| 1997 |
The T. gondii GPI glycolipid antigen bears a unique Glcα1-4GalNAc side branch on the conserved mannosyl core (structure: (ethanolamine-PO4)-Man-Man-(Glcα1-4GalNAcβ1-4)Man-GlcN-inositol-PO4-lipid), and only GPIs bearing this Glc-GalNAc side branch are immunogenic in humans; GPIs with GalNAc alone (without the terminal glucose) are not immunogenic. |
Exoglycosidase treatments, high pH anion-exchange chromatography, gel filtration, lectin affinity chromatography, 2D 1H-NMR, FAB-MS, methylation linkage analysis, and monoclonal antibody characterization |
Journal of molecular biology |
High |
9102470
|
| 2001 |
The GPI anchor of SAG1 (P30) exists in two glycoforms: one with GalNAcβ1,4 on the first mannose, and one with an additional terminal Glcα1,4 linked to GalNAc. Core GalNAc modification of the anchor takes place prior to transfer of the GPI to the protein, as inferred by comparison with free GPI precursor structures. |
Isolation of GPI anchor peptides from [3H]glucosamine-labeled purified P30, gel filtration, HPEIC, exoglycosidase treatment, MALDI-TOF-MS, FAB-MS, methylation linkage analysis, aminopropyl-HPLC |
Molecular and biochemical parasitology |
High |
11522346
|
| 2003 |
T. gondii encodes a functional UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T1), a type II membrane protein with GT1, Gal/GalNAc-T, and ricin lectin-like domains, that catalyzes transfer of GalNAc to Ser/Thr residues in polypeptides — the initial step of mucin-type O-glycosylation. When expressed in Drosophila S2 cells, purified recombinant protein exhibits transferase activity in vitro. |
cDNA library screening with degenerate PCR, stable expression in Drosophila S2 cells, in vitro transferase activity assay, Northern blot, genomic/cDNA sequence comparison |
Molecular and biochemical parasitology |
High |
14511808
|
| 2004 |
T. gondii encodes a family of five ppGalNAc-T isoforms (T1–T5). Three (T1, T2, T3) were fully cloned from a cDNA library, and two (T4, T5) were identified from genome sequence. All are type II membrane proteins with conserved GT1, Gal/GalNAc-T, and ricin lectin-like domains, establishing that mucin-type O-glycosylation in T. gondii is carried out by a multi-member enzyme family. |
cDNA library screening, genome sequence analysis, sequence alignment and domain characterization, exon structure determination |
International journal for parasitology |
Medium |
15003492
|
| 2004 |
T. gondii ppGalNAc-T3 is a functional 'follow-up' (glycopeptide) O-glycosyltransferase: when expressed in Drosophila S2 cells, it transfers GalNAc to glycopeptide acceptors but NOT to 'naked' (unglycosylated) peptide acceptors, distinguishing it functionally from initiating transferases. Its mRNA is expressed equivalently in tachyzoites and bradyzoites. |
cDNA cloning, stable expression in Drosophila S2 cells, in vitro glycosyltransferase activity assay with peptide vs. glycopeptide acceptors, RT-PCR for stage expression |
Archives of biochemistry and biophysics |
High |
15158673
|
| 2006 |
In T. gondii grown in human cells, the GalNAc-containing GPI (without terminal Glc) is predominantly used to anchor surface proteins, while the Glcα1,4GalNAc-containing GPI is found exclusively as free glycolipid (GIPL) on the parasite cell surface, enriched in lipid microdomains associated with proteins involved in motility and host cell invasion. |
Metabolic labeling, phase partitioning, serology with human sera, lipid microdomain fractionation, proteomic association |
The international journal of biochemistry & cell biology |
Medium |
16822699
|
| 2006 |
In the T. gondii GPI biosynthetic pathway, early GPI intermediates (GlcN-PI and GlcNAc-PI) are predominantly localized in the cytoplasmic leaflet of the ER membrane. More highly mannosylated and Glc-GalNAc-modified GPI intermediates are also preferentially in the cytoplasmic leaflet of the ER. |
Hypotonic permeabilization of T. gondii tachyzoites, metabolic labeling with UDP-[3H]GlcNAc, GDP-[3H]mannose, or UDP-[3H]glucose, PI-PLC treatment to assess membrane topology, electron microscopy, protease protection assays |
Eukaryotic cell |
Medium |
16896225
|
| 2007 |
In the T. gondii GPI biosynthetic pathway, inositol acylation (acyl-CoA-dependent) occurs before mannosylation and is inhibited by PMSF. Inositol deacylation of the fully mannosylated GPI intermediate (also inhibited by PMSF and DFP) must precede addition of the GalNAc side chain to the first mannose. The sn-2 position of the diacylglycerol lipid moiety in both free GPIs and GPI-anchored proteins contains almost exclusively a C18:1 acyl chain. |
In vitro GPI biosynthesis assays with radiolabeled substrates, use of serine protease inhibitors (PMSF, DFP) as pathway probes, structural characterization of GPI intermediates and products |
The Journal of biological chemistry |
High |
17804418
|
| 2017 |
ppGalNAc-T2 and ppGalNAc-T3 are the two isoforms responsible for O-GalNAc glycosylation of the cyst wall mucin-like glycoprotein CST1 in T. gondii, acting in a hierarchical manner: T2 performs the initial glycosylation of the mucin-like domain's threonine-rich tandem repeats, while T3 fills in subsequent neighboring acceptor sites. This glycosylation is required for CST1 to confer structural rigidity on brain cysts. Deletion of T2 or T3 produces glycosylation defects on multiple cyst wall glycoproteins. |
Individual CRISPR/Cas9 deletion of all five ppGalNAc-T isoforms, glycan analysis of cyst walls, cyst wall rigidity assays, mutagenesis-based assignment of hierarchical glycosylation, immunofluorescence |
mBio |
High |
28074022
|
| 2018 |
By coupling CRISPR/Cas9 gene editing with MS-based glycomics, T. gondii ppGalNAc-T activity was shown to be required for Tn-antigen (GalNAc-Ser/Thr) and a novel HexNAc-GalNAc mucin-type O-glycan. The study also identified and characterized an O-glycosylation pathway yielding novel nuclear O-Fuc-type and cell-surface Glc-Fuc-type O-glycans from separate glycosyltransferases, with the latter being important for in vitro growth. |
Double-CRISPR/Cas9 gene replacement of 17 glycogenes, MS-based glycomics, fibroblast infection growth assays |
The Journal of biological chemistry |
High |
30463938
|
| 2018 |
SRS13, a novel mucin-domain-containing SRS protein, is O-GalNAc glycosylated by ppGalNAc-T2 and T3, localizes to the cyst wall in bradyzoites, but is dispensable for normal cyst wall formation. |
Glycoepitope-specific antibody identification, immunofluorescence localization, genetic characterization, stage-specific expression analysis |
Parasitology research |
Medium |
29911257
|
| 2024 |
X-ray crystal structures of T. gondii ppGalNAc-T3 (TxgGalNAc-T3) reveal multiple features strictly conserved among apicomplexan homologues including a unique second metal ion that is coupled to substrate binding and enzymatic activity in vitro. This second metal is required for cyst wall O-glycosylation in T. gondii. The enzyme's structure diverges from host homologues, suggesting potential as a therapeutic target. |
X-ray crystallography, in vitro enzymatic activity assays with mutagenesis of metal-binding residues, cyst wall O-glycosylation assessment in T. gondii |
Nature communications |
High |
38710711
|
| 2024 |
Two glycosyltransferases responsible for building the GPI Glcα1,4GalNAcβ1 sidechain were identified: PIGJ adds the first GalNAc to the sidechain. Deletion of PIGJ abolishes the sidechain, increases parasite virulence, enhances scavenger receptor CD36 binding, and elevates galectin-3 binding to GIPLs, while antibody recognition of GPI-anchored SAGs and complement binding remain intact. Deletion of the second glycosyltransferase (adds Glc to GalNAc) leaves GalNAc-only sidechain with negligible effect on disease outcome. |
CRISPR/Cas9 deletion of PIGJ and second glycosyltransferase, serology, mass spectrometry for GPI structure confirmation, mouse virulence assays (WT and galectin-3 KO mice), CD36 binding assays, complement binding assays, brain cyst quantification |
mBio |
High |
39302131
|