Functional Characterization of 5-Oxoproline Transport via SLC16A1/MCT1

Background: acid Results: Na + -dependent and amino acid ABSTRACT Thyrotropin-releasing hormone is a tri-peptide that consists of 5-oxoproline, histidine and proline. The peptide is rapidly metabolized by various enzymes. 5-Oxoproline is produced by enzymatic hydrolysis in a variety of Uptake 5-[ with 15-min incubation in parallel in water-injected oocytes cRNA-injected oocytes, and SLC16A1-specific transport was calculated by subtracting the uptake in water-injected oocytes from the uptake in cRNA-injected oocytes. Uptake the absence presence of 5-oxoproline, Uptake uptake

TRH is rapidly metabolized and its mean half-life is 5 min (2). 5-Oxoproline (also known as pyroglutamic acid, glutimic acid or pyrrolidonecarboxylic acid) is found as an N-terminal modification in many neuronal peptides and hormones that also include the accumulating peptides in Alzheimer's disease and familial dementia (3). 5-Oxoproline is also generated by γ-glutamyl cyclotransferase, which converts γ-glutamyl amino acids into 5-oxoproline and corresponding free amino acids (4). The γ-glutamyl cycle in the brain influences amino acid transport indirectly through 5-oxoproline to maintain low concentrations of glutamate, aspartate and glycine in the brain (5). Grioli et al. reported that 5-oxoproline was effective in improving some verbal memory functions in subjects affected by age-related memory decline (6).
A recent study has shown that 5-oxoproline could become a possible biomarker for autism spectrum disorders (ASD) (7).
The present study was undertaken to determine the role of SLC16A1 (also known as a monocarboxylate transporter, MCT1) in the transport of 5-oxoproline. SLC16A1 and SLC16A3 are expressed in astrocytes (8).
Despite the fact that 5-oxoproline is an amino acid derivative, it exists predominantly as an anionic monocarboxylate, indicating that 5-oxoproline may be transported by SLC16A1.

Materials-L-[
Site-directed mutagenesis-The cDNA gene of SLC16A1, which was prepared from the human brain, was purchased from DNAFORM and cloned using a strategy similar to that described in a previous report on SLC16A3 (16). The molecular cloning of SLC16A3 was previously described (16). in Barth's solution as previously described (16). Oocytes were injected with 50 ng cRNA in a 50-nl volume and incubated for 3-6 days.
The transport function of heterologously expressed SLC16A1 was monitored at 25˚C.
We measured the uptake of radiolabeled compounds by using a liquid scintillation counter. Uptake of a radiolabeled compound in water-injected and cRNA-injected oocytes was performed, and oocytes were washed with ice-cold transport buffer as previously described (16). All data are given as

Immunohistochemistry-The
immunohistochemistry procedure used was the same as that previously described (16).  Significance was assigned at p<0.05.

RESULTS
The chemical structures of TRH and 5-oxoproline are shown in Figure 1. TRH is a tri-peptide that consists of 5-oxoproline, histidine and proline. 5-Oxoproline is a TRH metabolite in which the amino group of glutamic acid cyclized to form a lactam.
5-Oxoproline, which is an uncommon amino acid derivative, has a pK a value of 3.6 (18).
Hence, it exists mostly as a monovalent anion in the living body. On the other hand, histidine and proline are common amino acids and exist principally as multivalent ions at physiological pH.
We investigated the transport of 5-oxoproline via SLC16A1 using a Histidine and proline had no effect on SLC16A1-specific lactate uptake (p>0.05).
Therefore, we assessed SLC16A1-mediated 5-oxoproline transport by monitoring the uptake of 5-[ 3 H]oxoproline in the same heterologous expression system.

Uptake of 5-oxoproline in SLC16A1
cRNA-injected oocytes was 300-fold higher than that in water-injected oocytes ( Figure   3A). This transport activity was enhanced by a proton or protons. The accumulation of 5-oxoproline by SLC16A1-expressing oocytes was markedly reduced by alkalizing the buffer pH. The transport of 5-oxoproline via SLC16A1 was inhibited by substrates of the transporter ( Figure 3B). Histidine and proline did not suppress the transport process (p>0.05).
We also investigated the effects of ( Figures 6C and 6D). The proton concentration required to produce half-maximal activation of transport activity compared with that in the wild type ( Figure   6E). We also investigated the effect of the polymorphism on transport of lactate as a classical substrate via SLC16A1 (Table 3).
Lactate uptake via SLC16A1-D490E was   (Table 6). SLC16A1 and SLC16A3 were expressed in T98G cells ( Figure 9). However, SLC5A12 was not expressed in T98G cells but was expressed in BeWo cells used as a positive control (data not shown). Therefore, we speculated from these results that 5-oxoproline is taken up through SLC16A1 and/or SLC16A3.
Hence, we investigated the transport of 5-oxoproline using a Xenopus laevis oocyte expression system. The transporters were expressed in oocytes by injection of SLC16A1 or SLC16A3 cRNA.
Water-injected oocytes were used as controls.
The uptake of 5-oxoproline in SLC16A1 cRNA-injected oocytes was higher than that in water-injected oocytes ( Figure 10).
However, there was no significant difference in uptake between SLC16A3-expressing oocytes and water-injected oocytes. These results indicate that H + -coupled 5-oxoproline transport is mediated solely by SLC16A1.

DISCUSSION
In this study, we demonstrated transport . It is also expressed in neurons but not in astrocytes (23). Therefore, we speculated that the contribution of SLC16A1 to 5-oxoproline transport in astrocytes is large.
We have shown dramatic activity of SLC16A1-mediated 5-oxoproline transport by a heterologous expression system.

Expression of SLC16A1 in Xenopus laevis
oocytes induced pH-dependent transport of 5-oxoproline, which was suppressed by lactate. The K m value was 10-fold larger than the value of SLC5A8 (30). This value is appropriate because the human brain has 100 billion neurons and 10-to 50-fold more glial

TABLE 2.
Demonstration of proton-coupled electroneutral transport of 5-oxoproline via SLC16A1 in a Xenopus laevis oocyte expression system. Uptake of 5-[ 3 H]oxoproline (60 nM) was monitored with 15-min incubation, and SLC16A1-specific transport of 5-oxoproline was calculated by subtracting the uptake in water-injected oocytes from the uptake in cRNA-injected oocytes. For the Na + -free condition, Na + was replaced with NMDG. For the Cl --free condition, Clwas replaced with gluconate. Uptake measurements were made in the presence of KCl (50 mM) and CCCP (40 μM). Uptake measured in the absence of CCCP was taken as 100% (control), and uptake in the presence of CCCP is given as percent of this control value. Uptake measured in the presence of 2 mM KCl was taken as 100% (control), and uptake in the presence of 50 mM KCl is given as percent of this control value. Uptake measured in the presence of Na + was taken as 100% (control), and uptake in the absence of Na + is given as percent of this control value.
Uptake measured in the presence of Clwas taken as 100% (control), and uptake in the absence of Clis given as percent of this control value. The experiment was done in triplicate. Data Impact of polymorphism (rs1049434) on SLC16A1-mediated lactate uptake. Uptake of lactate (100 μM) was monitored in oocytes injected with SLC16A1-WT or the indicated mutants.
SLC16A1-specific transport of lactate was calculated by subtracting the uptake in water-injected oocytes from the uptake in cRNA-injected oocytes.