E protein was studied using specific anti E antibodies
E1 protein was studied using specific anti-E1 d-biotin australia in wild type cells and in transfected cells overexpressing the enzyme. The results revealed several unusual characteristics, which are unique to this ubiquitin-activating enzyme. The E1 was translated as a full-length protein and then quickly proteolytically processed to generate two major peptides: E1-68 (N-terminal) and the E1-47 (C-terminal) (the names according with their approximate molecular weight) (Fig. 2, Fig. 3). These proteins were observed in immunoblots from trophozoites, cyst, all encystation stages and in cells transformed with the tagged protein. In transformed trophozoites, that express a recombinant E1, the proteolytic event occurs as in normal cells since the full length protein was not detected, but an enhanced E1 activity was observed (reflected as a much higher Ub-conjugates production). The ubiquitination in vitro assay showed that E1 functionality was not affected by the proteolytic process of the enzyme. This processing of Giardia E1 in two polypeptides E1-68 and E1-47 strongly resembles the heterodimeric E1 proteins in the ubiquitination-like SUMO and NEED8 pathways. Members of the E1 family as SUMO- and NEED8-activating enzymes (E1s) are heterodimers as shown in Fig. 1 (Schulman and Harper, 2009) and have to be associated to generate the active E1. SAE1 (for SUMO) and NAE1 (for NEED8) are homologous to the N-terminal half of the E1 for Ub described here, and on the other hand UBA2 (for SUMO) and UBA3 (for NEED8) are homologous to the C-terminal half. Both subunits (MoeB or ThiF repeats) are important in generating the adenylation domain responsible for the Ub-like protein (Ubl) recognition (Lee and Schindelin, 2008, Schulman and Harper, 2009). By analogy to these heterodimeric E1 enzymes, and based in our results that show the absence of the complete protein both in normal and in transformed cells over-expressing E1, we suggest the possibility that Giardia's E1 peptides: E1-68 and E1-47, associate to generate the active enzyme. Immunofluorescence assays were performed to localize the enzyme during growth and differentiation to further characterize Giardia E1. As stated before, the assays showed that the antibodies generated recognized specific E1 forms (E1-114, E1-90 and E1-67 with anti-gNTE1; and E1-47 with anti-gE1CT); the patterns were very similar when using antibodies against the C or the N terminal fragments. The protein was localized at the cytoplasm in several small spots and not in a diffuse form (Fig. 4). The punctuate pattern may indicate E1 vesicular or membrane association. This specific pattern may also reflect regulation by ubiquitination in specifics compartments within the cell. When Giardia was induced to encyst, the protein presented a more diffuse pattern in the cytoplasm (Fig. 4). Encystation involves important molecular and cellular processes and regulation of the synthesis, sorting and transport of cyst wall components (Carranza and Lujan, 2010). The change in the localization pattern in encysting cells may be caused by association of ubiquitination to events involved in the process. Besides, we observed co-localization between some E1 signals and encystation-specific vesicles (ESVs) in encysting cells (Fig. 4), the ESVs are secretory granules which transport CWPs (cyst wall proteins) to the cell surface for release and assembly into the cyst wall (Carranza and Lujan, 2010). The protein was localized also as a diffuse pattern in the cyst cell body but, interestingly, also in the wall or very close to it. Such localization might be important because the only known antibodies to label the wall are those against CWPs. Other proteins had been localized in the cyst wall: A high cysteine non-variant cyst protein (HCNCp) and a group of (EGF)-like cyst proteins (EGFCPs), in these cases the authors used an epitope-tag approach for detecting those molecules in the cyst wall (Davids et al., 2006, Chiu et al., 2010). Localization in the cyst wall of E1 may show a relationship between ubiquitination and the dynamic formation and disruption of the cyst matrix, which is a poorly understood process.