EOGT Mouse
Description
Key Molecular Features:
Notch Signaling Regulation
EOGT-mediated O-GlcNAcylation modulates Notch receptor activity:
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Residual Signaling in Intestine: Mice lacking Pofut1 (O-fucosyltransferase) survive due to residual Notch signaling enabled by EOGT. Double knockout (Eogt:Pofut1 dKO) results in lethality by postnatal day 28, with severe intestinal defects (e.g., goblet/Paneth cell hyperplasia) .
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Retinal Angiogenesis: Eogt−/− mice exhibit delayed retinal vascularization and reduced Notch target gene (Hes1, Hey1) expression .
Phenotypic Comparison of Knockout Models:
| Genotype | Viability | Key Phenotypes |
|---|---|---|
| Eogt−/− | Viable | Mild vascular defects |
| Pofut1 cKO | Viable | Intestinal lineage imbalance |
| Eogt:Pofut1 dKO | Lethal (P28) | Severe weight loss, Hes1 downregulation |
Research Reagents and Applications
Commercially available EOGT Mouse proteins are used for:
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Enzyme Activity Assays: Measuring GlcNAc transfer to Notch EGF repeats .
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Structural Studies: Analyzing N-glycan roles in ER retention and protein maturation .
Product Comparison:
| Vendor | Catalog # | Source | Molecular Mass |
|---|---|---|---|
| Bio-Techne | 8116-GT | NS0 cells | 59 kDa |
| Prospec Bio | ENZ-946 | Sf9 cells | 60.4 kDa |
Future Directions
EOGT Mouse models are pivotal for dissecting O-GlcNAc’s role in developmental disorders (e.g., Adams-Oliver syndrome ) and cancer . Ongoing research focuses on:
Product Specs
Q&A
What is the primary role of EOGT in mice, and how is it studied experimentally?
Basic: EOGT (EGF domain-specific O-GlcNAc transferase) is an ER-resident enzyme that O-GlcNAcylates EGF domains of Notch receptors, modulating Delta-like ligand-mediated Notch signaling . Experimental models include CRISPR-generated Eogt knockout (KO) mice to study its role in developmental and pathological processes.
Advanced: EOGT’s function is tightly linked to N-glycosylation, which affects its maturation, ER localization, and enzymatic activity. Studies substitute N-glycosylation sites (e.g., N263Q/N354Q) to assess their impact on protein stability and Notch1 O-GlcNAc stoichiometry .
Methodological Note: Validate EOGT KO models via Western blot for EOGT protein and Notch1 O-GlcNAc levels. Use lectin blotting (e.g., ConA) to confirm N-glycan structures .
How do N-glycans influence EOGT’s function, and what experimental approaches identify their roles?
Basic: N-Glycans at Asn-263 and Asn-354 stabilize EOGT expression and ensure proper ER localization. Loss of both sites impairs protein maturation and reduces Notch1 O-GlcNAcylation .
Advanced: Single-site N-glycosylation (e.g., N263Q or N354Q) is sufficient for EOGT maturation and activity, but double-site loss disrupts ER distribution and Notch signaling .
Methodological Approach: Use endoglycosidase H (Endo H) digestion and mass spectrometry to confirm N-glycan types (e.g., oligomannose structures) . Compare subcellular localization via confocal microscopy of WT vs. mutant EOGT .
What are the key challenges in interpreting EOGT knockout data, and how are they addressed?
Basic: Eogt KO models may exhibit compensatory mechanisms or residual Notch signaling via alternative pathways.
Advanced: In Pofut1 cKO mice lacking EOGT, viability is lost by P28, highlighting EOGT’s role in residual Notch signaling . Confounding factors include off-target CRISPR effects; validate with RNA sequencing and rescue experiments.
Data Contradiction Analysis: Variability in Notch1 O-GlcNAc stoichiometry between studies may stem from differences in cell type, developmental stage, or detection methods (e.g., antibodies vs. lectin blots) .
How is EOGT’s interaction with Notch signaling studied in intestinal epithelial cells?
Basic: Eogt:Pofut1 double knockout (dKO) mice enable analysis of EOGT’s contribution to residual Notch signaling in the absence of protein O-fucosylation .
Advanced: Use lineage-tracing strategies (e.g., Villin-Cre) to assess EOGT’s role in intestinal stem cell maintenance. Quantify crypt-villus axis morphology and cell proliferation markers (e.g., Ki67) .
Methodological Table:
| Parameter | Control (Pofut1 cKO) | dKO (Eogt:Pofut1) |
|---|---|---|
| Survival | ≥6 months | Death by P28 |
| Intestinal Lineage | Altered representation | Severe dysplasia |
| Notch1 O-GlcNAc | Detectable | Undetectable |
What are best practices for designing EOGT-related olfactory studies in mice?
Basic: EOGT is not directly linked to olfaction, but olfactory testing methodologies (e.g., olfactometers) inform behavioral assays for Notch-related phenotypes.
Advanced: Address threshold variability by standardizing criteria (e.g., 85% correct responses over 340 trials) and controlling for extraneous stimuli (e.g., valve noise) .
Contradiction Resolution: Discrepancies in olfactory thresholds between studies often arise from differing session/block numbers and % correct thresholds. Replicate results across multiple mice and cohorts .
How do N-glycosylation site mutations in EOGT affect its enzymatic activity?
Basic: Mutations at S265A, S265L, or T356I (near N-glycosylation sites) may impair EOGT’s ability to O-GlcNAcylate Notch1.
Advanced: Substitution of Asn-263 or Asn-354 with Gln (N263Q/N354Q) reduces EOGT expression but not enzymatic activity. Single-site mutations (e.g., N263Q) retain activity, suggesting redundancy in N-glycan function .
Experimental Workflow:
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Transfect HEK293T cells with WT or mutant EOGT.
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Co-express Notch1 and perform lectin blotting (WGA, ConA) to assess O-GlcNAc levels.
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Quantify enzymatic activity via Notch1 O-GlcNAc stoichiometry .
What tools are optimal for detecting O-GlcNAc modifications on Notch1 in EOGT KO models?
Basic: Use O-GlcNAc-specific antibodies (e.g., RL2) or lectin-based assays (e.g., wheat germ agglutinin).
Advanced: Validate antibody specificity with O-GlcNAcase treatment or EOGT inhibition. For quantitative analysis, employ mass spectrometry to map O-GlcNAc sites on Notch1 .
Limitations: Antibody cross-reactivity with N-glycans or other glycan structures may confound results. Include negative controls (e.g., O-GlcNAcase-treated lysates) .
How does EOGT’s ER localization impact Notch signaling in vivo?
Basic: EOGT’s KDEL-like motif ensures ER retention, enabling O-GlcNAcylation of Notch1 during its synthesis .
Advanced: Disruption of ER localization (e.g., via N-glycosylation loss) misdirects EOGT to the Golgi, reducing Notch1 modification. Use subcellular fractionation and confocal microscopy to assess EOGT distribution .
Key Findings:
What are the implications of EOGT mutations in cancer?
Basic: EOGT mutations near N-glycosylation sites (e.g., S265A, T356I) are annotated in cancer databases but lack functional validation .
Advanced: Test these mutations in Eogt KO models to assess their impact on Notch signaling and tumorigenesis. Compare with WT EOGT’s role in maintaining Notch1 O-GlcNAc levels .
Data Gap: No studies have directly linked EOGT mutations to cancer progression. Prioritize functional characterization of cancer-associated variants .
How should researchers address conflicting data in EOGT studies?
Basic: Discrepancies often arise from differences in cell types, detection methods, or genetic models.
Advanced: Cross-validate findings using orthogonal approaches:
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Biochemical: Lectin blotting + mass spectrometry.
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Genetic: Rescue experiments with WT vs. mutant EOGT.
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Functional: Tissue-specific KO models to isolate phenotypes .
Example: Pofut1 cKO vs. dKO mice reveal EOGT’s role in residual Notch signaling .
What computational tools aid in analyzing EOGT’s N-glycosylation and O-GlcNAcylation data?
Basic: Use UniProt for N-glycosylation site prediction and NetNGlyc for site-specific analysis.
Advanced: Integrate glycomics data with proteomics using tools like Skyline or MaxQuant for quantitative analysis of O-GlcNAc modifications .
Workflow:
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Identify N-glycosylation sites via MS-based glycoproteomics.
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Validate with site-directed mutagenesis and lectin blotting.
How does EOGT’s interaction with Notch receptors differ from other O-GlcNAc transferases?
Basic: Unlike cytoplasmic/nuclear O-GlcNAc transferases, EOGT targets ER-localized Notch1 EGF domains .
Advanced: EOGT’s substrate specificity is determined by its ER localization and interaction with EGF repeats. Compare EOGT and TDP-43 (a cytoplasmic O-GlcNAc transferase) activity in Notch1 modification assays .
Key Insight: EOGT’s ER residency ensures spatial segregation from other O-GlcNAc transferases, enabling precise substrate targeting .
Product Science Overview
EGF Domain-Specific O-Linked N-Acetylglucosamine Transferase (EOGT) is an enzyme that plays a crucial role in the post-translational modification of proteins. This enzyme is responsible for the addition of N-acetylglucosamine (GlcNAc) to serine or threonine residues within the epidermal growth factor (EGF)-like domains of extracellular proteins. The mouse recombinant version of this enzyme is often used in research to study its function and role in various biological processes.
EOGT is a glycosyltransferase that catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in extracellular proteins. This modification results in the formation of a beta-linked N-acetylglucosamine (O-GlcNAc) on the target protein . The enzyme specifically glycosylates the threonine residue located between the fifth and sixth conserved cysteines of folded EGF-like domains .
The modification of proteins by O-GlcNAc is essential for various cellular processes, including intracellular signaling, endocytosis, transcription, and protein stability . EOGT’s activity is particularly important in the regulation of developmental signaling pathways. For instance, it modifies the Notch receptor, which is involved in cell differentiation, proliferation, and apoptosis .
The EOGT gene is located on chromosome 3p14.1 and encodes a protein that is 527 amino acids long . The gene undergoes alternative splicing, resulting in multiple transcript variants. Mutations in the EOGT gene have been associated with Adams-Oliver Syndrome 4, a genetic disorder characterized by congenital limb defects and scalp abnormalities .
The mouse recombinant version of EOGT is widely used in research to study its enzymatic activity and role in various biological processes. Researchers utilize this enzyme to investigate the mechanisms of protein glycosylation and its impact on cellular functions. Additionally, studies on EOGT can provide insights into the development of therapeutic strategies for diseases associated with glycosylation defects.
