Phospho-GYS1 (S641) Recombinant Monoclonal Antibody

What is GYS1 and why is its phosphorylation at S641 physiologically significant?

GYS1 is the rate-limiting enzyme in muscle glycogen synthesis, playing a central role in energy homeostasis . This isoform catalyzes the conversion of glucose to glycogen and is primarily expressed in muscle tissue . The phosphorylation status of GYS1 at specific residues, particularly S641, critically regulates its enzymatic activity.

GYS1 activity is regulated by two central mechanisms:

• Allosteric activation by glucose-6-phosphate (G6P)

• Inhibition by phosphorylation at multiple residues

Phosphorylation at S641 specifically leads to enzyme inhibition, serving as a key regulatory switch for glycogen synthesis in response to energy status . Under conditions requiring energy conservation, kinases including glycogen synthase kinase 3, AMPK, PKA, and casein kinase 2 phosphorylate GYS1 at S641, inhibiting glycogen synthesis .

What experimental applications can Phospho-GYS1 (S641) antibodies be used for?

Phospho-GYS1 (S641) antibodies have been validated for multiple research applications:

The antibody specifically recognizes human GYS1 when phosphorylated at S641, with most recombinant versions showing cross-reactivity with mouse and rat samples as well .

How does recombinant monoclonal Phospho-GYS1 (S641) antibody differ from polyclonal versions?

Recombinant monoclonal antibodies offer several advantages over polyclonal antibodies:

For experiments requiring high specificity and reproducibility, recombinant monoclonal antibodies like clone 1D1 are preferred due to their consistent performance across experimental replicates .

What are the optimal protocols for using Phospho-GYS1 (S641) antibodies in Western blotting?

For optimal Western blot results with Phospho-GYS1 (S641) antibodies:

• Sample preparation:

  • Harvest cells in ice-cold lysis buffer containing phosphatase inhibitors

  • Include sodium fluoride and sodium orthovanadate to preserve phosphorylation status

  • Use fresh tissue samples or flash-freeze immediately after collection

• Gel electrophoresis conditions:

  • Load 20-50 μg total protein per lane

  • Use 10% SDS-PAGE gels for optimal resolution around 42-85 kDa range

• Transfer and detection:

  • Transfer to PVDF membrane (preferred over nitrocellulose for phosphoproteins)

  • Block with 5% BSA (not milk) in TBS-T to prevent phosphatase activity

  • Incubate with primary antibody at 1:1000 dilution overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody and ECL detection system

• Controls and validation:

  • Include phosphatase-treated samples as negative controls

  • Use samples from cells treated with insulin (decreases phosphorylation) or adrenaline (increases phosphorylation)

  • Consider using tissues from GYS1 knockout models as specificity controls

The phosphorylated form of GYS1 typically appears as a band at approximately 42 kDa, though the calculated molecular weight of the full protein is around 84 kDa .

How can researchers verify the specificity of Phospho-GYS1 (S641) antibody binding?

Validating antibody specificity is critical for meaningful experimental results:

• Peptide competition assay:

  • Pre-incubate antibody with excess phosphorylated and non-phosphorylated peptides

  • Specific binding should be blocked by phospho-peptide but not by non-phospho-peptide

• Phosphatase treatment:

  • Treat half of your sample with lambda phosphatase before Western blotting

  • Signal should disappear in phosphatase-treated samples

• Genetic validation:

  • Use cells expressing GYS1 with S641A mutation (prevents phosphorylation)

  • No signal should be detected in mutant samples

• Correlation with total GYS1:

  • Always run parallel blots for phospho-GYS1 and total GYS1

  • Changes in phosphorylation status should be normalized to total protein

• Physiological validation:

  • Known stimuli that increase GYS1 phosphorylation include AMPK activation and PKA stimulation

  • Insulin treatment should decrease S641 phosphorylation through activation of protein phosphatase 1

What cell types or tissues express significant levels of phosphorylated GYS1?

GYS1 phosphorylation at S641 shows tissue-specific and condition-dependent patterns:

The phosphorylation status of GYS1 is highly dynamic and responds to nutritional status, hormonal signaling, and energy demands . For experimental design, consider that fresh tissue samples will more accurately reflect physiological phosphorylation states than cultured cell lines.

How can Phospho-GYS1 (S641) antibodies be used to study glycogen storage disorders?

Phospho-GYS1 (S641) antibodies are valuable tools for investigating glycogen storage disorders like Pompe disease:

• Differential phosphorylation analysis:

  • Studies show altered GYS1 phosphorylation patterns in Pompe disease models

  • Research reveals a 3-4 fold increase in inhibitory phosphorylation at S641 in Pompe disease fibroblasts

• Therapeutic intervention assessment:

  • Monitor changes in GYS1 phosphorylation in response to substrate reduction therapy

  • Compare effectiveness of small molecule inhibitors like MZ-101 versus phosphorylation-based regulation

• Mechanism elucidation:

  • Use phospho-specific antibodies to track the feed-forward loop of aberrant glycogen metabolism

  • Analyze how treatment with enzyme replacement therapy (ERT) affects GYS1 phosphorylation

• Cellular pathology correlation:

  • Correlate phospho-GYS1 levels with glycogen accumulation in lysosomes

  • Use co-staining with lysosomal markers to assess localization patterns

Data from mouse models of Pompe disease show that total glycogen stores are 2-3 fold higher than wild-type, with corresponding alterations in the phosphorylation status of GYS1 .

What is the interplay between glucose-6-phosphate (G6P) allosteric activation and S641 phosphorylation in regulating GYS1?

The dual regulation of GYS1 through phosphorylation and allosteric activation represents a sophisticated control mechanism:

• Opposing regulatory forces:

  • G6P allosterically activates GYS1 even when phosphorylated

  • S641 phosphorylation inhibits GYS1 activity but does not prevent G6P binding

• Concentration-dependent effects:

  • Increasing G6P from 0.15mM to 5mM shifts the IC50 of inhibitors by approximately 2-fold

  • The allosteric activation constant (Ka) for G6P is approximately 0.311 mM

• Structural insights:

  • Phosphorylation at S641 and other sites stabilizes an inactive conformation

  • G6P binding induces conformational changes that can partially overcome inhibition by phosphorylation

  • Recent structural studies have defined distinct activated and inactivated states

• Experimental implications:

  • When designing experiments with GYS1 inhibitors, consider varying G6P concentrations

  • The inhibitory potency of compounds like MZ-101 shows a non-competitive mechanism with G6P binding

This complex regulation allows for fine-tuning of glycogen synthesis based on both immediate cellular energy status (G6P levels) and longer-term regulatory signals (phosphorylation status).

How do experimental conditions affect the detection of GYS1 phosphorylation at S641?

The reliable detection of phosphorylated GYS1 requires careful attention to experimental conditions:

• Sample handling critical factors:

  • Rapid sample processing prevents phosphatase activity

  • Flash-freezing tissues immediately preserves physiological phosphorylation status

  • Phosphatase inhibitor cocktails must include both serine/threonine and tyrosine phosphatase inhibitors

• Culture conditions affecting phosphorylation:

  • High glucose medium increases GYS1 activity and may decrease phosphorylation

  • Serum starvation typically increases phosphorylation at S641

  • Cell density and confluence can affect phosphorylation patterns

• Tissue-specific considerations:

  • Muscle tissue shows rapid dephosphorylation post-mortem

  • Liver expresses predominantly GYS2 (another isoform) with different regulation

  • Antibody cross-reactivity between GYS1 and GYS2 must be considered in hepatic samples

• Recombinant protein studies:

  • Recombinant GYS1 expressed in insect cells shows substantially heavier phosphorylation than would be observed in intact tissues

  • The activity ratio (measured in the absence vs. presence of G6P) serves as an indicator of phosphorylation status

For quantitative studies, researchers should establish baseline phosphorylation levels in their specific experimental system and include appropriate controls to account for technical variations in phosphoprotein detection.

What therapeutic approaches targeting GYS1 are being developed for glycogen storage disorders?

Research into GYS1-targeted therapeutics is advancing on multiple fronts:

• Small molecule GYS1 inhibitors:

  • MZ-101 represents a breakthrough as a potent (IC50 0.041 μM) and selective inhibitor of GYS1 that doesn't affect GYS2

  • This compound maintains inhibitory activity across various G6P concentrations and phosphorylation states

  • In mouse models of Pompe disease, MZ-101 treatment reduced glycogen accumulation to levels comparable to enzyme replacement therapy (ERT)

• Substrate reduction therapy concepts:

  • Inhibiting GYS1 prevents further glycogen accumulation in lysosomes

  • This approach may complement existing ERT therapies

  • Combined treatment shows additive effects and could normalize muscle glycogen concentrations

• RNA interference approaches:

  • siRNA and phosphorodiamidate morpholino oligomers targeting GYS1 have shown efficacy in reducing glycogen accumulation

  • These genetic approaches face delivery challenges to affected tissues

• Experimental considerations:

  • Monitoring phospho-GYS1 (S641) levels during treatment helps assess target engagement

  • Balancing glycogen reduction against physiological needs remains challenging

  • Complete loss of muscle glycogen through GYS1 inhibition may lead to cardiac abnormalities

Preliminary data from mouse studies suggest that partial inhibition of GYS1 is well-tolerated and offers therapeutic benefits in glycogen storage disorders .

How can phospho-specific antibodies be used to evaluate small molecule inhibitors of GYS1?

Phospho-GYS1 (S641) antibodies provide valuable tools for characterizing novel GYS1 inhibitors:

• Target engagement verification:

  • Monitor changes in S641 phosphorylation as a direct readout of inhibitor binding

  • Compare Western blot results using both phospho-specific and total GYS1 antibodies

• Mechanism of action elucidation:

  • Determine if inhibitors like MZ-101 affect phosphorylation status or act through other mechanisms

  • Data suggests MZ-101 uses a non-competitive inhibition mechanism with respect to both G6P binding and UDP-glucose

• Cell-based assay development:

  • Establish high-throughput phospho-GYS1 detection assays for drug screening

  • Normalize phospho-signal to total GYS1 levels for accurate comparisons

• In vivo efficacy assessment:

  • Use tissue samples from treated animals to correlate inhibitor concentration with phospho-GYS1 levels

  • Relate phosphorylation changes to functional outcomes like glycogen accumulation

The implementation of these approaches has led to the identification of MZ-101 as a promising therapeutic candidate with an EC50 of approximately 500nM for reducing glycogen in fibroblasts .

What is the relationship between GYS1 phosphorylation and its interaction with glycogenin-1?

The functional relationship between GYS1 and glycogenin-1 (GN1) represents an important area of investigation:

• Complex formation:

  • GYS1 forms a stoichiometric complex with GN1 in vivo

  • This complex has been successfully expressed and purified using bicistronic vectors in insect cells

• Phosphorylation effects on complex formation:

  • Phosphorylation status may influence GYS1:GN1 interaction stability

  • Structural studies have revealed both phosphorylation-dependent and independent states

• Experimental approaches:

  • Co-immunoprecipitation with phospho-GYS1 (S641) antibodies can assess how phosphorylation affects complex formation

  • Size-exclusion chromatography combined with Western blotting helps determine complex stoichiometry

  • Recent high-resolution structural studies have elucidated activated and inactivated states of the complex

• Therapeutic implications:

  • Inhibitors targeting the GYS1:GN1 complex may offer alternative approaches to modulating glycogen synthesis

  • Understanding how phosphorylation affects complex formation could reveal new drug targets

Recent research suggests that the GYS1:GN1 complex undergoes structural transitions regulated both by G6P binding and phosphorylation status , creating opportunities for therapeutic intervention beyond direct enzyme inhibition.

What are the storage and handling recommendations for optimizing Phospho-GYS1 (S641) antibody performance?

Proper storage and handling are essential for maintaining antibody functionality:

To maximize antibody performance:

• Avoid repeated freeze-thaw cycles by creating single-use aliquots

• Centrifuge briefly after thawing to collect all liquid

• Store working dilutions at 4°C for no more than one week

• Include proper controls in each experiment to verify antibody performance

These precautions help ensure reliable and reproducible results when working with phospho-specific antibodies, which can be particularly sensitive to storage conditions.

What methodological approaches can resolve contradictory phospho-GYS1 (S641) results between different experimental techniques?

When facing discrepancies in phospho-GYS1 (S641) detection between methods:

• Western blot vs. immunocytochemistry discrepancies:

  • Consider epitope accessibility differences in fixed vs. denatured samples

  • Optimize fixation methods (paraformaldehyde concentration and duration)

  • Validate with phosphatase treatment controls in both techniques

• ELISA vs. Western blot inconsistencies:

  • Evaluate differences in sample preparation that might affect phosphoepitope presentation

  • Consider matrix effects in ELISA buffers that might interfere with antibody binding

  • Compare direct coating vs. sandwich ELISA approaches

• Cross-technique validation strategy:

  • Implement at least two independent methods to confirm phosphorylation status

  • Consider mass spectrometry as a definitive method for phosphorylation site verification

  • Correlate results with functional assays of GYS1 activity

• Common sources of error:

  • Rapid phosphorylation changes during sample processing

  • Cross-reactivity with other phosphorylated proteins

  • Interference from high glycogen content in samples

  • Differences in antibody lot performance