Subcellular Localization of the NAD + -Dependent Alcohol Dehydrogenase in Entamoeba histolytica Trophozoites

Abstract

The protozoan parasite Entamoeba histolytica is an ancient eukaryotic cell that shows morphologically atypical organelles and differs metabolically from higher eukaryotic cells. The aim of this study was to determine the subcellular localization of ameba NAD+-dependent alcohol dehydrogenase (ADH2). The enzyme activity was present in soluble and mainly in particulate material whose density was 1.105 in a sucrose gradient. By differential centrifugation, most of the ADH activity sedimented at 160,000 g (160,000-g pellet), similar to the Escherichia coli polymeric ADHE. In the Coomassie staining of the 160,000-g pellet analyzed by electrophoresis, a 96-kDa protein was more prominent than in other fractions; this band was recognized by antibodies against Lactococcus lactis ADHE. By gold labeling, the antibodies recognized the granular material that mainly constitutes the 160,000-g pellet and a material that sedimented along with the internal membrane vesicles. By negative staining, the 160,000-g fraction showed helical rodlike structures with an average length of 103 nm; almost no membrane vesicles were observed in this pellet. In internal membrane fractions, no rodlike structures were found, but protomerlike round structures were observed. These results indicate that the main amebic NAD+-dependent ADH2 activity is naturally organized as rodlike helical particles, similar to bacterial ADHE. Detection of ADH2 in membrane fractions might be explained by cosedimentation of the multimeric ADH during membrane purification.

Acetaldehyde/alcohol dehydrogenase-2 (EhADH2) and clathrin are involved in internalization of human transferrin by Entamoeba histolytica

“…In permeabilized amoebae, EhADH2 was shown to be distributed in the cell membrane and in the cytoplasm (Fig. 2a, red), as was reported by Flores et al, (1996) and Avila et al, (2002). In nonpermeabilized amoebae, EhADH2 was observed to be bound only on the cell membrane (Fig.…”

“…EhADH2 has been purified and studied by several groups (Avila et al, 2002;Bruchhaus & Tannich, 1994;Chen et al, 2004;Espinosa et al, 2001Espinosa et al, , 2004Espinosa et al, , 2009Flores et al, 1996;Yang et al, 1994) and has the ability to bind to extracellular matrix (ECM) proteins. Interestingly, it is a typical cytoplasmic enzyme, although it is also found in the cell membrane.…”

“…EhADH2 was purified and a rabbit antiserum was prepared against the 95 kDa protein band excised from the gel (Avila et al, 2002). To determine whether the purified EhADH2 and the immunoprecipitated EhTfbp were the same protein, they were separated by 10 % SDS-PAGE and transferred to a nitrocellulose membrane blocked with PBS-T-milk for 1 h at room temperature (RT).…”

“…When the polypeptide was analysed by MS/MS, and after searching in public databases with ProteinPilot software, two peptides, TRNPIVFSFHPSALK and KIFIVSDRM, were identified with 99 % confidence in two independent experiments. The two peptides belong to EhADH2 (ADH2_ENTHI with accession number Q24803) previously described by Yang et al, (1994), Bruchhaus & Tannich, (1994) and Avila et al, (2002). …”

Transferrin Binding Proteins as a Means to Obtain Iron in Parasitic Protozoa

“…The EhADH2 amino acid sequence and that predicted for the HsTFR are not similar, so the recognition of both proteins by the mAb could be explained by a structural connection. EhADH2 is an essential enzyme used for obtaining energy by glucose fermentation [Bruchhaus and Tannich, 1994b;Yang et al, 1994;Flores et al, 1996;Espinosa et al, 2001;Avila et al, 2002;Chen et al, 2004;Espinosa et al, 2004;Espinosa et al, 2009]. Due to the properties of EhADH2, such as its ability to bind to host extracellular matrix proteins, its presence on the cell membrane, and its requirement for iron, the blocking of this enzyme with iron chelators as a therapeutic strategy against E. histolytica is an interesting future perspective [Espinosa et al, 2009].…”