Toxoplasma gondiidense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif

Abstract

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory-secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine 'KKXX' endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

“…This explanation could be applied to GRA6 of which the N-terminal sequence was similar to that of GRA3 [28,29]. Hydrophobicity analysis predicted 2 putative TMDs in the middle of GRA3, which is different from the previous report [15]. GRA7 and GRA10 also have 2 TMDs in their polypeptide, in which the cell attachment sequence of RGD motif is present in the middle of TMDs [19,30], whereas no significant motifs in GRA3.…”

“…A second ATG 39 bp downstream of the first one was followed by TTCACA and a T at position +4. Two ways of translation were possible on the same in-frame of the sequence as noted by previous authors [15,16]. The first ATG was selected as the starting of translation by applying the consensus rule strictly in this study, which provided an ORF of 669 bp.…”

“…Downstream of the second TMD, a CM of Yxx appeared to sort the remaining C-terminal sequence to the cytoplasmic face of membrane [31,32]. In the Cterminal end, an ERM of KKxx [15,33] appeared to function of GRA3 in PVM-organelle association. With the consideration of STS, 2 TMDs and CM, it is suggested that GRA3 belongs to a type III transmembrane protein rather than type I.…”

“…During the screening of a T. gondii cDNA expression library, Tg556 had been found to bind to GRA3 of T. gondii of which the sequence was homologous to that revised by a previous author [15] but not to the first reported one [16]. In the yeast two-hybrid assay with GRA3 as a bait to the prey of HeLa cDNA expression library, GRA3 interacted with calcium modulating ligand (CAMLG), an ER-anchoring protein [17].…”

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

“…The endogenous dense granule marker GRA3 is secreted constitutively in a calcium-independent fashion using T. gondii NSF/SNAP/SNARE/Rab machinery that can interact functionally with their mammalian homologues [33]. Recently, previously published sequence for GRA3 is actually an artificial chimera of 2 proteins of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank [34]. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM.…”