A stress-responsive glyoxalase I from the parasitic nematode Onchocerca volvulus

Glyoxal, methylglyoxal and other physiological alpha -oxoaldehydes are form ed by the lipid peroxidation, glycation and degradation of glycolytic inter mediates. They are detoxified enzymically by the glyoxalase system. To inve stigate the physiological function of glyoxalase I in parasitic organisms, the cDNA for glyoxalase I from the filarial nematode Onchocerca volvulus (d esignated OvGloI) has been cloned and characterized. The isolated cDNA cont ains an open reading frame of 579 bp encoding a protein with a calculated m olecular mass of 21930 Da, Owing to the high degree of sequence identity (6 0 %) with human glyoxalase I, for which the X-ray structure is available, i t has been possible to build a three-dimensional model of Ov-GloI. The mode lled core of Ov-GloI is conserved compared with the human glyoxalase I; how ever, there are critical differences in the residues lining the hydrophobic substrate-binding pocket of Ov-GloI. A 22 kDa protein was obtained by hete rologous expression in Escherichia coli, A homogeneous enzyme preparation w as obtained by affinity purification and functional characterization of the recombinant enzyme included the determination of kinetic constants for met hylglyoxal and phenylglyoxal as well as inhibition studies. Gel filtration demonstrated a dimeric structure. To assess the role of Ov-GloI as a potent ial 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 insul t, namely plumbagin and xanthine/xanthine oxidase, on the gene transcript l evel of Ov-GloI were investigated. By using a semi-quantitative PCR ELISA i t was shown that Ov-GloI is expressed at elevated levels under conditions o f oxidative stress.