CHST6 Antibody

What is CHST6 and what is its function?

CHST6 is a sulfotransferase enzyme that catalyzes the transfer of sulfate to position 6 of non-reducing N-acetylglucosamine (GlcNAc) residues of keratan. It utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as a sulfonate donor for this reaction . The enzyme cooperates with B4GALT4 galactosyltransferase and B3GNT7 N-acetylglucosaminyltransferase to construct and elongate the sulfated disaccharide unit [->3Galbeta1->4(6-sulfoGlcNAcbeta)1->] within the keratan sulfate polymer .

CHST6 plays a critical role in biosynthesis of keratan sulfate in the cornea, significantly impacting proteoglycan fibril organization and corneal transparency . The enzyme is also involved in sulfation of endothelial mucins such as GLYCAM1 .

What is the relationship between CHST6 and corneal diseases?

CHST6 mutations are directly associated with Macular Corneal Dystrophy (MCD), a rare autosomal recessive disorder characterized by bilateral progressive stromal clouding and vision loss . MCD patients typically present with decreased vision, multiple irregular gray-white corneal opacities, and corneal thinning .

The pathogenesis involves defective sulfation of keratan sulfate due to CHST6 mutations, which disrupts corneal transparency . A comprehensive evaluation of 181 reported CHST6 variants in 408 MCD cases revealed that the vast majority (165 out of 181) could be classified as pathogenic or likely pathogenic according to ACMG guidelines . Corneal transplantation is often necessary for MCD patients .

What types of CHST6 antibodies are available for research?

Based on the available literature, researchers have access to polyclonal antibodies against CHST6. Specifically, a Rabbit Polyclonal CHST6 antibody (ab154332) has been documented, which targets a recombinant fragment protein within Human Carbohydrate sulfotransferase 6 amino acids 50-350 . This antibody has been cited in multiple publications, suggesting its reliability for research purposes .

The antibody's immunogen corresponds to a recombinant fragment protein within Human Carbohydrate sulfotransferase 6 amino acids 50-350, which encompasses a significant portion of the critical sulfotransferase domain (amino acids 42-356) .

What applications are CHST6 antibodies suitable for?

The documented CHST6 antibody has been validated for Western Blot (WB) applications with human samples . This allows researchers to detect and quantify CHST6 protein expression in tissue or cell lysates, assess protein degradation products, and evaluate post-translational modifications.

When selecting an antibody for specific applications, researchers should consider the validation status provided by the manufacturer. The antibody mentioned in the literature has been categorized according to validation status:

• Tested and working for the specific species and application (covered by product promise)

• Expected to work based on testing (covered by product promise)

• Predicted to work based on homology (not covered by product promise)

• Not recommended (not covered by product promise)

How can CHST6 antibodies be used to study macular corneal dystrophy (MCD)?

CHST6 antibodies represent valuable tools for investigating the molecular mechanisms underlying MCD through several methodological approaches:

What are the known pathogenic mutations in CHST6 and how can antibodies help characterize them?

A comprehensive evaluation of CHST6 variants has identified 181 reported variants in 408 MCD cases . These mutations include:

The majority of these variants (165 out of 181) were classified as pathogenic or likely pathogenic according to ACMG guidelines . Interestingly, the number of patients carrying CHST6 variants at each position was significantly correlated with the conservation score of the corresponding residue (Spearman's correlation coefficient = -0.311, P = 0.000012) .

CHST6 antibodies can help characterize these mutations by:

• Detecting truncated protein products resulting from nonsense mutations like p.Gln182Ter

• Assessing whether missense mutations affect protein stability by comparing expression levels

• Determining if mutations in the sulfotransferase domain (residues 42-356) affect enzymatic activity

What molecular mechanisms underlie CHST6 mutations in MCD pathogenesis?

The pathogenesis of MCD involves several molecular mechanisms that can be studied using CHST6 antibodies:

• Enzymatic activity disruption: CHST6 mutations can impair the enzyme's ability to sulfate keratan, affecting corneal proteoglycan structure . Antibodies can help correlate protein expression with enzymatic function.

• Domain-specific effects: Mutations in different domains may have distinct effects. For example, mutations within the 5'PB domain (like Arg205 or Asp203) may substantially reduce the ability to combine with PAPS, while other conserved mutations may impact enzymatic activity .

• Protein stability and processing: Some mutations may lead to misfolding and premature degradation of CHST6. Antibodies can help detect reduced protein levels or abnormal processing forms .

• Conservation-related impacts: The strong correlation between mutant frequency and residue conservation (Spearman's correlation coefficient = -0.311, P = 0.000012) suggests that mutations in highly conserved residues are more likely to cause disease . Antibodies can help validate the functional importance of these conserved regions.

The key sulfotransferase domain ranges from residue 42 to 356, and mutations within this domain are considered to have moderate evidence (PM2) for pathogenicity .

What are the optimal protocols for using CHST6 antibodies in Western blot applications?

For optimal Western blot results with CHST6 antibodies, researchers should consider the following protocol guidelines:

Sample preparation:

• Extract proteins from tissues or cells using appropriate lysis buffers with protease inhibitors

• For corneal tissues, use mechanical homogenization followed by sonication

• Determine protein concentration using standard assays (BCA or Bradford)

• Prepare samples in Laemmli buffer with reducing agent and heat at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Load 20-40 μg of protein per lane on 10-12% SDS-PAGE gels

• Include molecular weight markers (CHST6 is approximately 44 kDa)

• Transfer to PVDF membranes using standard conditions

Antibody incubation:

• Block membranes with 5% non-fat milk or BSA in TBST

• Incubate with CHST6 primary antibody at manufacturer-recommended dilution (typically 1:500-1:2000) overnight at 4°C

• Wash membranes thoroughly with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Wash thoroughly with TBST

Controls:

• Positive control: Normal corneal tissue lysate

• Negative control: Tissue known not to express CHST6

• Loading control: Housekeeping proteins like GAPDH or β-actin

The CHST6 antibody (ab154332) mentioned in the literature has been validated for Western blot applications with human samples , suggesting these protocols should work effectively with proper optimization.

What controls should be used when working with CHST6 antibodies?

When working with CHST6 antibodies, implementing appropriate controls is crucial for experimental validity:

Positive controls:

• Corneal tissue lysates or sections (known to express CHST6)

• Cell lines with confirmed CHST6 expression

• Recombinant CHST6 protein (for Western blot)

Negative controls:

• Tissues or cell lines not expressing CHST6

• Primary antibody omission (to detect secondary antibody non-specific binding)

• Isotype control antibodies (matched to CHST6 antibody class and concentration)

Specificity controls:

• If possible, samples from MCD patients with known CHST6 mutations that lead to protein absence

• Comparison with mRNA expression data (RT-PCR or RNA-seq)

Technical controls:

• Loading controls for Western blot (β-actin, GAPDH, etc.)

• Staining controls for immunohistochemistry

Since CHST6 is primarily expressed in corneal tissues , corneal epithelial cell lines or corneal tissue sections serve as ideal positive controls for antibody validation experiments.

How can CHST6 antibodies be used to detect different mutant forms of the protein?

CHST6 antibodies can be strategically employed to detect and characterize various mutant forms of the protein:

For truncation mutations:

• Use antibodies targeting N-terminal epitopes to detect truncated proteins resulting from nonsense mutations like p.Gln182Ter

• Compare band sizes on Western blots between wild-type (44 kDa) and truncated forms

• Consider using multiple antibodies targeting different regions to map the truncation site

For missense mutations:

• Assess protein stability by comparing expression levels between wild-type and mutant samples (e.g., p.Arg211Gly)

• Look for altered migration patterns that might indicate changes in post-translational modifications

• Correlate antibody detection with functional assays measuring sulfotransferase activity

Experimental approaches:

• Patient-derived samples: Compare antibody reactivity in tissues or cells from patients with different CHST6 mutations

• Recombinant expression: Express wild-type and mutant CHST6 in cell lines to directly compare protein levels and characteristics

• Subcellular localization studies: Assess if mutations alter normal Golgi localization using immunofluorescence

These approaches can provide insights into how different mutations affect CHST6 protein expression, stability, and potentially function, helping to establish genotype-phenotype correlations in MCD.

How to troubleshoot non-specific binding with CHST6 antibodies?

When encountering non-specific binding with CHST6 antibodies, researchers can implement these troubleshooting strategies:

For Western blot applications:

• Optimize blocking: Use 5% BSA instead of milk, or try commercial blocking reagents

• Adjust antibody concentration: Test a dilution series to find the optimal concentration that maximizes specific signal while minimizing background

• Increase washing stringency: Add more wash steps or increase detergent concentration in wash buffer

• Modify incubation conditions: Shorter primary antibody incubation times or higher temperatures may reduce non-specific binding

For immunohistochemistry/immunofluorescence:

• Optimize fixation: Overfixation can increase background; test different fixation times

• Enhance blocking: Include serum from the secondary antibody host species and/or add detergent to blocking buffer

• Reduce antibody concentration: Non-specific binding often increases with higher antibody concentrations

General strategies:

• Antibody validation: Confirm specificity using positive and negative controls

• Cross-adsorption: Consider using cross-adsorbed secondary antibodies

• Filter samples: Centrifuge lysates at high speed to remove particulates that cause non-specific binding

Since CHST6 is a membrane-associated protein primarily expressed in corneal tissues , special attention should be paid to extraction methods and buffer compositions to ensure specific detection.

What approaches can be used to quantify CHST6 expression levels in different tissues?

Researchers can employ several methodological approaches to accurately quantify CHST6 expression levels:

Protein-based quantification methods:

• Western blot densitometry:

  • Normalize CHST6 band intensity to housekeeping proteins (β-actin, GAPDH)

  • Use digital imaging software for precise quantification

  • Include standard curves if possible for semi-quantitative analysis

• Comparative analysis across tissues:

  • CHST6 is primarily expressed in corneal tissues

  • Lower expression may be found in other tissues requiring optimization of detection methods

  • Consider enrichment approaches for tissues with low expression

Transcript-based quantification methods (complementary):

• RT-qPCR:

  • Design primers spanning exon-exon junctions for specificity

  • Normalize to appropriate reference genes validated for the tissues being compared

  • Correlate with protein expression data when possible

Data analysis considerations:

• Statistical approach: Use appropriate statistical tests when comparing expression between normal and pathological samples

• Biological relevance: Consider that even small changes in CHST6 expression may have significant effects on keratan sulfate production

• Mutation impact: Different mutations may affect protein expression differently; some may reduce levels while others produce normal levels of dysfunctional protein

When studying MCD, it's particularly important to correlate CHST6 expression levels with specific mutations and clinical phenotypes to better understand genotype-phenotype relationships .

How to interpret contradictory results from different CHST6 antibodies?

When facing contradictory results from different CHST6 antibodies, researchers should follow this systematic approach to interpretation and resolution:

Analysis of antibody characteristics:

• Compare epitope locations:

  • Antibodies targeting different domains may yield different results

  • The CHST6 protein has a short cytosolic tail, a transmembrane domain, and a large Golgi luminal C-terminal domain

  • The sulfotransferase domain (residues 42-356) is critical for function

• Review antibody validation data:

  • Check if each antibody has been validated using appropriate controls

  • Assess cross-reactivity profiles with other sulfotransferase family members

  • Determine if validation was performed in relevant tissues/species

Resolution strategies:

• Correlation with orthogonal methods:

  • Compare antibody results with mRNA expression (RT-qPCR)

  • Use tagged recombinant proteins as controls

  • Consider the impact of mutations on epitope recognition

• Consensus approach:

  • Use multiple antibodies targeting different regions of CHST6

  • Weight results based on validation quality

  • Consider the specific research question when interpreting results

In MCD research, it's important to consider that different mutations might affect antibody binding differently, potentially leading to apparently contradictory results that actually reflect biological reality rather than technical issues .

How can CHST6 antibodies help in understanding keratan sulfate biosynthesis pathways?

CHST6 antibodies can provide valuable insights into keratan sulfate biosynthesis through several research approaches:

• Pathway component analysis:

  • CHST6 cooperates with B4GALT4 galactosyltransferase and B3GNT7 N-acetylglucosaminyltransferase in keratan sulfate synthesis

  • Antibodies can help visualize and quantify these pathway components in normal and disease states

• Structure-function studies:

  • The CHST6 protein contains an N-terminal with a short cytosolic tail, a transmembrane domain, and a large Golgi luminal C-terminal domain

  • The C-terminal domain has PAPS binding sites, a catalytic module, and a carbohydrate specificity determination area

  • Antibodies targeting specific domains can help understand their functional roles

• Disease mechanism investigation:

  • In MCD, defective CHST6 leads to impaired sulfation of keratan sulfate

  • Antibodies can help track how different mutations affect protein localization, stability, and interactions

• Corneal development studies:

  • CHST6 is crucial for corneal transparency

  • Antibodies can help monitor CHST6 expression during development and in response to corneal injuries or treatments

The CHST6 protein functions in the formation of sulfated keratan sulfate, which is transported to the extracellular matrix where it forms components of the corneal stroma . Antibodies can help track this process and understand how it is disrupted in diseases like MCD.

What novel applications might emerge for CHST6 antibodies in corneal disease research?

CHST6 antibodies have potential for several innovative applications in corneal disease research:

• Biomarker development:

  • CHST6 protein levels or modifications might serve as biomarkers for early MCD detection

  • Antibodies could be used in diagnostic assays to complement genetic testing

• Therapeutic monitoring:

  • As gene therapies for MCD are developed, antibodies could monitor restoration of CHST6 expression

  • Quantitative assessment of CHST6 levels could correlate with clinical improvement

• Structure-based drug design:

  • Antibodies could help validate binding of small molecules designed to enhance residual CHST6 function in missense mutations

  • Conformational antibodies might reveal druggable pockets in the protein structure

• Personalized medicine approaches:

  • Different mutations have varying effects on CHST6 protein

  • Antibodies could help categorize patients based on protein expression patterns, potentially guiding treatment decisions

The significant correlation between mutant frequency and residue conservation in CHST6 (Spearman's correlation coefficient = -0.311, P = 0.000012) suggests that further antibody-based studies of conserved residues could reveal critical insights into protein function and disease mechanisms.

What methodological improvements would enhance CHST6 antibody research?

Several methodological advancements could significantly enhance CHST6 antibody research:

• Antibody development:

  • Generation of monoclonal antibodies against specific CHST6 domains

  • Development of antibodies that can distinguish between wild-type and common mutant forms

  • Creation of antibodies specific to different conformational states of the protein

• Advanced imaging techniques:

  • Super-resolution microscopy to better visualize CHST6 localization in the Golgi apparatus

  • Live-cell imaging approaches to track CHST6 trafficking and activity

  • Correlative light and electron microscopy to relate CHST6 distribution to ultrastructural features

• Functional assays:

  • Development of activity-based probes that could be used with antibodies to correlate CHST6 localization with enzymatic activity

  • High-throughput screening methods to assess how mutations affect antibody binding and protein function

• Model systems:

  • Patient-derived corneal organoids to study CHST6 expression in a more physiologically relevant context

  • CRISPR-engineered cell lines with specific CHST6 mutations for antibody validation

These methodological improvements would help address current limitations in CHST6 research, particularly the challenges in correlating genotype with phenotype and immunophenotype in MCD patients .