Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

Abstract

ABSTRACT:Serine hydroxymethyltransferase (SHMT) provides activated onecarbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N 5 ,N 10 -methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N 5 -CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.Folates carry the chemically activated single carbons at N 5 and/or N 10 positions and are required for the biosynthesis and metabolism of nucleic acid, protein, amino acid, methyl group, neurotransmitter, and vitamins. Its vital role in nucleotide biogenesis has led to the development of many anticancer drugs targeting folate-requiring enzymes. Among them, methotrexate (MTX) is one of the most widely used anticancer agents to date. It blocks de novo nucleotide synthesis by depleting reduced tetrahydrofolates mainly through inhibition of dihydrofolate reductase (DHFR) and thymidylate synthase (Fig. 1, enzymes 2 and 3, respectively). However, resistance to MTX often emerges and becomes the major impediment to its curative potential.To overcome this obstacle and prevent MTX-associated toxicity, high-dose MTX combined with leucovorin rescue is administered and has become an important regimen in the treatment of a variety of cancers (Frei et al., 1980). Despite these preventive measures, MTXinduced resistance and toxicity continue to occur, although infrequently. Mechanisms including elevated DHFR, decreased thymidylate synthase, impaired folate/antifolate transportation, and decreased polyglutamylation on MTX have been pr...

Recombinant Zebrafish γ-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme

“…Using the zebrafish model for drug discovery is foreseen to significantly speed up the translation of drug or treatments from bench research to bedside reality by revolutionizing the cell-based assays to embryo-based screening (Kari et al, 2007). In previous studies, we have shown that zebrafish folate enzymes are comparable with their human and mammalian counterparts for their catalytic and structural properties, as well as susceptibility to antifolate compounds (Chang et al, 2006(Chang et al, , 2007Kao et al, 2008). This purified recombinant z␥GH will allow for further investigation to unravel the role of this key enzyme in regulating the intracellular availability of folates and antifolate drugs and to use zebrafish as an in vivo model for folate-related studies and antifolate drug discovery.…”

“…We had carefully examined the coding sequence for accuracy and codon usage and found several rarely used codons in E. coli. However, the use of the Rosetta strain for expression should have overcome this potential problem (Chang et al, 2007). We also tested for inclusion bodies and culture broth with SDS-PAGE for the possibility of aggregation or export of expressed z␥GH because secretion of this protein had been reported in mammalian tumor cell lines (Gourdon et al, 2008).…”

“…Well established tools of molecular biology for gene manipulation have also significantly assisted progress with zebrafish. We have started an extensive study of folate-mediated one-carbon metabolism in zebrafish (Chang et al, 2006(Chang et al, , 2007Kao et al, 2008). In this study, we cloned the coding sequence of zebrafish ␥GH (z␥GH) from a zebrafish cDNA library and developed an efficient protocol for expression and purification of the recombinant z␥GH.…”

Ethanol-Induced Upregulation of 10-Formyltetrahydrofolate Dehydrogenase Helps Relieve Ethanol-Induced Oxidative Stress

“…This "closed and isolated system" enables the intracellular fluctuation among different folate derivatives to be observed. Many zebrafish folate enzymes have been shown to be comparable to mammalian orthologs, supporting the appropriateness of modeling the impact of ethanol on folate-mediated OCM with zebrafish (7)(8)(9)(10)(11). Here, we report how the embryonic folate status and the expression of folate enzymes are altered when embryos are exposed to ethanol.…”

“…For gene expression characterization and folate content measurement, ethanol was added to 3-hpf embryos for 6 h before mRNA extraction. For promoter activity assays in cells, zebrafish liver epithelium cells (ZLE) were cultured in Leibovitz's 15 medium supplemented with 10% FBS at 28°C as previously described (8). Cells were seeded (1 ϫ 10 6 cells/ 6-cm dish) and transfected with promoter constructs.…”