ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein-protein interactions with HPRT1

Abstract

Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers.

Fig. 1

Production and transport of uric acid in epithelial cells. Uric acid is the final product of adenosine monophosphate (AMP) catabolism. Initially, AMP can be phosphorylated back to ADP and ATP by adenylate kinase and also can stimulate ATP production by stimulation of AMP kinase, glycolysis and fat oxidation. Alternatively, AMP can enter in the purine degradation pathway which is initiallized by its deamination to inosine monophosphate (IMP) by adenosine monophosphate deaminase (AMPD). Elimination of the ribose from IMP by 5⁰nucleotidase forms inosine which is then metabolized to hypoxanthine by purine nucleoside phosphorylase (PNP). Inosine can also be produced from the deamina-tion of adenosine by adenosine deaminase, Alternatively, adenosine can also enter the route through its phosphorylation to AMP by adenosine kinase. Hypoxanthine is sequentially converted to xanthine and uric acid by xanthine oxidase. A salvage pathway exists whereby hypoxanthine can be converted back to IMP through hypoxanthine phosphoribosyltransferase 1 (HPRT1). Extracellular uric acid levels can also be influenced by modulating its transport rates through importers: uric acid transporter 1 (URAT1) and organic anion transporter 4 (OAT4) and exporters: glucose transporter 9 (glut9), ATP-binding cassette sub-family G member 2 (ABCG2), organic anion transporters 1 and 3 (OAT1 and OAT3), multidrug resistance protein 4 (MRP4) and sodium-phosphate transporter 1 (NPT1).

Fig. 2

ALDH16A1 gene structure and ALDH16A1 protein in human cell lines. (A) Schematic illustration of the human ALDH16A1 gene and its spliced variant ALDH16A1_v2 that lacks an exon (shown by the dashed box and arrow). The location and change caused by the ALDH16A1*2 SNP associated with gout in the recent Icelandic study [34] is depicted by the blue box and arrow below each figure. PCR forward (F) and reverse (R) primers pair were designed to amplify an 470-bp and 314-bp fragment from ALDH16A1 and ALDH16A1_v2 mRNA, respectively. Semi-quantitative PCR analysis of ALDH16A1 variants (B) and Western blotting analysis (C) of human ALDH16A1 in human cell lines. These included proximal tubule epithelial (HK-2), hepatoma (HepG-2), embryonic kidney (HEK-293), adenocarcinomic alveolar basal epithelial (A549), neuroblastoma (SH-SY5Y), retinal pigment epithelial (D407) and retinal pigment epithelial (ARPE-19) cell lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Clustering dendrogram of ALDH16 proteins. Putative ALDH16 amino acid sequences were aligned, and then a dendrogram was generated using a maximum likelihood approach with 1000× bootstrap resampling (labeled at nodes). The ALDH16family consists so far of 4 subfamilies 16A(mammals), 16B (amphibians and lower animals), 16C (bacteria) and 16D (fish).

Fig. 4

Human ALDH16A1 protein structure and alignment. (A) Predicted conserved domains in the human ALDH16A1 protein by alignment and homology with ALDH2 [33]. In addition the P527R mutation is noted. The two ALDH protein domains (aldehyde DH) are shown as orange bars with a line between them representing the flexible linker. Numbers indicate the amino acid positions of the domains. Additional predicted motifs are shown reflecting four predicted trans-membrane domains (TM), the predicted coiled-coil motif (C-C), as well as the location of the deleted exon in human ALDH16A1v2 (v2 del). (B) Amino acid sequence alignment of several ALDH proteins around Cys-302 of the active site. Note that this region encodes part of the exon missing from ALDH16A1v2. Dashes in sequences correspond to gaps. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Molecular modeling of human and frog ALDH16 proteins. The secondary structure of human ALDH16A1 (hALDH16A1, left) and frog ALDH16B1 (fALDH16B1, right) are presented as ribbon diagrams (outside figures) with alpha-helices in red, beta-sheets in blue, and coils in green. Space filling models (inner panels) show the substrate-binding pocket (top panels), and cofactor (NAD⁺) binding pocket (bottom panels). Labeled amino acids correspond to residues that are important in substrate or NAD⁺ binding. The substrate binding and NAD⁺ binding pockets are located on opposite faces of the protein, and tunnels between the two is indicated by a white arrow (frog ALDH16B1 only). Residues highlighted in purple (all figures) indicate critical substrate binding amino acids. The location of the deletion that is homologous to Cys-302 in hALDH16A1 is labeled Cys^⁄. Amino acid residues critical to NAD⁺ binding are highlighted in red (inner panels only, not shown in ribbon diagrams). Details on the amino acid positions compared to hALDH3A1 numbering are provided in Table 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Homology modeling of two splice variants of human ALDH16A1. Homology models of the long (A) and short (B) forms of human ALDH16A1 consist of globular N-terminal (colored blue in the long form and magenta in the short form) and C-terminal (orange) domains separated by a flexible linker (green). The region of the long form missing from the short form (i.e., Glu253–Pro304) is highlighted in yellow. Pro527 and Pro476 are depicted as yellow spheres in (A) and (B), respectively. Lower figures provide a perspective after 180° rotation of the molecule. Representative energy minimized structures from the predicted protein-protein interaction analysis indicating the capacity for the N- and C-terminal domains of the long (C) and short (D) forms of ALDH16A1 to associate together into a closed conformation. The numbers under each figure are the calculated interaction energies of the respective complexes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Predicted ALDH16A1 and HPRT1 protein complexes. Energy minimized protein complexes of wild-type (A) ALDH16A1 long (N-terminus = blue, linker = green, C-terminus = orange) and (B) ALDH16A1 short (N-terminus = magenta, linker = green, C-terminus = orange) homology models with the crystal structure of human HPRT1 (PDB ID: 1BZY; purple). Molecules of ImmucillinGP, the bound inhibitor co-crystallized with HPRT1, are represented as yellow sticks to indicate the positions of the four active sites of HPRT1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)