The human hGSTA5 gene encodes an enzymatically active protein.
BACKGROUND
Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional.


METHODS
Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity.


RESULTS
Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity.


CONCLUSIONS
hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme.


CONCLUSIONS
Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.
Singh
Sharda P
SP
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Zimniak
Ludwika
L
Zimniak
Piotr
P
D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004586:Electrophoresis; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D020014:K562 Cells; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D000447:Aldehydes; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D005978:Glutathione; C027576:4-hydroxy-2-nonenal