Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk

Abstract

In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid) in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs), i.e., 421C>A (major) and 376C>T (minor), in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

Figure 1

Chemical structures of purine and its derivatives, i.e., adenine, guanine, hypoxanthine, xantine, theobromine, caffeine, uric acid, and isoguanine.

Figure 2

Metabolic pathways of uric acid formation from nucleotide monophosphates. AMP, adenosine monophosphate; IMP, inosine monophosphate; XMP, xanthine monophosphate, GMP, guanine monophosphate.

Figure 3

Schematic illustration of renal re-absorption and excretion of uric acid. Uric acid filtrated from the renal glomerular body is first re-absorbed by human renal proximal tubule cells. Thereafter, uric acid is eliminated, in part, from the blood circulation to urine by renal transporters. Arrows in the figure indicate the direction of transport of uric acid. Dotted and solid lines indicate re-absorption and secretion, respectively.

Figure 4

Schematic illustration of human ABCG2 and its non-synonymous polymorphisms. The ABCG2 protein expressed in the plasma membrane is a homodimer linked via a cysteinyl disulfide bond. The cysteine residue corresponding to Cys603 of human ABCG2 is involved in the homodimer formation, whereas Cys592 and Cys608 form an intra-molecular disulfide bond that is important for N-linked glycan formation at Asn596. The SNP 421C>A is a non-synonymous polymorphism that leads to amino acid substitution; Gln to Lys (Q141K) in the intracellular loop containing an ATP-binding cassette (ABC).

Figure 5

Effect of the SNP variant (Q141K) on the protein expression level and degradation of ABCG2. (A) The ABCG2 wild type (WT) protein has glutamine residue at amino acid position 141. To assess the effect of Q141K variant on the protein expression level, Flp-In-293 cells expressing WT and the Q141K variant were incubated in the absence or presence of MG132 (2 μM) for 24 h. ABCG2 WT and Q141 variant proteins were analyzed by immunoblotting with the ABCG2-specific monoclonal antibody (BXP-21) after PNGase F treatment. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level was analyzed by GAPDH-specific antibody. The signal intensity ratio (ABCG2/GAPDH) was normalized to the control level. Data are expressed as means ± S.D. in triplicate experiments [33]. (B) Correctly processed ABCG2 WT protein is destined to reach the plasma membrane and is then degraded by the endosome-lysosome pathway after remaining in the plasma membrane domain for a certain period. In contrast, the ABCG2 Q141K variant protein is recognized as a misfolded form and then undergoes ubiquitination-mediated proteasomal degradation. Bafilomycin A₁ (BMA) and MG132 inhibit lysosomal and proteasomal degradation, respectively.

Figure 6

The allele frequencies of 421C (WT) and 421A (Q141K) among different ethnic populations. Data are calculated from Ishikawa et al. [36].

Figure 7

Rapid genetic testing to assess gout risk by the SmartAmp method. (A) Schematic illustration of the human ABCG2 gene located on chromosome 4q22. The SNP 421C>A resides in exon 5. (B) Detection of SNP 421C>A in the human ABCG2 gene by the SmartAmp method. Three panels depict the time-courses of the SmartAmp assay reactions with ABCG2 allele–specific primers carrying WT (421C) or SNP (421A) alleles; namely, C/C homozygote, C/A heterozygote, and A/A homozygote. After genomic DNA in peripheral blood samples (5 μL) was denatured at 98 °C for 3 min, the genotyping reactions were allowed to proceed isothermally at 60 °C for 60 min in a Mx3000P PCR system (Agilent Technologies, Santa Clara, CA, USA) [48]. DNP amplification was continuously monitored by detecting the fluorescence of DNA-intercalated SYBR Green I dye in the reaction mixture.