Table 1.
Comparison of NADP-dependent alcohol dehydrogenase activity in lysates of E. dispar, E. histolytica HM-1∶IMSS, and E. histolytica HM-1∶IMSS overexpressing EhADH3 (HAO).
Figure 1.
2D-DIGE comparison of two E. histolytica HM-1∶IMSS strains.
2852 spots were identified in this gel representing whole cell lysates from two strains of E. histolytica HM-1∶IMSS separately prepared. One was maintained in Saint Louis, Missouri, USA, and the other in London, England. Using a three-fold cutoff, only 6 labeled protein spots were found to fluoresce at different levels, suggesting limited biological variation exists between preparations and isolates. White spots are indicative of identical protein amounts; blue represents increased abundance in the Saint Louis isolate, while yellow represented increased abundance in the London isolate.
Figure 2.
Representative 2D-DIGE gel of E. histolytica and E. dispar lysates demonstrating the extent of differences between species.
One representative gel image of DIGE comparisons between E. histolytica and E. dispar highlights the measured differences in fluorescently labeled protein abundance between these two species. Yellow identifies protein spots that were proportionately higher in E. dispar; blue represents increased abundance in E. histolytica; white represents equal signal. Proteins were identified as follows (see Table 1): 1: ADH2- Higher in E. histolytica 6.1×; 2: ADH3- Higher in E. histolytica 5.8×; 3: Grainin 2- Higher in E. dispar 9.6×; 4: LIM domain protein- Higher in E. histolytica 12.6×.
Figure 3.
Peptide data from observed proteomic differences between E. histolytica and E. dispar.
Figure 4.
The gel area and spot representing ADH3 from one representative DIGE gel.
The right panel is a fluorescent intensity scan of E. histolytica; the identical region from E. dispar is on the left. The outlined spot was identified as ADH3 by mass spectrometry. The demarcated region was used to calculate the signal fold difference between the species' ADH3 protein abundance, which was 5.82-fold higher in E. histolytica than E. dispar in this gel.
Figure 5.
Anti-ADH3 Western blot confirms the difference between ADH3 protein abundance between species.
Polyclonal antibodies developed against recombinant EhADH3 were generated in rabbits, and used to stain amebic lysates on a 1D Western blot. Densitometric analysis, normalized against the amount of actin present in each species' sample, results in 5.4-fold more ADH3 in E. histolytica compared to E. dispar. To the right of each image is MagicMark XP (Invitrogen, Carlsbad, CA) marking the following ascending molecular weights: 20 kD, 30 kD, 40 kD, 50 kD, 60 kD, 80 kD, 100 kD, and 120 kD.
Figure 6.
Recombinant EhADH3 prefers straight chain alcohols as a substrate.
An enzymatic substrate preference assay was conducted to determine the optimal substrate for recombinant EhADH3. Butanol was demonstrated to be the preferred substrate, followed by shorter straight-chain alcohols. Branched alcohols were not detectably processed by recombinant EhADH3.
Figure 7.
Live immuofluoresent surface staining of EhADH3 reveals its presence on the plasma membrane surface of E. histolytica HM-1∶IMSS.
Amebae were harvested at 4°C, then blocked with blocking buffer for 10 min prior to staining with rabbit polyclonal anti-EhADH3 antibodies (panels A,B,C) or staining with antibodies pre-incubated with a molar excess of recombinant EhADH3 (panels D,E,F). Panels A and D show staining with the AlexaFlour secondary antibody, panels B and E the brightfield image, and panels C and F are a merge of the two. Magnification 63×.
Figure 8.
Western blot confirmation of EhADH3 overexpression in transfected E. histolytica.
E. histolytica strain E. histolytica HM-1∶IMSS was transfected to overexpress EhADH3 Lysates from the parent strain (E. histolytica HM-1∶IMSS), the pNEO control transfectant (HN), and the transfectants overexpressing EhADH3 (HAO) were separated on an SDS-PAGE gel, blotted to PVDF, and stained with polyclonal anti-EhADH3 antibodies (top panel) or anti-actin antibodies (bottom panel). A similar experiment was performed for transfectants overexpressing EhADH3 in E. histolytica Rahman (right upper and lower panels). Lysates from the parent E. histolytica Rahman strain (Rahman), the pNEO control transfectant (RN) and tranfectants overexpressing EhADH3 were processed as above.