C1GALT1 Antibody

What is C1GALT1 and why is it significant in glycobiology research?

C1GALT1 (Core 1 β1,3-galactosyltransferase 1) is an essential inverting glycosyltransferase that catalyzes the generation of the core 1 O-glycan structure (Gal-β1-3GalNAc-α1-Ser/Thr), commonly known as the T antigen. This structure serves as a crucial precursor for many extended O-glycans in glycoproteins .

The enzyme plays central roles in numerous biological processes, including:

• Angiogenesis

• Thrombopoiesis

• Kidney homeostasis development

• Cell signaling

• Immune responses

C1GALT1 research is significant because altered O-glycan structures can significantly impact cellular functions and contribute to various pathological conditions, making it a valuable target for understanding disease mechanisms .

How does C1GALT1 function in the glycosylation pathway?

C1GALT1 functions at a critical juncture in the O-glycosylation pathway:

• The pathway begins when GalNAc transferase adds GalNAc to serine/threonine residues, forming the Tn antigen (GalNAc α1-Ser/Thr)

• C1GALT1 then catalyzes the addition of galactose (Gal) from UDP-Gal to the Tn antigen, creating the core 1 structure (T antigen)

• This core 1 structure serves as the foundation for extension into more complex O-linked glycans

Importantly, C1GALT1 requires the molecular chaperone COSMC (C1GALT1C1) for proper folding and activity. In the endoplasmic reticulum, COSMC facilitates the conversion of C1GALT1 into its active dimeric form before it enters the Golgi apparatus, where it competes with other glycosyltransferases (C3GnT and ST6GalNAC-I/II) to initiate O-linked mucin glycan formation .

What criteria should be considered when selecting a C1GALT1 antibody for specific research applications?

When selecting a C1GALT1 antibody, researchers should consider:

Application compatibility:

• Verify the antibody has been validated for your specific application (WB, IHC, IF, IP, ELISA)

• Review published research using the antibody in your intended application

• Check if the antibody works in your sample type (human, mouse, etc.)

Antibody type considerations:

• Monoclonal antibodies (like the F-31 clone) offer high specificity but may recognize only a single epitope

• Polyclonal antibodies may provide greater sensitivity by recognizing multiple epitopes, but with potential for higher background

Validated experimental parameters:

• For WB: Confirm predicted band size (typically 42 kDa for C1GALT1)

• For IHC: Review staining patterns (typically cytoplasmic localization)

• Optimal dilution ranges for different applications (e.g., 1:500 for WB, 1:100 for IHC)

Immunogen information:

• Antibodies raised against recombinant fragments (e.g., within aa 1-150 of human C1GALT1) may have different recognition properties than those against full-length protein

How can researchers validate the specificity of a C1GALT1 antibody?

Methodological approach to antibody validation:

• Positive and negative controls:

  • Use tissues/cells known to express C1GALT1 (heart, kidney, liver, placenta) as positive controls

  • Include tissues/samples where C1GALT1 expression is minimal as negative controls

  • Consider using C1GALT1 knockdown cells (shRNA or siRNA) to confirm specificity

• Multiple detection methods:

  • Confirm findings with at least two independent detection techniques (e.g., WB and IHC)

  • Compare results from antibodies targeting different epitopes of C1GALT1

• Western blot validation:

  • Verify the antibody detects bands at the expected molecular weight (42 kDa)

  • Look for minimal non-specific bands

  • Perform peptide competition assays to confirm specificity

• Correlation with mRNA expression:

  • Compare antibody staining patterns with mRNA expression data from RT-PCR or RNA-seq

  • Concordance between protein and mRNA levels supports antibody specificity

What are the optimal protocols for detecting C1GALT1 in tissue samples using immunohistochemistry?

IHC-P Protocol for C1GALT1 Detection:

Sample preparation:

• Fix tissues in 10% neutral buffered formalin (24-48 hours)

• Process and embed in paraffin

• Section at 4μm thickness

Antigen retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Microwave or pressure cooker method (20 minutes)

Staining procedure:

• Block endogenous peroxidase (3% H₂O₂, 10 minutes)

• Protein blocking (5% normal serum, 30 minutes)

• Primary antibody incubation:

  • Recommended dilution: 1:100 for C1GALT1 antibodies

  • Incubate overnight at 4°C or 60 minutes at room temperature

• Detection system:

  • Use HRP-polymer detection systems for better signal-to-noise ratio

  • DAB chromogen for visualization

  • Hematoxylin counterstaining

Scoring method:

• Record percentage of C1GALT1 positive tumor cells (0-100% in 10% increments)

• Assess staining intensity (absent/faint/moderate/intense)

• Calculate expression score by multiplying percentage by intensity

• C1GALT1 shows primarily cytoplasmic localization

Quality control measures:

• Include known positive controls (kidney, liver tissues)

• Consider cores adequate if at least 25% of tumoral tissue is available for scoring

How can C1GALT1 antibodies be used to investigate altered glycosylation in disease models?

C1GALT1 antibodies can be employed in multiple ways to study glycosylation alterations:

Comparative expression analysis:

• Compare C1GALT1 expression levels between normal and pathological samples using IHC or WB

• Correlate with clinicopathological data to establish prognostic relationships

• Example: In neuroblastoma, high C1GALT1 expression correlates with differentiated tumor histology and better survival outcomes

Functional studies:

• Knockdown/knockout experiments:

  • Use C1GALT1 antibodies to confirm successful knockdown efficiency after siRNA/shRNA treatment

  • Monitor resulting changes in glycosylation patterns using lectin-based assays

  • Example: C1GALT1 depletion in ECC-1 endometrial cancer cells mimics an aggressive cancer phenotype

• Glycoprotein analysis:

  • Immunoprecipitate specific glycoproteins, then probe with C1GALT1 antibodies

  • Use lectin pull-down experiments (e.g., Vicia villosa agglutinin beads) followed by C1GALT1 antibody detection

  • Example: In neuroblastoma, C1GALT1 knockdown increases TrkA pulled down by VVA beads, indicating altered O-glycosylation

• Downstream signaling effects:

  • Monitor changes in signaling pathways (AKT, ERK) following C1GALT1 modulation

  • Example: C1GALT1 knockdown enhances AKT phosphorylation but attenuates ERK phosphorylation in neuroblastoma cells

How is C1GALT1 expression linked to IgA nephropathy pathogenesis?

C1GALT1 plays a central role in IgA nephropathy (IgAN) pathogenesis through the following mechanisms:

Expression abnormalities:

• C1GALT1 mRNA is significantly downregulated in B lymphocytes of IgAN patients compared to healthy controls

• This downregulation directly correlates with aberrant IgA1 glycosylation

Quantitative relationship:

• In a study of 30 IgAN patients and 30 healthy controls, C1GALT1 expression levels in B cells were significantly lower in IgAN patients (1.01 ± 0.19 vs 1.43 ± 0.11, p = 0.04)

• Meta-analysis of multiple studies confirmed reduced C1GALT1 gene expression in IgAN patient B lymphocytes (weighted mean difference, 0.39 [95% CI 0.08 to 0.69], p = 0.01)

Correlation with Gd-IgA1 levels:

• Galactose-deficient IgA1 (Gd-IgA1) levels are significantly elevated in IgAN patients

• C1GALT1 mRNA levels inversely correlate with Gd-IgA1 levels (r = −0.33, p < 0.001)

• In IgAN patients, Gd-IgA1 ranges from 8.55 to 14.48 U/mL compared to 3.97 to 12.15 U/mL in healthy controls

Enzymatic activity:

• Not only is C1GALT1 expression reduced, but β1,3Gal-T enzymatic activity is decreased in B cells of IgAN patients

• This reduced activity directly impacts the galactosylation of IgA1 O-glycans

Therapeutic implications:

• Targeting C1GALT1 expression or activity represents a potential therapeutic approach for IgAN

• Strategies to enhance C1GALT1 function might help normalize IgA1 glycosylation patterns

What is the significance of C1GALT1 expression in cancer progression and prognosis?

C1GALT1 exhibits context-dependent roles in cancer with divergent prognostic implications:

Neuroblastoma (favorable prognosis):

• High C1GALT1 expression correlates with differentiated tumor histology and predicts better survival outcomes

• In a cohort of 134 neuroblastoma patients, C1GALT1 high expression was significantly associated with differentiated histology (p < 0.001)

• Multivariate analysis confirmed C1GALT1's role as an independent prognostic factor

• 5-year survival probability was significantly higher in patients with high C1GALT1 expression

Mechanistic insights in neuroblastoma:

• C1GALT1 modulates O-glycans on TrkA, a neurotrophin receptor associated with favorable outcomes

• C1GALT1 knockdown reduces TrkA expression and promotes malignant phenotypes

• Overexpression of C1GALT1 increases TrkA protein levels and promotes neuronal differentiation

Endometrial cancer (unfavorable implications):

• C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype

• Low C1GALT1 expression in aggressive endometrial cancer was confirmed by IHC

• SILAC proteomics identified 100 dysregulated proteins in cell extracts and 144 in secretomes following C1GALT1 depletion

Ewing sarcoma (tumor-promoting role):

• C1GALT1 promotes EWSR1::FLI1 expression in Ewing sarcoma

• Mechanistically, C1GALT1 O-glycosylates Smoothened (SMO), stabilizing it and stimulating the Hedgehog pathway

• Inhibition of C1GALT1 reduces EWSR1::FLI1 levels and suppresses tumor growth in xenograft models

Colorectal cancer considerations:

• C1GALT1 plays a role in abnormal glycosylation and cancer progression

• It catalyzes formation of the T antigen, which serves as a precursor for complex O-glycans

• Altered O-glycan structures contribute to colorectal cancer pathogenesis

How can researchers effectively address contradictory findings related to C1GALT1 expression across different cancer types?

The contradictory roles of C1GALT1 in different cancers require careful methodological approaches:

Standardized quantification methods:

• Implement consistent scoring systems for C1GALT1 immunostaining

  • Use the 11-tiered scale (0-100% in 10% increments) for percentage of positive cells

  • Apply 4-tiered intensity scoring (absent/faint/moderate/intense)

  • Calculate expression scores by multiplying percentage by intensity

• Validate antibody specificity in each cancer type to ensure comparable measurements

• Include multiple antibody clones targeting different epitopes to confirm findings

Context-specific analysis:

• Evaluate C1GALT1 in relation to tissue-specific glycosylation targets

  • In neuroblastoma: Focus on TrkA O-glycosylation

  • In Ewing sarcoma: Examine Smoothened (SMO) O-glycosylation

• Determine downstream glycoproteins relevant to each cancer type

• Analyze tissue-specific expression patterns of competing glycosyltransferases

Functional validation approaches:

• Perform comparative knockdown/overexpression studies across multiple cancer cell lines

• Use rescue experiments to confirm phenotype specificity

• Complement in vitro findings with appropriate in vivo models for each cancer type

Integration with multi-omics data:

• Correlate C1GALT1 protein expression with transcriptomic datasets

• Perform glycoproteomic analysis to identify cancer-specific O-glycosylation targets

• Use SILAC quantitative proteomics to compare protein dysregulation patterns across cancer types

Reconciliation strategies:

• Consider tissue-specific C1GALT1 isoforms and their functional differences

• Investigate potential differences in C1GALT1C1 (COSMC) expression or mutation status

• Examine cancer-specific differences in O-glycosylation substrate availability

What advanced experimental approaches can detect subtle changes in C1GALT1 activity beyond expression levels?

Enzyme activity assays:

• Measure β1,3-galactosyltransferase activity using:

  • Synthetic glycopeptide substrates (e.g., P1-P7 design series)

  • Radioisotope-labeled UDP-Gal transfer assays

  • Fluorescent-based kinetic activity measurements

• Compare enzyme kinetics (Km, Vmax) between experimental conditions

O-glycan structural analysis:

• Mass spectrometry-based O-glycomics to profile glycan structures

• Lectin microarrays to detect specific glycan modifications

• High-sensitivity detection using acridinium-conjugated lectins instead of biotin-labeled lectins (approximately 10-fold increased sensitivity)

Substrate-specific approaches:

• Lectin pull-down experiments (e.g., Vicia villosa agglutinin beads for GalNAc-containing proteins)

• GalNAc-specific monoclonal antibodies (e.g., KM55) to examine Gd-IgA1 levels

• Monitor changes in O-glycosylation of specific proteins (e.g., TrkA, SMO)

Live cell imaging techniques:

• FRET-based biosensors to monitor C1GALT1-substrate interactions in real-time

• Proximity ligation assays to detect C1GALT1 interactions with specific target glycoproteins

• Subcellular localization tracking of C1GALT1 using confocal microscopy

Competitive glycosyltransferase dynamics:

• Assess relative activities of competing enzymes (C1GALT1, C3GnT, ST6GalNAC-I/II)

• Measure ratios of different glycan structures (core 1 vs. core 3 vs. sialylated structures)

• Determine rate-limiting steps in the O-glycosylation pathway

How can researchers establish causality between altered C1GALT1 function and disease phenotypes?

Genetic manipulation strategies:

• CRISPR/Cas9 approaches:

  • Generate precise C1GALT1 knockout cell lines

  • Create knockin models with specific mutations or tagged versions

  • Perform genome-scale CRISPR/Cas9 screens to identify C1GALT1-dependent pathways

• Inducible expression systems:

  • Establish tetracycline-inducible C1GALT1 expression models

  • Create temporal control of C1GALT1 activity to assess acute vs. chronic effects

  • Determine threshold levels required for phenotypic changes

Rescue experiments:

• Reintroduce wild-type or mutant C1GALT1 into knockout backgrounds

• Test domain-specific contributions through truncation or point mutations

• Assess whether catalytically inactive mutants can rescue phenotypes

Pharmacological interventions:

• Use specific inhibitors (e.g., itraconazole for C1GALT1 in Ewing sarcoma)

• Apply small molecule modulators of glycosylation pathways

• Compare pharmacological inhibition with genetic knockdown to confirm specificity

In vivo validation:

• Generate tissue-specific C1GALT1 knockout mouse models

• Use xenograft models with C1GALT1-modulated cells

• Perform patient-derived xenograft studies to validate clinical relevance

Clinical correlation studies:

• Analyze C1GALT1 expression in large patient cohorts with comprehensive clinical data

• Perform multivariate analyses to control for confounding factors

• Use Cox proportional hazards models to establish independent prognostic value