A stress-responsive glyoxalase I from the parasitic nematode Onchocerca volvulus.
Glyoxal, methylglyoxal and other physiological alpha-oxoaldehydes are formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates. They are detoxified enzymically by the glyoxalase system. To investigate the physiological function of glyoxalase I in parasitic organisms, the cDNA for glyoxalase I from the filarial nematode Onchocerca volvulus (designated Ov-GloI) has been cloned and characterized. The isolated cDNA contains an open reading frame of 579 bp encoding a protein with a calculated molecular mass of 21930 Da. Owing to the high degree of sequence identity (60%) with human glyoxalase I, for which the X-ray structure is available, it has been possible to build a three-dimensional model of Ov-GloI. The modelled core of Ov-GloI is conserved compared with the human glyoxalase I; however, there are critical differences in the residues lining the hydrophobic substrate-binding pocket of Ov-GloI. A 22 kDa protein was obtained by heterologous expression in Escherichia coli. A homogeneous enzyme preparation was obtained by affinity purification and functional characterization of the recombinant enzyme included the determination of kinetic constants for methylglyoxal and phenylglyoxal as well as inhibition studies. Gel filtration demonstrated a dimeric structure. To assess the role of Ov-GloI as a potential vaccine candidate or serodiagnostic tool, the serological reactivity of the recombinant Ov-GloI was analysed with sera from microfilaria carriers and specific IgG1 antibodies were detected. The effects of oxidative insult, namely plumbagin and xanthine/xanthine oxidase, on the gene transcript level of Ov-GloI were investigated. By using a semi-quantitative PCR ELISA it was shown that Ov-GloI is expressed at elevated levels under conditions of oxidative stress.
Sommer
A
A
Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.
Fischer
P
P
Krause
K
K
Boettcher
K
K
Brophy
P M
PM
Walter
R D
RD
Liebau
E
E
D000595:Amino Acid Sequence; D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000201:enzymology*; D018384:Oxidative Stress; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D007791:Lactoylglutathione Lyase