| 1994 |
GRA3 is stored as a soluble protein in dense granules and, following secretion into the parasitophorous vacuole, inserts directly into the vacuole membrane in an oligomeric form via hydrophobic interactions; it cannot be removed by sodium carbonate extraction, is not labeled by [1-14C]acetate, and cross-links to itself (forming a ladder on SDS-PAGE) but no other parasite or host proteins co-precipitate with cross-linked GRA3. |
Sucrose flotation gradients, Triton X-114 phase partitioning, sodium carbonate extraction, chemical cross-linking, SDS-PAGE, [1-14C]acetate labeling |
The Journal of biological chemistry |
High |
8195173
|
| 1994 |
GRA3 cDNA encodes a 30 kDa protein with an N-terminal signal sequence (sufficient to target GFP to dense granules and PV) and four short hydrophobic regions insufficient to span the membrane; Southern blot indicates a single-copy gene; GRA3 localizes to dense granules, the PVM, and PVM extensions protruding into the host cytoplasm. |
cDNA cloning, Northern blot, Southern blot, in vitro translation with dog pancreas microsomes, immunofluorescence, immunoblot with affinity-purified antibodies |
Molecular and biochemical parasitology |
High |
7739670
|
| 1993 |
Dense granule exocytosis (releasing GRA2 and GRA3) occurs in the apical area of the tachyzoite within minutes of invasion; dense granule contents appear first as dense material between parasite and vacuole membranes before the vacuolar network or PVM are immunolabeled; GRA3 antibodies label the PVM and strands extending from PVs into the host-cell cytoplasm. |
Immunofluorescence assay (IFA), immunoelectron microscopy, serial sectioning, freeze-fracture studies at defined post-invasion time points |
Parasitology research |
High |
8415546
|
| 1999 |
GRA3 release from dense granules is constitutive and calcium-independent; the NSF/SNAP/SNARE/Rab machinery mediates dense granule exocytosis, as shown by inhibition with N-ethylmaleimide and Rab-GDI, and augmentation with recombinant NSF and α-SNAP in streptolysin O-permeabilized parasites. |
Endogenous GRA3 secretion assay in intact and SLO-permeabilized parasites, recombinant protein addition, pharmacological inhibitors (NEM, Rab-GDI), GTPγS and cAMP treatments |
The Journal of biological chemistry |
High |
9891012
|
| 2005 |
The originally published GRA3 sequence is an artificial chimera of two proteins. The authentic GRA3 (24 kDa, recognized by mAb 2H11) contains an N-terminal secretory signal sequence and a transmembrane domain consistent with PVM insertion, plus a C-terminal dilysine (KKXX) ER retrieval motif. The signal peptide alone is necessary and sufficient to target GFP to dense granules and the PV. A Neospora caninum homologue was identified. |
Sequence analysis, recombinant antibody production, immunoblot of ESA, GFP fusion/targeting assay, bioinformatics identification of KKXX motif |
Parasitology |
High |
16149193
|
| 2007 |
Within the parasite and the PV soluble fraction, GRA2 and GRA5 form high-molecular-weight oligomeric complexes with multiple GRA proteins; affinity purification of HA-FLAG-tagged GRA2 from ΔeGRA2 complemented parasites co-purified GRA3, GRA6, and GRA7 as the major partners. After membrane insertion, GRA2 associates with GRA5 and GRA7 but not GRA3. |
Affinity chromatography (HA-FLAG tag), conventional chromatography, complementation of KO parasites with tagged proteins, co-purification/MS |
Molecular and biochemical parasitology |
High |
17959262
|
| 2007 |
Targeted deletion of GRA3 in a type II strain shows no effect on cell culture growth rate or bradyzoite formation, and does not alter localization of other dense granule proteins or association with host cell organelles; however, at ~4× the LD50, ΔeGRA3 mice survived acute infection, and complementation restored and even enhanced virulence, demonstrating GRA3 contributes to acute virulence in vivo. |
Homologous recombination gene knockout, plaque assay, bradyzoite differentiation assay, IFA for GRA protein localization, mouse survival experiments, genetic complementation |
Molecular and biochemical parasitology |
High |
17418907
|
| 2008 |
PVM-inserted GRA3 physically interacts with CAMLG (calcium modulating ligand), a type II transmembrane protein of the host cell ER, as established by yeast two-hybrid screening of a HeLa cDNA library, GST pull-down, and co-immunoprecipitation; GRA3 and CAMLG co-localize at the PVM in infected cells, suggesting GRA3 mediates ER recruitment to the PVM. |
Yeast two-hybrid (GRA3 ORF vs HeLa cDNA library), GST pull-down assay, co-immunoprecipitation, co-transfection/co-localization by fluorescence microscopy, IFA in infected cells |
The Korean journal of parasitology |
Medium |
19127325
|
| 2008 |
GRA7 strands in the host cytosol (formed within 10 min of invasion) also contain GRA1 and GRA3, indicating GRA3 is present in cytosolic strand-like structures alongside rhoptry proteins ROP2 and ROP4 in infected host cells. |
IFA with co-labeling of GRA7, GRA1, GRA3, ROP2, ROP4 at defined post-invasion time points |
Infection and immunity |
Medium |
18809661
|
| 1995 |
Sporozoites of T. gondii lack GRA3 protein (not detected by IFA or Western blot in sporozoite-infected cells 2–15 h post-inoculation); GRA3 expression first appears 15 h post-inoculation and is present in all intracellular parasites at 24 h, coinciding with expression of the tachyzoite marker SAG1 and onset of replication. |
Indirect immunofluorescence, Western blot of sporozoite extracts, time-course monitoring of GRA3 and SAG1 expression |
Molecular and biochemical parasitology |
Medium |
8720177
|
| 2019 |
Deletion of PVM-associated GRA3 in the type II Prugniaud strain induced severe defects in chronic cyst development in vivo without affecting parasite growth rate or in vitro cyst differentiation; acute virulence of PruΔgra3 was reduced but not abolished. |
Targeted gene deletion (Δgra3) in type II Pru strain, in vivo mouse cyst burden assay, in vitro bradyzoite differentiation, plaque assay |
mBio |
High |
31266861
|
| 2019 |
TgGRA3 interacts with host Golgi membranes, oligomerizes, and promotes the formation of tubules and entry of host Golgi material into the PV; disruption of the TgGRA3 gene delays engulfment of host Golgi; GRA3 dysregulates anterograde transport in the host cell. |
Gene disruption (ΔgGRA3), live imaging/fluorescence microscopy of host Golgi, biochemical binding assay of GRA3 to isolated Golgi membranes, oligomerization analysis |
Biology open |
Medium |
30814066
|
| 2022 |
GRA3 (Wh6 allele) induces neuronal apoptosis via ER stress by activating the PERK pathway; transfection of N2a cells with GRA3Wh6 increased GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP; PERK inhibitor GSK2656157 and caspase-12 inhibitor Z-ATAD-FMK reduced GRA3-induced apoptosis. |
Plasmid transfection of N2a cells with pEGFP-GRA3Wh6, flow cytometry (apoptosis), immunoblotting for ER stress/apoptosis markers, pharmacological inhibitors (GSK2656157, Z-ATAD-FMK) |
Parasites & vectors |
Medium |
35918751
|
| 2023 |
GRA3-deficient T. gondii cysts showed significantly enhanced elimination by anti-cyst CD8+ T cells (perforin-mediated) compared to wild-type; cysts that persisted in the presence of CD8+ T cells had significantly greater GRA3 mRNA levels than the total cyst population; GRA3 mRNA levels correlated with successful cyst persistence during active T-cell immunity. |
GRA3-deficient parasite strain, in vivo cyst burden measurement in BALB/c mice with CD8+ T-cell immunity, qRT-PCR of GRA3 mRNA in persisting vs. total cysts, correlation analysis |
Frontiers in immunology |
Medium |
37868957
|
| 2019 |
Deletion of GRA3 (PVM-associated) did not affect the antitumor immunity activated by uracil auxotrophic T. gondii mutants, in contrast to deletions of GRA2, GRA12, GRA24, ROP5, ROP17, ROP18, and ROP35, which severely abrogated the antitumor response. |
Targeted gene deletion in Δku80 vaccine strain, tumor model (established ovarian cancer), flow cytometry, cytokine measurement |
PLoS genetics |
Medium |
27447180
|
| 2026 |
GRA3 interacts with host STING, promotes STING oligomerization, and nuclear translocation of phosphorylated IRF3, enhancing IFN-β production via the cGAS-STING pathway; GRA3 also promotes STAT6 phosphorylation; the net effect is enhanced parasite proliferation mediated by ISG56 and STAT6 rather than IFN-β itself. |
Co-immunoprecipitation (GRA3-STING interaction), STING oligomerization assay, nuclear fractionation (p-IRF3 translocation), gene KO/overexpression, IFN-β ELISA, ISG56/STAT6 inhibitor/KO experiments, parasite proliferation assays |
PLoS neglected tropical diseases |
Medium |
41712610
|
| 2025 |
CRISPR/Cas9-generated ME49Δgra3 parasites show reduced plaque number, a lower proportion of PVs with ≥4 tachyzoites, and significantly increased host mortality in C57BL/6 mice (70% vs 10% at 35 days), demonstrating that GRA3 promotes intracellular replication and virulence in the ME49 type II strain. |
CRISPR/Cas9 gene disruption, Western blot confirmation of protein absence, crystal violet plaque assay, Giemsa staining of PV sizes, mouse survival experiment |
Zhongguo xue xi chong bing fang zhi za zhi |
Medium |
40730529
|
| 2019 |
GRA3 (along with GRA2, GRA4, GRA5, GRA7, GRA12) was found in a proteomic characterization of purified cyst wall fragments from in vitro T. gondii cysts, establishing GRA3 as a component of the cyst wall. |
Percoll gradient enrichment of cyst wall fragments, CST1 immunoprecipitation, LC-MS/MS proteomic analysis |
mBio |
Medium |
31040239
|
| 2019 |
Deletion of GRA3 increased MHC-I antigen presentation of PV-localized parasite antigens to CD8+ T cells in infected macrophages and/or dendritic cells, indicating that GRA3 suppresses antigen presentation; this suppression operated independently of IFN-γ or IRG effectors (unlike GRA7/ROP5/ROP18). |
Targeted GRA3 deletion, MHC-I antigen presentation assay with CD8+ T cells, macrophage and dendritic cell infection, ±IFN-γ conditions, comparison with IRG-dependent effectors |
Frontiers in immunology |
Medium |
31555296
|