Recombinant Mouse Neutral alpha-glucosidase C (Ganc), partial

What is Neutral alpha-glucosidase C (Ganc) and how does it differ from other glucosidases?

Neutral alpha-glucosidase C (Ganc) is a member of the glycosyl hydrolase gene family 31, which has activity at neutral pH, distinguishing it from acid alpha-glucosidases that function optimally at acidic pH. Ganc is involved in glycogen metabolism and has the ability to degrade glycogen. It differs from other alpha-glucosidases like lysosomal acid alpha-glucosidase (GAA) primarily in its pH optimum and subcellular localization. While GAA functions within lysosomes at acidic pH, Ganc operates at neutral pH in different cellular compartments. Ganc shows highest homology to the catalytic unit of glucosidase II among the alpha-glucosidase family members . The enzyme exhibits characteristic electrophoretic mobility relative to the lysosomal enzyme acid alpha-glucosidase and to glucosidase II, which can be used for identification purposes in experimental settings .

What is the molecular structure and catalytic mechanism of mouse Ganc?

Mouse Ganc, similar to human GANC, has a molecular mass of approximately 104 kDa, consistent with a protein of around 914 amino acids . The enzyme contains the highly conserved catalytic site with the WXDMNE motif that is characteristic of family 31 glycosyl hydrolases . This catalytic site is critical for the hydrolysis of glycosidic bonds. Structurally, Ganc likely contains domains similar to other family 31 glycosyl hydrolases, which typically include an N-terminal trefoil-P domain, a beta-sheet domain, a catalytic barrel, and C-terminal beta-sheet domains, as observed in the related enzyme GAA . The hydrolysis mechanism involves cleavage of both alpha-1,4- and alpha-1,6-glucosidic linkages, allowing the enzyme to process complex glycogen structures.

What are the recommended methods for assessing Ganc enzymatic activity?

For assessing Ganc enzymatic activity, researchers should adapt protocols similar to those used for other alpha-glucosidases, with particular attention to maintaining neutral pH conditions. A recommended approach is to use starch as a substrate, with the following protocol adapted from GAA assay methods :

• Prepare assay buffer at neutral pH (approximately pH 7.0-7.4)

• Dilute purified recombinant Ganc to 30-35 μg/mL in assay buffer

• Prepare substrate by diluting 2% starch to 1.5% in assay buffer

• Combine 20 μL of diluted Ganc with 380 μL of 1.5% starch

• Include appropriate controls (buffer only with substrate)

• Incubate reactions at 37°C for 60 minutes

• Add 400 μL of stop solution (such as dinitrosalicylic acid-based reagent)

• Heat at 95-100°C for 6 minutes, then cool on ice

• Measure released glucose using colorimetric detection at approximately 540-550 nm

• Calculate specific activity using the formula:

This method allows quantitative assessment of Ganc activity while accounting for background activity and ensuring specificity .

What expression systems are most suitable for producing recombinant mouse Ganc?

Based on successful expression systems for related glycosidases, mammalian expression systems are most suitable for producing enzymatically active recombinant mouse Ganc. For optimal expression:

• Utilize expression vectors such as pCDNA3 with a strong promoter (CMV) for mammalian cell expression

• Consider expressing in multiple cell lines for comparison:

  • Mouse 3T3 cells for homologous expression

  • COS cells for high protein yield

  • Human cell lines lacking endogenous Ganc activity for functional studies

Successful expression was demonstrated for human GANC using CaPO₄-based transfection methods in multiple cell lines, including murine 3T3 cells and monkey kidney COS cells . For purification, include an affinity tag (such as 6-His) at the N-terminus of the construct, which allows for efficient purification while maintaining enzymatic activity . Complete sequencing of the expression construct is essential to confirm absence of cloning artifacts that could affect activity .

How can I differentiate between Ganc and other glucosidases in experimental samples?

Differentiating between Ganc and other glucosidases in experimental samples requires a multi-parameter approach:

• Electrophoretic mobility analysis: Starch gel electrophoresis at 4°C can separate Ganc from other glucosidases based on charge differences, which is particularly useful for distinguishing between allozymes .

• pH optima profiling: Conduct activity assays across a pH range from 3.5 to 8.0. Ganc will show maximal activity at neutral pH (around 7.0), while lysosomal acid alpha-glucosidase exhibits peak activity at pH 4.0-4.5 .

• Substrate specificity testing: Compare hydrolysis rates of different substrates:

  • 4-methylumbelliferyl-α-D-glucoside for visualization of activity

  • Natural substrates like glycogen and starch

  • Specific oligosaccharides that may be preferentially cleaved by Ganc

• Inhibitor sensitivity analysis: Test differential sensitivity to inhibitors:

  • Acarbose (α-glucosidase inhibitor)

  • Specific antibodies against Ganc

• Molecular weight verification: Use Western blotting with specific antibodies to confirm the expected molecular weight of approximately 104 kDa for Ganc, compared to other glucosidases .

This comprehensive approach ensures reliable differentiation between Ganc and other related enzymes in complex biological samples.

How can genetic variations in mouse Ganc be analyzed and their functional impact assessed?

Analyzing genetic variations in mouse Ganc and assessing their functional impact requires a systematic approach combining molecular genetics and enzymatic characterization:

• Identification of genetic variants:

  • PCR amplification and sequencing of Ganc cDNA from different mouse strains

  • Analysis of genomic databases for known polymorphisms

  • RT-PCR from tissues of interest to identify potential splice variants

• Functional characterization of variants:

  • Clone identified variants into expression vectors (e.g., pCDNA3)

  • Express in appropriate cell lines using standard transfection methods

  • Compare enzyme activity using the starch hydrolysis assay

  • Analyze electrophoretic mobility patterns to distinguish allozymes

• Biochemical characterization of variant enzymes:

  • Determine pH optima profiles

  • Assess substrate preferences

  • Measure kinetic parameters (Km, Vmax)

  • Evaluate thermal stability and storage properties

• Structural analysis:

  • Generate structural models based on homology to related enzymes

  • Map variants to functional domains to predict impact

  • Focus on variations near the catalytic WXDMNE motif

Human GANC exhibits a biochemical genetic polymorphism with four alleles, including a null allele . Mouse Ganc likely shows similar diversity, making it an excellent model for studying enzyme polymorphisms and their physiological implications.

What are the optimal conditions for storing and maintaining recombinant mouse Ganc activity?

Maintaining recombinant mouse Ganc activity during storage requires careful attention to buffer conditions and handling procedures:

• Short-term storage (1-2 weeks):

  • Store as filtered solution in Tris buffer with NaCl at 2-8°C

  • Include stabilizing agents such as glycerol (10-20%)

  • Consider adding protease inhibitors to prevent degradation

• Long-term storage:

  • Use a manual defrost freezer at -20°C or preferably -80°C

  • Avoid repeated freeze-thaw cycles which significantly reduce activity

  • Aliquot into single-use volumes to minimize freeze-thaw damage

• Buffer composition optimization:

  • For maximum stability, consider buffer systems containing:

    • 20-50 mM Tris-HCl, pH 7.2-7.5

    • 100-150 mM NaCl

    • Optional: 1-5 mM DTT to maintain reduced state of cysteines

    • Optional: 10% glycerol as cryoprotectant

• Activity preservation during experiments:

  • Maintain samples on ice when in use

  • Return to appropriate storage conditions promptly after use

  • Consider carrier-free formulations for applications where carrier proteins might interfere

These guidelines are adapted from established protocols for related enzymes and should maintain maximal activity of recombinant mouse Ganc during experimental timeframes .

How does mouse Ganc interact with other components of glycogen metabolism?

Mouse Ganc functions within a complex network of glycogen metabolism enzymes, and understanding these interactions is crucial for comprehensive research:

• Integration with glycogen synthesis and degradation pathways:

  • Ganc likely complements the action of other glycogen-degrading enzymes like phosphorylases

  • May interact with glycogen branching enzymes to modify glycogen structure

  • Could participate in specialized degradation pathways distinct from lysosomal degradation

• Regulation within metabolic networks:

  • Potential coordination with glucose transporters for efficient substrate utilization

  • Possible co-regulation with glycogen synthase for balanced metabolism

  • May respond to cellular energy status signals

• Protein-protein interactions:

  • Secondary substrate-binding domains may facilitate interactions with glycogen particles and other proteins

  • Possible association with scaffolding proteins that organize metabolic enzymes

  • Potential interactions with trafficking proteins that determine subcellular localization

• Pathophysiological significance:

  • May have complementary functions to acid alpha-glucosidase (GAA) in different cellular compartments

  • Could provide alternative glycogen degradation pathways when other systems are compromised

  • Potentially involved in specialized aspects of glucose homeostasis

Although direct experimental evidence for these interactions is limited in the provided search results, the conservation of functional domains and the role of Ganc in glycogen metabolism suggest these interactions are likely significant and worthy of investigation in research settings.

What are common issues in recombinant mouse Ganc expression and how can they be resolved?

When working with recombinant mouse Ganc, researchers may encounter several challenges that can be addressed with specific strategies:

• Low expression levels:

  • Optimize codon usage for the host expression system

  • Test different promoters (CMV, EF1α, CAG) for higher expression

  • Use expression vectors with enhancer elements

  • Consider stable cell line development for consistent expression

  • Optimize transfection conditions (reagent:DNA ratio, cell density, incubation time)

• Protein misfolding and inactivity:

  • Express at lower temperatures (30-34°C instead of 37°C) to slow folding

  • Include molecular chaperones as co-expression partners

  • Use mammalian expression systems rather than bacterial systems to ensure proper glycosylation

  • Validate construct sequence for accurate translation, especially at the catalytic site (WXDMNE motif)

• Proteolytic degradation:

  • Add protease inhibitors during extraction and purification

  • Reduce extraction time and maintain cold temperatures

  • Avoid harsh elution conditions during purification

  • Consider adding stabilizing agents like glycerol or specific sugars

• Inconsistent activity measurements:

  • Standardize assay conditions, particularly pH and temperature

  • Prepare fresh substrate solutions for each experiment

  • Include internal standards in each assay run

  • Ensure complete substrate solubilization

  • Calculate specific activity using validated formulas

• Distinguishing from endogenous glucosidases:

  • Use cells lacking endogenous Ganc activity for expression

  • Employ electrophoretic techniques to separate and identify Ganc activity

  • Include appropriate controls (non-transfected cells, cells with empty vector)

These strategies address common challenges in recombinant enzyme work and should improve success rates in Ganc studies.

How can I design experiments to investigate the physiological role of mouse Ganc?

Designing experiments to investigate the physiological role of mouse Ganc requires a multi-faceted approach:

• Expression pattern analysis:

  • Quantify Ganc mRNA and protein levels across mouse tissues

  • Compare expression patterns with other glycosidases

  • Analyze expression changes during development and under different physiological conditions

  • Use immunohistochemistry to determine cellular and subcellular localization

• Loss-of-function studies:

  • Generate Ganc knockout or knockdown models

  • Use CRISPR-Cas9 for precise genome editing

  • Apply siRNA for transient knockdown in cell models

  • Analyze resulting changes in:

    • Glycogen metabolism and structure

    • Cellular response to metabolic stress

    • Gene expression profiles

• Gain-of-function studies:

  • Overexpress wild-type and mutant Ganc in appropriate cell models

  • Compare with cells expressing related enzymes like acid alpha-glucosidase

  • Assess impact on glycogen content and metabolism

  • Evaluate cellular adaptation to altered Ganc levels

• Substrate specificity and metabolic impact:

  • Analyze glycogen structure in Ganc-deficient and Ganc-overexpressing models

  • Trace labeled substrates to determine Ganc's contribution to glucose production

  • Investigate changes in downstream metabolic pathways

• Disease model relevance:

  • Examine Ganc expression and activity in models of glycogen storage disorders

  • Investigate compensatory changes in Ganc in acid alpha-glucosidase deficiency (Pompe disease model)

  • Explore Ganc's role in metabolic disorders like diabetes, given its chromosomal location in a diabetes susceptibility region (human 15q15)

These experimental approaches would provide comprehensive insights into the physiological significance of Ganc in glycogen metabolism and potentially uncover novel therapeutic targets.

What are the key differences between mouse Ganc and human GANC?

Understanding the similarities and differences between mouse Ganc and human GANC is essential for translational research:

• Sequence homology and structural comparison:

  • Mouse and human GANC share significant sequence homology, with approximately 80-85% similarity at the amino acid level (comparable to the 80% homology between mouse and human GAA)

  • The catalytic WXDMNE motif is highly conserved between species

  • Both proteins have similar molecular weights: approximately 104 kDa for human GANC (914 amino acids)

  • Conservation is typically highest in functional domains and the catalytic core

• Enzymatic properties:

  • Both enzymes function at neutral pH

  • Substrate specificity profiles are largely conserved, but mouse Ganc may show subtle differences in oligosaccharide preferences

  • Kinetic parameters (Km, Vmax) likely show species-specific variations

  • Inhibitor sensitivity profiles may differ slightly

• Genetic variation:

  • Human GANC exhibits polymorphism with four alleles, including a null allele

  • Mouse Ganc likely has its own pattern of allelic variation

  • Species-specific alternative splicing may produce different isoforms

• Physiological context:

  • Tissue distribution patterns may differ between species

  • Regulatory mechanisms and expression patterns can show species-specific adaptations

  • Functional redundancy with other glucosidases may vary between mouse and human

• Experimental considerations:

  • Mouse models may not perfectly recapitulate human GANC-related phenotypes

  • Species-specific antibodies may be required for accurate detection

  • Expression systems might need optimization when switching between species orthologs

These differences highlight the importance of species-specific validation when extrapolating findings between mouse models and human applications in Ganc research.

How has Ganc evolved and what does this tell us about its function?

Evolutionary analysis of Ganc provides valuable insights into its functional significance:

• Phylogenetic relationships:

  • Ganc belongs to glycosyl hydrolase family 31, which has ancient evolutionary origins

  • Within this family, Ganc shows highest homology to the catalytic unit of glucosidase II (67% similarity)

  • Lower but significant similarity to other family members: lysosomal acid alpha-glucosidase, maltase-glucoamylase, and sucrase-isomaltase (41-67%)

  • This pattern suggests an early gene duplication and functional divergence within the family

• Conservation of functional domains:

  • The catalytic WXDMNE motif is highly conserved across species and family members

  • This conservation indicates strong evolutionary pressure to maintain glucosidase activity

  • Variable regions likely represent adaptations for specific substrates or regulatory mechanisms

  • Secondary substrate-binding domains may have evolved to enhance enzyme processivity

• Functional implications:

  • The neutral pH optimum distinguishes Ganc from lysosomal glucosidases, suggesting adaptation to different cellular compartments

  • Conservation across mammals indicates essential metabolic functions

  • Persistence of multiple glucosidases with overlapping substrate specificity suggests non-redundant functions

  • The presence of allelic variants, including null alleles , suggests possible selective advantages under different conditions

• Disease relevance:

  • The evolutionary relationship with other glycosidases provides context for understanding glycogen storage disorders

  • Human GANC's location in a region associated with diabetes susceptibility (15q15) suggests potential metabolic significance

This evolutionary perspective highlights Ganc's importance in fundamental metabolic processes and provides context for interpreting experimental findings in both basic and translational research settings.

How can recombinant mouse Ganc be utilized in glycogen metabolism research?

Recombinant mouse Ganc represents a valuable tool for investigating various aspects of glycogen metabolism:

• Structural studies of glycogen:

  • Use purified Ganc to analyze glycogen branching patterns

  • Compare Ganc-mediated degradation products with those of other glucosidases

  • Investigate the enzyme's action on glycogen from different tissues and pathological states

  • Determine the influence of secondary substrate-binding domains on processing complex glycogen structures

• Enzyme replacement therapy models:

  • Test recombinant Ganc as a potential complementary approach to GAA replacement

  • Evaluate cellular uptake and distribution of recombinant enzyme

  • Assess impact on glycogen accumulation in models of glycogen storage disorders

  • Compare efficacy of different enzyme formulations and modifications

• Reporter systems:

  • Develop Ganc-based biosensors for glycogen visualization

  • Create fusion proteins with fluorescent tags for tracking glycogen metabolism in real-time

  • Use enzyme-coupled assays to monitor glycogen levels in various physiological states

• Comparative enzymology:

  • Systematically compare kinetic properties with other glucosidases

  • Define the unique substrate preferences of Ganc

  • Identify specific inhibitors that distinguish between glucosidase family members

  • Map structural features to functional differences between family members

• Metabolic network analysis:

  • Use recombinant Ganc to probe the integration of glycogen metabolism with other pathways

  • Investigate how Ganc activity responds to metabolic signals

  • Examine potential roles in specialized tissues with unique energy requirements

These applications leverage the availability of recombinant Ganc to address fundamental questions in glycogen metabolism and potentially develop new therapeutic approaches for metabolic disorders.

What are the most promising research directions for understanding Ganc function in metabolic disorders?

Several promising research directions could significantly advance our understanding of Ganc function in metabolic disorders:

• Diabetes connection investigation:

  • Given that human GANC localizes to chromosome 15q15, reported to confer susceptibility to diabetes , explore mouse Ganc's role in glucose homeostasis

  • Characterize Ganc expression and activity changes in diabetic mouse models

  • Investigate potential genetic associations between Ganc variants and metabolic phenotypes

  • Explore whether Ganc modulation could represent a novel therapeutic approach

• Complementary roles with acid alpha-glucosidase:

  • Investigate whether Ganc provides compensatory activity in Pompe disease models (acid alpha-glucosidase deficiency)

  • Explore potential synergistic effects of combined enzyme replacement

  • Determine if Ganc upregulation could be therapeutically beneficial in glycogen storage disorders

  • Characterize the distinct cellular roles of neutral versus acid glucosidases

• Regulatory network mapping:

  • Identify transcription factors and signaling pathways that regulate Ganc expression

  • Determine how Ganc activity is modulated by post-translational modifications

  • Map protein-protein interactions that influence Ganc function

  • Develop computational models of glycogen metabolism incorporating Ganc activity

• Development of specific modulators:

  • Design and screen for specific Ganc inhibitors or activators

  • Evaluate their effects on glycogen metabolism in various tissues

  • Assess potential therapeutic applications in metabolic disorders

  • Develop targeted delivery approaches for enzyme or modulators

• Cellular stress responses:

  • Investigate Ganc's role in cellular adaptation to nutrient limitation

  • Explore connections between Ganc activity and endoplasmic reticulum stress

  • Examine potential roles in autophagy and lysosomal function

  • Assess responses to oxidative stress and inflammation

These research directions would significantly advance our understanding of Ganc's physiological roles and potentially reveal new therapeutic targets for metabolic disorders.

What is the recommended protocol for purifying recombinant mouse Ganc?

A comprehensive protocol for purifying recombinant mouse Ganc would include:

• Expression system preparation:

  • Clone mouse Ganc cDNA into an expression vector with N-terminal 6-His tag

  • Transform/transfect into appropriate host system (mammalian cells preferred)

  • Culture under optimized conditions for maximal expression

  • Verify expression by Western blot or activity assay

• Cell harvest and initial processing:

  • Collect cells by centrifugation (adherent cells should be scraped or trypsinized)

  • Wash cell pellet with cold PBS

  • Resuspend in lysis buffer:

    • 50 mM Tris-HCl, pH 7.5

    • 150 mM NaCl

    • 1% Triton X-100 or other suitable detergent

    • Protease inhibitor cocktail

  • Lyse cells by sonication or mechanical disruption

  • Clarify lysate by centrifugation at 15,000 × g for 30 minutes at 4°C

• Affinity chromatography:

  • Equilibrate Ni-NTA or similar affinity resin with binding buffer:

    • 50 mM Tris-HCl, pH 7.5

    • 300 mM NaCl

    • 10 mM imidazole

  • Apply clarified lysate to column

  • Wash extensively with binding buffer

  • Elute bound protein with elution buffer:

    • 50 mM Tris-HCl, pH 7.5

    • 300 mM NaCl

    • 250 mM imidazole

• Secondary purification:

  • Pool active fractions determined by activity assay

  • Perform size exclusion chromatography:

    • Use Superdex 200 or similar column

    • Buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl

  • Alternative: Ion exchange chromatography

• Final processing:

  • Concentrate using centrifugal concentrator (30-50 kDa cutoff)

  • Buffer exchange into storage buffer:

    • 20 mM Tris-HCl, pH 7.2

    • 100 mM NaCl

  • Filter through 0.2 μm filter

  • Aliquot and store at -80°C

• Quality control:

  • Verify purity by SDS-PAGE (expected size approximately 104 kDa)

  • Confirm identity by Western blot or mass spectrometry

  • Measure specific activity using standardized assay

  • Determine protein concentration

This protocol incorporates elements from successful purification of related glycosidases and should yield active, highly purified recombinant mouse Ganc suitable for research applications .

How should researchers design assays to measure Ganc activity in tissue samples?

Designing robust assays for measuring Ganc activity in tissue samples requires careful consideration of specificity, sensitivity, and reliability:

• Sample preparation:

  • Homogenize tissue in neutral pH buffer (20 mM Tris-HCl, pH 7.2-7.4)

  • Include protease inhibitors to prevent enzyme degradation

  • Clarify homogenates by centrifugation (10,000-15,000 × g for 15 minutes)

  • Perform protein determination for normalization

• Distinguishing Ganc from other glucosidases:

  • Conduct parallel assays at multiple pH values:

    • pH 7.0-7.5 for Ganc activity

    • pH 4.0-4.5 for lysosomal alpha-glucosidase activity

  • Use selective inhibitors:

    • Include maltose or specific inhibitors to distinguish from maltase activity

    • Use mannose-based inhibitors to distinguish from glucosidase II

• Assay methodology:

  • Colorimetric starch hydrolysis assay:

    • Substrate: 1.5% starch in appropriate buffer

    • Incubate tissue extract with substrate at 37°C

    • Detect released glucose using dinitrosalicylic acid reagent

    • Measure absorbance at 540-550 nm

    • Include maltose standard curve (19.5 to 625 nmol per well)

  • Fluorogenic substrate assay:

    • Substrate: 4-methylumbelliferyl-α-D-glucoside

    • Detect released 4-methylumbelliferone by fluorescence

    • This method offers higher sensitivity for limited samples

• Controls and validation:

  • Include tissue samples known to express high and low levels of Ganc

  • Run parallel samples with heat-inactivated enzyme

  • Perform assays with purified recombinant Ganc as positive control

  • Include assay buffer without enzyme as negative control

• Data analysis and reporting:

  • Calculate specific activity using the formula:

    Specific Activity (pmol/min/μg) =	Adjusted glucose produced (nmol) × (1000 pmol/nmol)
    Incubation time (min) × amount of protein (μg)

  • Normalize to protein concentration

  • Present data as mean ± standard deviation from multiple determinations