Identification of a Novel Nucleobase-Ascorbate Transporter Family Member in Fish and Amphibians

Nucleobase-Ascorbate Transporter (NAT) family includes ascorbic acid, nucleobases, and uric acid transporters: With broad evolutionary distribution. In vertebrates, four members have been previously recognized, the ascorbate transporters Slc23a1 and Slc3a2, the nucleobase transporter Slc23a4 and an orphan transporter Slc23a3. Using phylogenetic and synteny analysis, we identify a fifth member of the vertebrate slc23 complement (slc23a5), present in neopterygians (gars and teleosts) and amphibians, and clarify the evolutionary relationships between the novel gene and known slc23 genes. Further comparative analysis puts forward uric acid as the preferred substrate for Slc23a5. Gene expression quantification, using available transcriptomic data, suggests kidney and testis as major expression sites in Xenopus tropicalis (western clawed frog) and Danio rerio (zebrafish). Additional expression in brain was detected in D. rerio, while in the Neoteleostei Oryzias latipes (medaka) slc23a5 expression is restricted to the brain. The biological relevance of the retention of an extra transporter in fish and amphibians is discussed.

substrate specificity [1,[11][12][13]. Thus, these transporters can be divided into three functional 58 groups. Slc23a1, Slc23a2 and Slc23a3 belong to the ascorbate group and harbour a conserved 59 proline (P) in the first amino acid position of the signature motif [1]. A glutamate (E) residue 60 defines the uracil group, including bacterial anion symporters and Slc23a4, while a glutamine (Q) 61 is present in the bacterial uric acid and/or xanthine anion symporters [1,[11][12][13]. Topology 62 predictions, using proteins from the distinct groups, suggest a cytosolic location of the motif [1, 63 11,12]. Here we revise the portfolio of NAT family members in vertebrates.  Table 1 (Table S1).

Synteny 74
Slc23 genes were mapped onto the respective species genomes, using the latest genome 75 assemblies available in GenBank database (https://www.ncbi.nlm.nih.gov/). Genes flanking 76 Slc23a1, Slc23a2, Slc23a3, Slc23a4 and the novel Slc23a-like gene were identified and mapped using 77 the human loci as reference. If the target gene was not found, the syntenic genomic region was 78 retrieved using neighboring genes as reference.

2.3Phylogenetic Analysis 80
The retrieved amino acid sequences were aligned using the MAFFT software web service 81 (http://mafft.cbrc.jp/alignment/software/) in default automatic settings, with L-INS-I refinement 82 method [14]. The alignment was stripped from columns with at least 20% of gaps resulting in an 83 alignment with 75 sequences and 526 positions. The output alignment was used to construct a 84 phylogenetic tree using PhyML 3.0 with Smart Model Selection web service (http://www.atgc-85 montpellier.fr/phyml-sms/) in default settings, which selected the LG +G+I+F model [15].

86
Bayesian-like transformation of aLRT was selected to assess branch support. An additional 87 phylogenetic tree, with automatic bootstrapping (552 bootstraps) and using the default hill- (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/287870 doi: bioRxiv preprint first posted online Mar. 23, 2018; To assess the relative gene expression of slc23 genes from the selected species, reference 108 sequences and the corresponding annotations were collected from NCBI and Ensembl [18]    including the previously reported slc23a4 and the novel slc23a5 genes, both outgrouped by 154 invertebrate sequences. A third cluster, which outgroups the remaining slc23 genes, included the 155 highly divergent slc23a3 genes, mirrored by the long branch lengths (Figure 1).

156
. CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/287870 doi: bioRxiv preprint first posted online Mar. 23, 2018; CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

180
. CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/287870 doi: bioRxiv preprint first posted online Mar. 23, 2018; CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/287870 doi: bioRxiv preprint first posted online Mar. 23, 2018; preference: Q -Uric acid/xanthine group; P -Ascorbate group and E -Uracil group. The mirror 186 Q in the Slc23a3 group is also highlighted by a black box.  CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

232
Besides its scaffolding role, NHERF family members were also shown to modulate the activity of 233 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/287870 doi: bioRxiv preprint first posted online Mar. 23, 2018; protein assemblages [24][25][26] 246 oculatus, appear to lack the full complement of renal urate transporters (Table S6). A similar 247 scenario is observed for birds and reptiles; however, their excretory system represents a particular 248 adaptation for uric acid excretion [27,28]. Besides simple excretion, and given that high CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/287870 doi: bioRxiv preprint first posted online Mar. 23, 2018; shown to be correlated with their swimming activity [45]. Neuronal cell populations are 286 particularly sensitive to oxidative stress, thus, uric acid could participate in the neutralization of 287 oxidative species to by-pass oxidative stress, notably in GLO-deficient fish. Yet, information on 288 neuronal uric acid levels in fish is currently absent. Wright, P.A. Nitrogen excretion: three end products, many physiological roles. J. Exp. 316 Biol. 1995, 198, 273-281. 317 6. CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  Fish Biol. 1976, 8, 45-53, doi:10.1111/j.1095-8649.1976 43. Parenti, L.R.; Grier, H.J. Evolution and phylogeny of gonad morphology in bony fishes. 424 Integr. Comp. Biol. 2004, 44, 333-348, doi . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.     CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.