Identification of a stress-responsive Onchocerca volvulus glutathione S-transferase (Ov-GST-3) by RT-PCR differential display.
The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.
Liebau
E
E
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. liebau@bni.uni-hamburg.de
Eschbach
M L
ML
Tawe
W
W
Sommer
A
A
Fischer
P
P
Walter
R D
RD
Henkle-Dührsen
K
K
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000201:enzymology* / Q000502:physiology; D018384:Oxidative Stress; D018510:RNA, Helminth / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D018076:DNA, Complementary; D018510:RNA, Helminth; D012333:RNA, Messenger; D005982:Glutathione Transferase