CHST14 Antibody

What is CHST14 and what is its biological significance?

CHST14 (Carbohydrate Sulfotransferase 14), also known as Dermatan 4-sulfotransferase 1 (D4ST1), is an enzyme that catalyzes the transfer of sulfate to position 4 of the N-acetylgalactosamine (GalNAc) residue of dermatan sulfate. It plays a pivotal role in the formation of 4-0-sulfated IdoA blocks in dermatan sulfate. The enzyme transfers sulfate more efficiently to GalNAc residues in -IdoUA-GalNAc-IdoUA- sequences than in -GlcUA-GalNAc-GlcUA-sequences and has a preference for partially desulfated dermatan sulfate .

CHST14 is critical for:

• Formation of dermatan sulfate proteoglycans in extracellular matrix

• Proper collagen fibril assembly

• Cerebellar neural network development during postnatal brain development

• Vascular development and perinatal survival

What are the common applications for CHST14 antibodies in research?

CHST14 antibodies are primarily employed in these methodologies:

When selecting an application, researchers should consider that CHST14 antibodies have been validated with multiple human tissues including placenta, brain, kidney, and spleen .

How should samples be prepared for optimal CHST14 detection in western blotting?

For successful western blot detection of CHST14:

• Sample preparation:

  • Use tissues with known CHST14 expression (e.g., HEK-293 cells, HepG2 cells)

  • Employ standard protein extraction with protease inhibitors

  • Target loading 20-50 μg of total protein per lane

• Technical parameters:

  • Recommended antibody dilution: 1:500-1:1000

  • Expected molecular weight: 45-50 kDa (observed) , 43 kDa (calculated)

  • Use reducing conditions with SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

• Controls:

  • Positive controls: HEK-293 cells, HepG2 cells

  • Negative controls: tissues or cells with CHST14 knockdown

  • Loading control: housekeeping proteins (e.g., β-actin, GAPDH)

• Visualization:

  • Use standard ECL detection or fluorescent secondary antibodies

  • Signal strength varies with expression level; adjust exposure accordingly

What are the critical considerations for immunohistochemical detection of CHST14?

Successful IHC detection of CHST14 requires attention to several parameters:

• Tissue preparation and fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are suitable

  • Optimal section thickness: 4-6 μm

  • Validated human tissues: placenta, brain, kidney, spleen

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: citrate buffer pH 6.0

  • Heat-induced epitope retrieval (pressure cooker or microwave)

• Antibody incubation parameters:

  • Recommended dilution: 1:20-1:200 or 1:500-1:1000 (antibody-dependent)

  • Optimal incubation time: overnight at 4°C or 1-2 hours at room temperature

  • Use a humidity chamber to prevent section drying

• Detection systems:

  • DAB (3,3'-Diaminobenzidine) for brightfield microscopy

  • Fluorescent-conjugated secondary antibodies for fluorescence microscopy

  • Controls should include primary antibody omission and known positive tissues

How do CHST14 mutations affect dermatan sulfate biosynthesis and contribute to mcEDS-CHST14?

CHST14 mutations found in musculocontractural Ehlers-Danlos syndrome (mcEDS-CHST14) significantly impact dermatan sulfate biosynthesis through several mechanisms:

• Molecular pathophysiology:

  • CHST14 mutations lead to loss of dermatan 4-O-sulfotransferase activity

  • This results in impaired dermatan sulfate (DS) biosynthesis

  • DS is essential for proper assembly of collagen fibrils through decorin, a DS proteoglycan

  • Patient fibroblasts show absence of DS and increased chondroitin sulfate production

• Structural consequences:

  • Intracellular retention of collagen types I and III

  • Lack of decorin and thrombospondin fibrils

  • Disorganized collagen fibrils not properly integrated into fibers

  • Dispersed fiber bundles in the ground substance

• Clinical manifestations:

  • Characteristic facial appearance, asthenic build

  • Hyperextensible and bruisable skin

  • Joint instability

  • Recurrent large subcutaneous hematomas

  • Vascular fragility and abnormalities

The relationship between DS loss and collagen disorganization represents the primary pathophysiological mechanism underlying the connective tissue fragility observed in mcEDS-CHST14 patients.

What experimental approaches are most effective for studying CHST14 function in disease models?

Based on published research, these experimental approaches have proven valuable for investigating CHST14 function:

• Gene modification models:

  • CRISPR/Cas9-generated knockout mice using sgRNAs targeting regions downstream of the translation start site

  • Chst14-/- mouse models for studying developmental and vascular phenotypes

  • Patient-derived iPSC-based models for human disease investigation

• Functional characterization methods:

  • Quantification of dermatan sulfate and chondroitin sulfate by disaccharide analysis

  • Electron microscopy for examining collagen fibril structure and organization

  • Analysis of basement membrane structure in vascular tissues

  • Histological examination of affected tissues (placenta, skin, vasculature)

• Experimental challenges:

  • Perinatal lethality in Chst14-/- mice, limiting adult studies

  • Placental analysis serves as a useful alternative model for vascular studies

  • Heterozygous mouse models may not fully recapitulate human disease phenotypes

• Translational approaches:

  • Patient iPSC-derived models allow investigation of human-specific disease phenotypes

  • Examination of specific tissues affected in human disease (skin, joints, vasculature)

  • Focus on vascular phenotypes to understand hematoma formation

What is the emerging role of CHST14 in cancer biology and potential therapeutic implications?

Recent studies have revealed significant associations between CHST14 and cancer:

How can researchers validate the specificity of CHST14 antibodies?

Ensuring antibody specificity is critical for CHST14 research. A comprehensive validation approach should include:

• Genetic validation approaches:

  • CHST14 knockdown using siRNA (e.g., siCHST14 sequence: GCAGGCGACGAUGUCACAUTT, AUGUGACAUCGUCGCCUGCTT)

  • CHST14 knockout cell lines or tissues as negative controls

  • Overexpression systems for positive control validation

• Cross-validation with multiple antibodies:

  • Testing multiple antibodies targeting different epitopes

  • Comparing commercial antibodies from different vendors:

    • Abcam (ab235056) - Recombinant fragment within human CHST14 aa 50-150

    • Proteintech (17749-1-AP) - CHST14 fusion protein Ag12014

    • Sigma-Aldrich (HPA071601) - Immunogen sequence: GILAEMKPLPLHPPGREGTAWRGKAPKPGGLSLRAGDADLQVRQDVRNRTLRAVCGQPGMPRDPWDLPV

• Technical validation controls:

  • Positive tissue controls: human placenta, brain, kidney, spleen

  • Blocking peptide competition assays

  • Western blot confirmation of appropriate molecular weight (45-50 kDa observed)

  • Consistent results across multiple applications (WB, IHC, ICC-IF)

• Orthogonal validation:

  • Correlation with mRNA expression data

  • Mass spectrometry confirmation of identified protein

  • Functional validation through enzyme activity assays

What are the most common technical challenges when working with CHST14 antibodies and how can they be addressed?

Researchers should be aware of these common challenges and their solutions:

• Nonspecific binding issues:

  • Challenge: Background staining in IHC or multiple bands in Western blot

  • Solutions:

    • Optimize blocking conditions (5% BSA or milk)

    • Increase wash steps duration and number

    • Titrate primary antibody concentration

    • Use validated antibody dilutions (WB: 1:500-1:1000; IHC: 1:20-1:200)

• Epitope accessibility problems:

  • Challenge: Poor or inconsistent signal in FFPE tissues

  • Solutions:

    • Test multiple antigen retrieval methods

    • Primary recommendation: TE buffer pH 9.0

    • Alternative: citrate buffer pH 6.0

    • Extend antigen retrieval time for challenging tissues

• Variability across applications:

  • Challenge: Antibody may perform well in WB but poorly in IHC or vice versa

  • Solutions:

    • Review validation data for specific applications

    • Consider application-specific antibodies

    • Optimize protocols for each application independently

• Tissue-specific expression variations:

  • Challenge: Variable CHST14 expression across tissues

  • Solutions:

    • Use appropriate positive controls (e.g., HEK-293 cells, HepG2 cells for WB)

    • Adjust exposure/development time based on expected expression levels

    • Consider tissue-specific protocol modifications

How do CHST14 knockout models contribute to our understanding of vascular abnormalities in mcEDS-CHST14?

CHST14 knockout models have provided critical insights into vascular pathophysiology:

• Placental vascular phenotypes in Chst14-/- mice:

  • Reduced placental weight

  • Alterations in vascular structure

  • Ischemic and/or necrotic-like changes

  • Abnormal basement membrane structure in placental villus capillaries

• Translational significance:

  • Placental models serve as proxies for studying vascular manifestations in mcEDS-CHST14

  • Findings help explain mechanisms behind large subcutaneous hematomas in patients

  • Demonstrate essential role of CHST14 in vascular development and integrity

• Limitations and considerations:

  • Perinatal lethality limits studies in adult Chst14-/- mice

  • Heterozygous mice show limited phenotypes

  • Compensatory mechanisms may differ between mouse and human models

• Future directions:

  • Tissue-specific conditional knockout models

  • Human iPSC-derived vascular models

  • Targeted examination of specific vascular beds affected in patients

What methods are used to evaluate CHST14 enzyme activity and its impact on dermatan sulfate production?

Assessing CHST14 enzymatic function involves several complementary approaches:

• Direct enzyme activity assays:

  • In vitro sulfotransferase assays measuring transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate to GalNAc residues

  • Quantification using radioactive or fluorescent labeling

  • Comparing activity between wild-type and mutant CHST14 proteins

• Dermatan sulfate characterization:

  • Disaccharide composition analysis

  • High-performance liquid chromatography

  • Mass spectrometry of glycosaminoglycans

  • Comparing DS/CS ratios in patient vs. control samples

• Functional consequences assessment:

  • Collagen fibril structure by electron microscopy

  • Decorin binding assays

  • Proteoglycan isolation and characterization

  • Mechanical testing of extracellular matrix properties

• Cell-based functional assays:

  • Patient-derived fibroblast studies

  • iPSC-based differentiation models

  • Rescue experiments with wild-type CHST14

  • Knockdown studies using siRNA (siCHST14)

How can CHST14 autoantibodies serve as biomarkers in osteoarthritis and other conditions?

The presence of CHST14 autoantibodies has emergent value in disease biomarker development:

• Autoantibody detection methods:

  • Protein array technologies:

    • Antigen microarrays

    • Nucleic acid programmable protein arrays (NAPPA)

  • ELISA validation of array findings

  • Bead-based arrays for high-throughput screening

• Disease associations:

  • Validated as autoantibody target in osteoarthritis (OA)

  • Differential autoantibody profiles between OA, rheumatoid arthritis (RA), and healthy controls

  • Potential for early diagnostic applications

• Technical considerations:

  • Need for standardized detection methods

  • Importance of appropriate control populations

  • Cut-off determination for clinical significance

  • Correlation with disease severity and progression

• Future applications:

  • Potential for monitoring disease progression

  • Evaluation of therapeutic responses

  • Integration into multi-biomarker panels

  • Stratification of patient populations for clinical trials