A Giardia-specific protein family denominated as ␣-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite Giardia lamblia. One of its members, ␣19-giardin, carries an N-terminal sequence extension of MGCXXS, which in many proteins serves as a target for dual lipid conjugation: myristoylation at the glycine residue after removal of the methionine and palmitoylation at the cysteine residue. As the first experimental evidence of a lipid modification, we found ␣19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of ␣19-giardin with giardial N-myristoyltransferase (NMT) in Escherichia coli, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in Saccharomyces cerevisiae, ␣19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type Giardia trophozoites with tubulin provide evidence that ␣19-giardin exclusively localizes to the ventral pair of the giardial flagella. A mutant in which the putatively myristoylated N-terminal glycine residue was replaced by alanine lost this specific localization. Our findings suggest that the dual lipidation of ␣19-giardin is responsible for its specific flagellar localization.

In a previous study, we reported that the novel annexin XX1 (annexin E1), identical to alpha14-giardin, is specifically localized to the flagella and to the median body of the trophozoites. However, the mode of interaction and the direct partners involved remained unclear. In the present study, we show that alpha4-giardin obviously does not evenly distribute over the full length of the axonemes, but rather, resides at local slubs near the proximal part and the ends of the flagella. In immunocytochemical co-localization studies, the anti-giardin primary antibody exclusively reacted with distinct regions of the flagella in permeabilized cells, whereas the anti-tubulin antibody bound to all areas of the axonemes in the cells and to isolated cytoskeletons. Isolated cytoskeletons did not react with anti-giardin antibodies. alpha14-Giardin itself is able to assemble to multimeric structures. Taken together, our findings suggest that alpha14-giardin adheres to microtubules of the flagella via self-assembly that may regulated by Ser/Thr-phosphorylation.

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The genome of Entamoeba histolytica contains two genes encoding inhibitors of cysteine proteases of the chagasin family. In contrast to that of EhICP1, the derived primary structure of the second inhibitor, EhICP2, possesses a typical N-terminal signal sequence. Processed EhICP2 is as weakly related to amoebiasin-1 (27% identity) as to chagasin (identity 30%), indicating a different evolutionary origin of both amebic genes. By Northern blots, we confirmed the expression of the ehicp2 gene, and in Western blots, the presence of the 11.5-kDa protein in trophozoite extracts was demonstrated. The inhibitor localized to large intracellular structures clearly differs from those containing EhICP1 as shown by indirect immunofluorescence. Recombinant EhICP2 significantly inhibited the cysteine protease activity of the amebic cell extract but with a lower extent than EhICP1. An overlay assay using a crude trophozoite extract demonstrated binding affinity of the amebic cysteine protease EhCP1 to EhICP2.

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