Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase

Chang, Wei-Ju; Tsai, Jia-Lun; Chen, Bing Hung; Fu, Tzu Fun

doi:10.1016/j.pep.2005.08.027

Cited by 17 publications

(28 citation statements)

References 29 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Kao¹,

Chang²,

Wu³

et al. 2008

Drug Metab Dispos

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ABSTRACT:A cDNA encoding for zebrafish ␥-glutamyl hydrolase (␥GH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-z␥GH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant z␥GH was similar to mammalian ␥GH. Folate is an essential B vitamin and participates in the biosynthesis and metabolism of nucleic acids, proteins, several amino acids, methyl groups, many neurotransmitters, and some vitamins. Mammalian cells are unable to synthesize folates de novo and therefore depend on their food for the supply of folates. Naturally occurring folates are synthesized as poly-␥-glutamate forms (folylpolyglutamate) but are absorbed and transported most efficiently as folylmonoglutamates. The conversion of folylpolyglutamates in dietary food to folylmonoglutamates is catalyzed by carboxypeptidase II (EC 3.4.22.12) in mammals. In a recent study, ␥-glutamyl hydrolase (␥GH, EC 3.4.19.9), a lysosomal cysteine peptidase, was reported to be the enzyme responsible for hydrolyzing dietary folate in rat small intestine (Shafizadeh and Halsted, 2007). After entering cells, these folylmonoglutamates are elongated to folylpolyglutamates by folylpoly-␥-glutamate synthetase (EC 6.3.2.17) for more effective retention and cofactor activity. As part of a salvage pathway, ␥GH catalyzes the hydrolysis of Glu-␥-Glu bonds to form folylmonoglutamates, enabling folate cofactors to be exported from cells and enter circulation again (Suh et al., 2001). Therefore, folylpoly-␥-glutamate synthetase and ␥GH are crucial for the maintenance and regulation of intracellular folate homeostasis. Nevertheless, the mechanism of turnover and control of cellular folylpolyglutamate levels by ␥GH remain unclear. A better understanding of this enzyme and an efficient assay system is prerequisite to answer this question.Consistent with this notion, the activity of ␥GH to hydrolyze the ␥-glutamyl peptide bonds of folylpolyglutamates has rendered this enzyme a potential target of antifolate chemotherapy and, at the same time, a primary component in regulating the intracellular levels of some antifolate drugs. Antifolate drugs, such as methotrexate, owe much of their effectiveness to being substrates for both folylpoly-␥-glutamate synthetase and ␥GH. Removal of ␥-linked glutamate residues decreases the retention and activity of these drugs. A polymorphism resulting in reduced catalytic activity of ␥GH was observed to be associated with greater accumulation of long-chain methotrexate polyglutamate forms (Cheng et al., 2004). In contrast, higher ␥GH activity has been connected to the development of drug resistance (Rhee et al., 1993). Therefore, alteration to ␥GH availability or activity seems to have functional and pharmacological consequences and even to be a potential tumor marker (Schneider and Ryan, 2006). Having pu...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Kao¹,

Chang²,

Wu³

et al. 2008

Drug Metab Dispos

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“…Increasing studies also demonstrate comparable features between zebrafish and human in many biological pathways and pathogenesis, including organ development, angiogenesis, hemostasis, heart function and circulation, apoptosis and proliferation, carcinogenesis, drug abuse and addiction, and toxicology and teratogenicity (Kari et al, 2007). However, folate-requiring one-carbon metabolism in zebrafish remains unexplored territory, albeit its critical role in oncogenesis and fetal development in vertebrates.Our previous study on the recombinant zebrafish cSHMT revealed strong similarities with mammalian orthologs, suggesting the appropriateness of using zebrafish as a model for folate-related studies (Chang et al, 2006). In the present report, we clone and characterize zebrafish mitochondrial SHMT (zmSHMT), the other isoform of SHMT, which is less understood in mammals.…”

mentioning

confidence: 85%

“…The pET43.1a plasmid and all the E. coli strains for cloning and expression were obtained from Novagen (Madison, WI). The materials and methods for the general cloning procedures were as described previously (Chang et al, 2006).Cloning of zmSHMT from Zebrafish cDNA Library by PCR-Based Cloning Strategy. A PCR-based approach with degenerate primers was used for the amplification and cloning of SHMT-encoding sequences from a zebrafish cDNA mixture.…”

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confidence: 99%

“…The same principle was used to determine the stoichiometry of PLP bound per molecule of enzyme with an extinction coefficient of 5580 M Ϫ1 cm Ϫ1 for thiazolidine (Ulevitch and Kallen, 1977). The quaternary structure was determined on a Superdex 200 size-exclusion column as described previously (Chang et al, 2006). The molar absorptivity coefficient was determined as stated previously by Gill and von Hippel (1989).…”

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

et al. 2007

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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...

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Folate polyglutamylation eliminates dependence of activity on enzyme concentration in mitochondrial serine hydroxymethyltransferases from Arabidopsis thaliana

Wei

Sun

Sandoval

et al. 2013

Archives of Biochemistry and Biophysics

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Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase

Cited by 17 publications

References 29 publications

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

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

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

Folate polyglutamylation eliminates dependence of activity on enzyme concentration in mitochondrial serine hydroxymethyltransferases from Arabidopsis thaliana

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