the Rosetta strain. Cells were spotted on a minimal medium plate containing 50 μM IPTG using a multi-prong replicator device, and 50 μg of HAP was spotted onto the filter paper at the center of each plate. The plates were incubated overnight at 37°C. C. Protection of the wild type strain EK1 from HAP-induced mutagenesis by human ITPA and its variants. Spot-tests were performed as in Fig. 4B on a minimal medium with or without ITPG. The cells were then replica-plated onto LB plates containing rifampicin and incubated overnight at 37°C.

Fig. 5. ITPA-P32T is functional in yeast Saccharomyces cerevisiae

Suppression of HAP-induced mutagenesis in ham1 mutant transformed with plasmid expressing the yeast HAM1 gene, human ITPA gene and its mutant alleles ITPA-P32T and ITPA-E44L. Transformants with empty vector pESC-URA were used as a control. For frequency determination we have used six independent cultures. The experiment was repeated three times and the data appeared to be homogenous. Medians of mutant frequencies for each data point were then found. Error bars represent 95% confidence limits.

Fig. 6. Diminished levels of P32T ITPA in human fibroblasts

Upper panel represents the results of two independent western blots with extracts of human fibroblasts with anti-ITPA antibodies. Bottom panel serves as a loading control and illustrates that the level of unrelated enzyme, GAPDH, is similar in the two cell lines.