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A stress-responsive glyoxalase I from the parasitic nematode Onchocerca volvulus
A Sommer1, P Fischer, K Krause
1Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.
Abstract:
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.
Insights
Glyoxalase I (GloI) from the parasitic nematode Onchocerca volvulus (Ov-GloI) was cloned and characterized. This enzyme is upregulated under oxidative stress and shows potential as a vaccine candidate or diagnostic tool for filarial infections.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- Alpha-oxoaldehydes like glyoxal and methylglyoxal are toxic byproducts of metabolism.
- The glyoxalase system, particularly glyoxalase I (GloI), detoxifies these compounds.
- Understanding parasitic GloI is crucial for developing treatments and diagnostics for diseases like onchocerciasis.
Purpose of the Study:
- To clone and characterize glyoxalase I (Ov-GloI) from Onchocerca volvulus.
- To investigate the structural and functional properties of Ov-GloI.
- To evaluate Ov-GloI as a potential target for vaccines or diagnostics against filarial parasites.
Main Methods:
- Cloning and sequencing of Ov-GloI cDNA.
- Heterologous expression in E. coli and protein purification.
- 3D modeling based on human GloI structure.
- Enzyme kinetics and inhibition studies.
- Serological analysis using patient sera.
- Gene expression analysis under oxidative stress using RT-PCR.
Main Results:
- The Ov-GloI cDNA encodes a 21.9 kDa protein.
- A 3D model revealed conserved core structure but distinct substrate-binding pocket residues compared to human GloI.
- Recombinant Ov-GloI exhibited enzymatic activity and a dimeric structure.
- Specific IgG1 antibodies were detected in sera from microfilaria carriers, indicating serological reactivity.
- Ov-GloI gene expression was significantly upregulated under oxidative stress conditions.
Conclusions:
- Ov-GloI plays a role in detoxifying alpha-oxoaldehydes in Onchocerca volvulus.
- The structural differences in the substrate-binding pocket may be exploited for targeted drug development.
- Ov-GloI is a promising candidate for serodiagnostic tools and potentially vaccines against onchocerciasis.