HGSNAT Antibody

What is HGSNAT and what is its biological function?

HGSNAT (heparan-alpha-glucosaminide N-acetyltransferase) is a lysosomal membrane enzyme that catalyzes a critical transmembrane acetylation reaction in the degradation pathway of heparan sulfate. Specifically, it transfers an acetyl group from acetyl-CoA to the terminal glucosamine residue of heparan sulfate, forming N-acetylglucosamine . This acetylation step is essential for the proper degradation of heparan sulfate in lysosomes. HGSNAT has a calculated molecular weight of approximately 22 kDa based on its 206 amino acid sequence, though the observed molecular weight in experimental conditions is typically around 70 kDa, likely due to post-translational modifications .

What applications can HGSNAT antibodies be used for in research?

HGSNAT antibodies, such as the 12399-1-AP polyclonal antibody, can be utilized in multiple experimental applications:

Each application requires specific optimization for optimal results, and the antibody should be titrated in each testing system to achieve the best signal-to-noise ratio .

What species reactivity can be expected from HGSNAT antibodies?

The commercially available HGSNAT antibodies, specifically the 12399-1-AP antibody, have been validated to show reactivity with human and mouse samples . This cross-species reactivity makes these antibodies valuable for comparative studies between human disease models and mouse models. Researchers should note that while these antibodies have been confirmed for these species, proper validation should be conducted when using them with tissues or cells from other organisms.

What are the optimal protocols for using HGSNAT antibody in Western blot applications?

For Western blot applications using HGSNAT antibody, researchers should follow these methodological considerations:

• Sample preparation: Use HepG2 cells, mouse liver tissue, A549 cells, or HeLa cells as positive controls

• Dilution range: Use the antibody at 1:500-1:3000 dilution

• Incubation conditions: Room temperature for 1.5 hours has been validated

• Expected band size: Approximately 70 kDa, though the calculated molecular weight is 22 kDa

• Buffer conditions: Standard PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

The observed molecular weight (70 kDa) differs significantly from the calculated weight (22 kDa), which is an important consideration when analyzing results . This discrepancy may be due to post-translational modifications, multimerization, or the membrane-associated nature of the protein.

What considerations are important for immunohistochemistry with HGSNAT antibody?

When performing immunohistochemistry with HGSNAT antibody, researchers should consider:

• Antigen retrieval: Suggested methods include TE buffer at pH 9.0 or alternatively, citrate buffer at pH 6.0

• Dilution: Use a range of 1:50-1:500, optimizing specifically for your tissue type

• Positive control: Human lung cancer tissue has shown reliable results

• Specificity verification: Include proper negative controls to ensure signal specificity

• Visualization system: Compatible with standard detection systems based on the selected secondary antibody

The antigen retrieval step is particularly critical for exposing the HGSNAT epitopes, which may be masked in formalin-fixed, paraffin-embedded tissues .

How should HGSNAT antibody be stored and handled to maintain its activity?

For optimal performance, HGSNAT antibodies should be stored and handled as follows:

• Storage temperature: -20°C for long-term storage

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability: Typically stable for one year after shipment under recommended storage conditions

• Aliquoting: Not necessary for -20°C storage with the glycerol-containing buffer

• Working solution preparation: Thaw completely before use and mix gently to ensure homogeneity

Note that some preparations (particularly 20μl sizes) may contain 0.1% BSA as a stabilizer . Repeated freeze-thaw cycles should be avoided even though the glycerol in the storage buffer provides some protection.

What is known about the structural characteristics of HGSNAT protein?

Recent structural studies have provided significant insights into HGSNAT architecture:

The structure of human HGSNAT in the acetyl-CoA bound state has been determined by high-resolution single-particle cryo-electron microscopy. This landmark study revealed the first detailed architecture of this family of integral membrane enzymes . The structure shows:

• HGSNAT functions as an integral membrane enzyme with a specific binding pocket for acetyl-CoA

• The high-resolution and isotropic single-particle cryoEM map provides clear density for the acetyl-CoA ligand

• The atomic model derived from this density map is well-justified by the experimental data

• The structure reveals the mode of acetyl-CoA binding, which is crucial for understanding the enzyme's mechanism

This structural information provides a foundation for understanding how disease-causing mutations might disrupt protein function, though experimental validation of the impact of specific mutations remains incomplete .

How can researchers effectively assay HGSNAT enzymatic activity?

Researchers have developed several methods to assay HGSNAT enzymatic activity, with recent innovations improving specificity and throughput:

• Traditional method: Using radiolabeled oligosaccharides derived from heparan sulfate, which is complex and requires specialized handling of radioisotopes

• Simplified radiometric method: Using commercially available [14C]-labeled glucosamine, which still requires radioactive materials

• Novel direct fluorometric assay: Utilizing glucosamine labeled at position C1 with BODIPY as a substrate

The novel BODIPY-glucosamine based assay offers several advantages:

• It's a direct method without the need for radioactive materials

• It relies on the conversion of positively charged substrate into neutral product (BODIPY-N-acetyl-glucosamine)

• The products can be separated using cation exchange chromatography

• Quantification uses standard fluorometer plate readers

• The assay has demonstrated specificity for HGSNAT activity, as confirmed with MPS IIIC patient fibroblasts

• Competitive inhibition profiles are similar to traditional substrates

• It's potentially suitable for high-throughput screening applications

How does HGSNAT catalytic activity relate to its membrane localization?

HGSNAT's function is intricately linked to its localization in the lysosomal membrane:

• Transmembrane acetylation: HGSNAT catalyzes a unique transmembrane acetylation reaction where the substrate (terminal glucosamine residue of heparan sulfate) is located in the lysosomal lumen while the acetyl donor (acetyl-CoA) is in the cytoplasm

• Structural adaptation: The enzyme's structure accommodates this transmembrane catalysis, with specific domains for binding acetyl-CoA on the cytoplasmic side and substrate interaction regions facing the lysosomal lumen

• Biological significance: This arrangement allows for degradation of heparan sulfate within lysosomes while utilizing acetyl-CoA from the cytoplasmic pool

• Disruption consequences: Mutations that affect membrane insertion, topology, or stability can disrupt enzymatic function without necessarily affecting the catalytic domain directly

The membrane localization is crucial for understanding disease mechanisms, as many pathogenic mutations may affect trafficking to lysosomes or proper membrane insertion rather than directly impacting catalytic activity .

How is HGSNAT deficiency associated with Mucopolysaccharidosis IIIC (Sanfilippo C Syndrome)?

Mucopolysaccharidosis IIIC (MPS IIIC) is an autosomal recessive lysosomal storage disorder directly caused by HGSNAT deficiency:

• Pathophysiology: Deficiency in HGSNAT activity leads to accumulation of partially degraded heparan sulfate in lysosomes

• Clinical manifestations: The disease is characterized by:

  • Deterioration of the central nervous system

  • Coarse facial features

  • Developmental delay

  • Macrocrania (abnormally large head)

  • Motor retardation

• Molecular basis: Over 54 variants of the HGSNAT gene have been identified in MPS IIIC patients, including 22 missense mutations

• Biochemical diagnosis: Patients with MPS IIIC show profound deficiency of HGSNAT activity compared to normal controls and patients with other MPS subtypes (such as MPS IIIA and MPS IIID)

The disease primarily affects the central nervous system due to the importance of proper heparan sulfate degradation in neuronal function and development.

What functional impacts do HGSNAT gene mutations have on protein expression and activity?

Functional analysis of HGSNAT mutations has revealed diverse effects on protein expression and enzymatic activity:

In a comprehensive study, 20 of the 22 known missense mutations were introduced into the cDNA of HGSNAT and exogenously expressed in cell culture. The results showed:

• Severe expression defects: 16 of the 20 mutations resulted in negligible HGSNAT protein production and activity

• Minimal impact: 4 mutations produced protein levels and function similar to wild-type HGSNAT

• Synergistic effects: Some mutations, such as c.1209G>T (p.W403C) and c.1843G>A (p.A615T), may work together to abolish HGSNAT activity, even though p.A615T alone has negligible effect on expression

These findings indicate that most disease-causing missense mutations lead to HGSNAT deficiency through protein misfolding, instability, or trafficking defects rather than direct catalytic impairment .

How can researchers interpret the discrepancy between calculated and observed molecular weights of HGSNAT?

The significant difference between the calculated molecular weight (22 kDa) and observed molecular weight (70 kDa) of HGSNAT is an important research consideration:

• Potential explanations:

  • Post-translational modifications such as glycosylation

  • Membrane protein properties affecting migration in SDS-PAGE

  • Protein complex formation or oligomerization

  • Potential cleavage of a precursor protein

• Experimental verification approaches:

  • Deglycosylation experiments to assess contribution of glycans

  • Size exclusion chromatography to determine native molecular weight

  • Mass spectrometry analysis for precise molecular weight determination

  • Domain-specific antibodies to identify processing events

• Research implications:

  • Studies of protein trafficking should consider post-translational processing

  • Mutation analysis should account for effects on processing

  • Structural predictions should incorporate these modifications

Researchers working with HGSNAT should be aware of this discrepancy when designing experiments and interpreting results, particularly in Western blot applications .

What are emerging therapeutic approaches targeting HGSNAT for MPS IIIC treatment?

Based on molecular understanding of HGSNAT, several therapeutic approaches are being investigated:

• Pharmacological chaperones: Compounds that can stabilize mutant HGSNAT protein, potentially rescuing function in patients with missense mutations that affect protein folding rather than catalytic activity

• High-throughput screening: The development of the novel BODIPY-glucosamine assay facilitates screening for HGSNAT inhibitors, which paradoxically can function as pharmacological chaperones at sub-inhibitory concentrations

• Enzyme replacement therapy: Although challenging due to the transmembrane nature of HGSNAT, approaches to deliver functional enzyme to lysosomes are being explored

• Gene therapy: Delivery of functional HGSNAT gene to affected cells, particularly in the central nervous system

The search for more potent pharmacological chaperones has been identified as a promising direction that could lead to development of therapeutic options for MPS IIIC .

How can structural insights into HGSNAT inform research on disease-causing mutations?

The recently determined structure of human HGSNAT in the acetyl-CoA bound state provides critical information for understanding disease mechanisms:

• Structure-function correlations: The structure reveals how acetyl-CoA binds to the enzyme, allowing researchers to predict how mutations near this binding site might affect enzyme function

• Mutation mapping: Disease-causing mutations can be mapped onto the structure to determine if they affect:

  • Substrate binding regions

  • Catalytic residues

  • Protein folding and stability

  • Membrane insertion

  • Protein-protein interaction interfaces

• Rational drug design: The structure enables structure-based design of pharmacological chaperones that could stabilize specific mutant forms of HGSNAT

• Mechanistic understanding: The structure provides insights into the unique transmembrane acetylation mechanism

While the structural work is highly convincing, with a high-resolution cryo-EM map and well-justified atomic model, experimental validation of the molecular mechanisms and mutation impacts remains to be completed .

What methodological approaches can distinguish between different MPS disorders in research and diagnostic settings?

Differentiating between MPS subtypes, particularly MPS III variants (A-D), requires specific enzymatic and molecular approaches:

• Enzymatic activity profiling:

  • MPS IIIC (HGSNAT deficiency) shows specifically reduced N-acetyltransferase activity

  • MPS IIIA and D patients have normal HGSNAT activity

  • The novel BODIPY-glucosamine assay provides a specific method for HGSNAT activity determination

• Biochemical signatures:

  • Analysis of accumulated heparan sulfate fragments can show specific patterns

  • Different MPS subtypes may have distinct biomarkers in body fluids

• Genetic testing:

  • Sequencing of specific genes (SGSH for MPS IIIA, NAGLU for MPS IIIB, HGSNAT for MPS IIIC, GNS for MPS IIID)

  • Next-generation sequencing panels covering all MPS-related genes

• Confirmatory testing workflow:

  • Initial screening with the appropriate enzymatic assay

  • Confirmation with genetic testing

  • Functional studies for novel or variants of uncertain significance

The specificity of the novel direct HGSNAT assay using BODIPY-glucosamine has been validated using cultured fibroblasts from MPS IIIC patients, which showed profound deficiency compared to normal controls and patients with other MPS subtypes .