Recombinant Mouse Starch-binding domain-containing protein 1 (Stbd1), partial

What is the general function of Stbd1 in cellular metabolism?

Stbd1 functions primarily as a cargo receptor for glycogen, facilitating the delivery of glycogen to lysosomes through an autophagic pathway termed "glycophagy" . This process involves the recognition, binding, and transport of glycogen to lysosomes for degradation. Stbd1 is a transmembrane, glycogen-binding protein that predominantly localizes to the endoplasmic reticulum (ER) and is also found at ER-mitochondria contact sites where it may participate in ER-mitochondria tethering . The protein contains a carbohydrate-binding module (CBM20) that enables it to interact with glycogen and an Atg8-family interacting motif (AIM) that mediates its interaction with the autophagy protein Gabarapl1 . These structural features collectively enable Stbd1 to function as a selective autophagy receptor, though definitive evidence for this specific role remains under investigation.

What are the structural characteristics of recombinant mouse Stbd1?

Recombinant mouse Stbd1 is typically expressed as a protein of approximately 36 kDa, detectable in most mouse tissues . The protein contains several functionally important domains that contribute to its cellular activities:

• An N-terminal hydrophobic segment essential for proper subcellular localization

• A carbohydrate-binding module (CBM20) responsible for glycogen interaction

• An Atg8-family interacting motif (AIM) that mediates interaction with autophagy proteins

Research has demonstrated that mutation or deletion of these domains significantly affects Stbd1 function. For example, mutant Stbd1 lacking the N-terminal hydrophobic segment displays a diffuse distribution throughout the cell rather than concentrating in perinuclear regions . Similarly, point mutations in the CBM20 domain do not alter the perinuclear localization of Stbd1 but prevent glycogen from concentrating in this compartment .

What expression systems are commonly used for recombinant mouse Stbd1 production?

The primary expression system used for recombinant mouse Stbd1 production is Escherichia coli. As detailed in the available research, His-tagged mouse Stbd1 (mStbd1) can be efficiently expressed in E. coli after induction with isopropyl β-D-thiogalactoside (IPTG) . Typically, protein expression is induced at lower temperatures (around 18°C) overnight to enhance proper folding and solubility. The recombinant protein can then be purified using nickel-affinity chromatography (Ni²⁺-NTA-agarose), with step-wise elution using imidazole gradients ranging from 40-200 mM . Mouse Stbd1 is primarily eluted in fractions containing 100-200 mM imidazole. Post-purification, the protein is commonly dialyzed against an appropriate buffer (such as 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5% 2-mercaptoethanol) and stored at -80°C with 15% glycerol to maintain stability and activity .

How is recombinant Stbd1 typically characterized after purification?

Following purification, recombinant Stbd1 is commonly characterized using several analytical techniques:

• SDS-PAGE analysis to confirm purity and molecular weight (typically showing a band at approximately 36 kDa)

• Western blotting using specific antibodies against Stbd1 or epitope tags

• Functional assays to assess glycogen binding capacity

• Subcellular localization studies using immunofluorescence microscopy when expressed in cell lines

For glycogen binding assays, purified recombinant Stbd1 (typically at 2.5 μg/ml) is incubated with glycogen or amylopectin (0.25 mg/ml) at 4°C for approximately 1 hour, followed by ultracentrifugation to separate bound and unbound fractions . The resulting pellets and supernatants are then analyzed by Western blotting to determine the proportion of Stbd1 bound to the carbohydrate substrate.

What experimental models are available for studying Stbd1 function in vivo?

Several sophisticated experimental models have been developed to investigate Stbd1 function in vivo, with knockout mouse models representing the most comprehensive systems:

• Stbd1 Knockout (SKO) Mice: Generated by replacing exon 2 of the Stbd1 gene with a neo cassette on a C57BL/6J genetic background . These mice provide a valuable model for studying the physiological functions of Stbd1 in isolation.

• GAA/Stbd1 Double Knockout (dKO) Mice: Created by crossing Stbd1 knockout mice with acid alpha-glucosidase (GAA) knockout mice, which serve as a model for Pompe disease . This double knockout model is particularly valuable for investigating the role of Stbd1 in lysosomal glycogen trafficking across various tissues, as glycogen accumulation due to GAA deficiency provides a quantifiable indicator of glycogen transport into lysosomes.

• Tissue-specific Stbd1 Knockdown Models: Various studies have employed tissue-specific knockdown approaches using techniques such as lentiviral shRNA delivery to examine the function of Stbd1 in specific cell types or tissues .

These models offer powerful tools for dissecting the physiological roles of Stbd1 and its potential contributions to disease pathology, particularly in the context of glycogen storage disorders.

How does Stbd1 respond to ER stress, and what is its role in this context?

Stbd1 exhibits a remarkable response to endoplasmic reticulum (ER) stress, showing significant upregulation during ER stress conditions. In C2C12 mouse myoblasts treated with tunicamycin (TM, an ER stress inducer), Stbd1 protein levels progressively increase over time, paralleling the increase in BiP, an early unfolded protein response (UPR) chaperone . This upregulation appears to play a critical functional role in the cellular response to ER stress.

The primary function of Stbd1 during ER stress appears to be the promotion of glycogen clustering. Research indicates that Stbd1 is both necessary and sufficient for the formation of glycogen clusters in response to ER stress in mouse myoblasts . This process involves:

• Recruitment of glycogenin (GN) and glycogen synthase 1 (GS1) to the ER membrane

• Formation of organized glycogen-containing structures at the ER

• Potential protection against ER stress-induced apoptosis

Importantly, knockdown of Stbd1 in C2C12 myoblasts prevents the formation of glycogen clusters during ER stress, and failure to induce glycogen clustering is associated with enhanced activation of apoptotic pathways . This suggests that Stbd1-mediated glycogen clustering may represent a cellular survival mechanism during ER stress conditions.

What are the current controversies regarding Stbd1's role in glycogen trafficking to lysosomes?

There exists significant scientific controversy regarding the precise role of Stbd1 in glycogen trafficking to lysosomes, particularly in the context of glycogen storage disorders such as Pompe disease. The contradictory findings can be summarized as follows:

Supporting a central role in glycophagy:

• Stbd1 contains both glycogen-binding domains (CBM20) and autophagy-related protein interaction motifs (AIM)

• Stbd1 co-localizes with glycogen, late endosomal/lysosomal markers (LAMP1), and autophagy proteins (GABARAPL1)

• These characteristics led to the proposition that Stbd1 functions as a cargo receptor for glycogen delivery to lysosomes via a selective autophagy pathway termed "glycophagy"

Challenging this role:

• Studies using GAA knockout mice (a model for Pompe disease) showed that significant knockdown of Stbd1 in skeletal muscle did not reduce lysosomal glycogen accumulation, suggesting Stbd1 may not be involved in lysosomal glycogen trafficking in this tissue

• To address this controversy more definitively, researchers generated Stbd1/GAA double knockout mice, providing an ideal system for investigating the role of Stbd1 in transporting glycogen into lysosomes across various tissues

These conflicting results highlight the complexity of glycogen metabolism regulation and suggest that Stbd1's role may vary depending on tissue type, developmental stage, or physiological conditions. The controversy underscores the need for continued investigation using diverse experimental approaches and model systems.

What methodological approaches are recommended for studying Stbd1-glycogen interactions?

For comprehensive investigation of Stbd1-glycogen interactions, researchers should employ multiple complementary methodological approaches:

In vitro binding assays:

• Recombinant Stbd1 protein (2.5 μg/ml) can be incubated with glycogen or amylopectin (0.25 mg/ml) at 4°C

• Following ultracentrifugation (100,000 × g at 4°C for 90 minutes), the pellets contain bound Stbd1-glycogen complexes

• Western blotting of both supernatant and pellet fractions allows quantification of binding efficiency

Microscopy-based approaches:

• Immunofluorescence microscopy using specific antibodies against Stbd1 (typically at 1:200 dilution) and glycogen

• Co-localization studies with organelle markers such as calnexin (ER), LAMP1 (lysosomes), and autophagy proteins (GABARAPL1, LC3)

• Fixed cell preparations using 4% paraformaldehyde with subsequent permeabilization using 0.2% Triton X-100

Genetic manipulation approaches:

• Overexpression of wild-type Stbd1 to induce glycogen clustering

• Expression of Stbd1 mutants lacking specific domains (N-terminal hydrophobic segment, CBM20 mutations) to assess their functional importance

• Knockdown using shRNA or CRISPR-Cas9 approaches to evaluate loss-of-function effects

Stress response evaluation:

• Treatment with ER stress inducers such as tunicamycin

• Assessment of glycogen structure formation during stress responses

• Correlation with cell survival metrics to determine functional significance

These multifaceted approaches provide complementary data that together offer a more complete understanding of Stbd1-glycogen interactions and their physiological significance.

What are the critical factors to consider when designing experiments involving recombinant mouse Stbd1?

When designing experiments with recombinant mouse Stbd1, researchers should carefully consider several critical factors:

Protein expression and purification:

• Expression temperature significantly affects protein solubility and proper folding; lower temperatures (18°C) typically yield better results than standard 37°C conditions

• Inclusion of reducing agents (such as 2-mercaptoethanol) in purification buffers helps maintain protein stability

• Glycerol (typically 15%) should be included for long-term storage at -80°C

Functional domain preservation:

• The integrity of the CBM20 domain is crucial for glycogen-binding function

• The N-terminal hydrophobic segment is essential for proper subcellular localization

• The AIM motif is necessary for interactions with autophagy proteins

Expression systems compatibility:

• E. coli-expressed Stbd1 is suitable for in vitro binding assays and antibody production

• Mammalian expression systems are preferable for localization and interaction studies

• Viral vectors (AAV) can be used for in vivo expression studies

Experimental controls:

• For binding assays, include Stbd1 without polysaccharide as a negative control

• For localization studies, include mutant Stbd1 variants with known localization patterns

• For knockdown studies, use scrambled shRNA sequences as controls

Careful attention to these factors will enhance experimental reproducibility and the validity of results obtained when working with recombinant mouse Stbd1.

How should researchers approach the quantification of Stbd1-mediated glycogen clustering?

Quantification of Stbd1-mediated glycogen clustering requires systematic approaches to ensure reliable and reproducible results:

Microscopy-based quantification:

• Immunofluorescence with co-staining for Stbd1 and glycogen

• Counterstain with organelle markers (calnexin for ER, LAMP1 for lysosomes)

• Acquisition of high-resolution z-stack images using confocal microscopy

• Analysis of glycogen cluster size, number, and distribution using image analysis software

• Quantification of co-localization coefficients (Pearson's or Manders' coefficients)

Biochemical fractionation approach:

• Cell homogenization under conditions that preserve glycogen-protein interactions

• Differential centrifugation to separate subcellular fractions

• Analysis of glycogen content in each fraction using enzymatic assays

• Western blotting of fractions for Stbd1 and organelle markers

• Comparison of glycogen distribution across fractions between experimental conditions

Standardization considerations:

• Establish clear criteria for defining a "glycogen cluster" (size, intensity, morphology)

• Use consistent imaging parameters across all experimental conditions

• Include appropriate positive controls (tunicamycin treatment) and negative controls (Stbd1 knockdown)

• Apply blinded analysis to prevent bias in quantification

• Employ appropriate statistical methods for comparing clustering between conditions

This multifaceted approach provides robust quantification of Stbd1-mediated glycogen clustering and enables meaningful comparisons between experimental conditions.

What considerations are important when generating or using Stbd1 knockout or knockdown models?

When generating or utilizing Stbd1 knockout or knockdown models, researchers should consider several important factors to ensure valid and interpretable results:

For germline knockout models:

• Confirm complete absence of Stbd1 protein using Western blotting with validated antibodies

• Assess potential developmental compensation mechanisms

• Consider tissue-specific effects, as Stbd1 function may vary across tissues

• Use appropriate congenic background strains (e.g., C57BL/6J) and include sufficient backcrossing generations (at least 5) to minimize genetic background effects

• Generate and maintain proper control groups (littermate controls whenever possible)

For knockdown models:

• Validate knockdown efficiency at both mRNA and protein levels

• Use multiple shRNA or siRNA sequences to control for off-target effects

• Include scrambled sequence controls (shScr) for comparison

• Consider potential differences between transient and stable knockdown approaches

For disease model combinations:

• When combining Stbd1 deficiency with disease models (e.g., GAA-KO for Pompe disease), carefully control for genetic background effects

• Use breeding schemes that generate all relevant genotypes as littermates (e.g., GAA−/−/Stbd1+/+ and GAA−/−/Stbd1−/−)

• Consider age-dependent effects, especially in progressive diseases

• Analyze multiple tissues to account for tissue-specific variability in phenotypes

Phenotypic analysis:

• Employ comprehensive phenotyping approaches covering multiple physiological systems

• Examine glycogen content and distribution using both biochemical and histological methods

• Assess potential compensatory mechanisms by examining related proteins

• Consider stress conditions (e.g., ER stress) that may reveal phenotypes not evident under basal conditions

Careful consideration of these factors will enhance the reliability and interpretability of results obtained from Stbd1 knockout or knockdown models.

What are the common challenges in purifying recombinant Stbd1 and how can they be addressed?

Researchers frequently encounter several challenges when purifying recombinant Stbd1, but these can be addressed with specific optimizations:

Solubility issues:

• Challenge: Recombinant Stbd1 often forms inclusion bodies when expressed at standard conditions.

• Solution: Induce protein expression at lower temperatures (18°C instead of 37°C) overnight and use reduced IPTG concentrations (0.4 mM) . This slower expression promotes proper folding and increases solubility.

Protein stability concerns:

• Challenge: Stbd1 may degrade during purification and storage.

• Solution: Include protease inhibitors during purification and add reducing agents (5% 2-mercaptoethanol) to buffers. For storage, use 15% glycerol and maintain at -80°C .

Purity limitations:

• Challenge: Achieving high purity (>90%) can be difficult with single-step purification.

• Solution: Implement step-wise elution with an imidazole gradient (40-200 mM) during Ni²⁺-NTA-agarose chromatography, collecting and analyzing individual fractions. Pooling the optimal fractions (often 100-200 mM imidazole) provides higher purity .

Functional verification:

• Challenge: Ensuring the purified protein maintains its glycogen-binding capacity.

• Solution: Perform functional binding assays using glycogen or amylopectin substrates, followed by ultracentrifugation and Western blotting analysis to confirm activity .

By implementing these optimized approaches, researchers can significantly improve the yield, purity, and functional quality of recombinant Stbd1 preparations.

What strategies can improve the detection and localization of Stbd1 in different experimental systems?

Effective detection and localization of Stbd1 requires tailored strategies depending on the experimental system:

For cell culture systems:

• Optimized fixation: Use 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100 to preserve Stbd1 localization while enabling antibody access

• Blocking optimization: Use 5% BSA in PBS to reduce background signal

• Antibody selection: Use affinity-purified antibodies against mouse Stbd1 at 1:200 dilution for optimal signal-to-noise ratio

• Co-localization markers: Include organelle markers such as calnexin (ER), LAMP1 (lysosomes), and GABARAPL1 (autophagy) at appropriate dilutions (1:50-1:1000) to contextualize Stbd1 localization

• Signal amplification: Consider using fluorophore-conjugated secondary antibodies with bright fluorophores (Alexa488, Texas Red) at 1:400 dilution

For tissue sections:

• Antigen retrieval: Implement citrate buffer-based antigen retrieval to enhance detection in fixed tissues

• Autofluorescence reduction: Use Sudan Black B treatment to reduce tissue autofluorescence

• Confocal microscopy: Utilize confocal microscopy with appropriate z-sectioning to improve spatial resolution

• Signal enhancement: Consider tyramide signal amplification for low-abundance detection

For biochemical detection:

• Extraction optimization: Use buffers containing mild detergents (0.2% Triton X-100) to maintain Stbd1 solubility

• Western blotting enhancement: Transfer proteins at lower voltage for longer periods to improve transfer of membrane-associated proteins

• Loading controls: Include appropriate loading controls specific to the cellular compartment being analyzed

• Subcellular fractionation: Implement differential centrifugation protocols to isolate and enrich specific subcellular compartments before analysis

These strategies significantly improve the sensitivity and specificity of Stbd1 detection across different experimental systems.

How can researchers effectively design and interpret experiments to resolve contradictions in Stbd1 function?

To address contradictions in the literature regarding Stbd1 function, researchers should implement a systematic experimental approach:

Comprehensive experimental design:

• Multiple model systems: Employ both cell culture systems and animal models to provide complementary insights

• Tissue specificity consideration: Examine Stbd1 function across multiple tissues, as its role may vary between tissue types

• Temporal dynamics: Assess Stbd1 function at different time points to capture dynamic processes

• Stress conditions: Include both basal and stress conditions (particularly ER stress), as Stbd1 function may be more evident under stress

Mechanistic dissection:

• Domain-specific mutations: Generate and analyze Stbd1 variants with specific domain mutations to dissect the contribution of each functional domain

• Interaction network mapping: Use proteomic approaches to comprehensively map Stbd1 interaction partners

• Conditional knockout models: Develop inducible or tissue-specific knockout models to avoid developmental compensation

Integration of multiple readouts:

• Glycogen quantification: Measure glycogen levels using both biochemical and histological methods

• Autophagic flux assessment: Monitor autophagy markers and flux in parallel with Stbd1 studies

• Physiological outcomes: Connect molecular observations to physiological outcomes (e.g., muscle function in Pompe disease models)

Addressing contradictions directly:

• Replication with attention to conditions: Carefully replicate published experiments with particular attention to methodological details

• Side-by-side comparisons: Perform direct comparisons of competing models under identical conditions

• Identification of reconciling factors: Systematically test variables that might explain contradictions (tissue type, developmental stage, stress conditions)

By implementing these approaches, researchers can design experiments specifically targeted at resolving contradictions in the literature regarding Stbd1 function, ultimately advancing understanding of this protein's role in glycogen metabolism and stress responses.

What are the most promising therapeutic applications of Stbd1 research?

While currently not at the clinical application stage, Stbd1 research shows significant therapeutic potential in several areas:

Glycogen storage disorders:

• Stbd1's role in glycogen trafficking makes it particularly relevant for conditions like Pompe disease, where abnormal glycogen accumulation occurs in lysosomes

• Initial expectations positioned Stbd1 as a potential therapeutic target for Pompe disease, though contradictory findings complicate this application

• Further elucidation of tissue-specific roles may clarify therapeutic relevance

• The development of Stbd1/GAA double knockout mice provides a valuable platform for evaluating therapeutic strategies

ER stress-related conditions:

• The newly discovered role of Stbd1 in glycogen clustering during ER stress and its potential protective effect against apoptosis suggests therapeutic relevance for conditions involving ER stress

• Diseases with prominent ER stress components, including certain neurodegenerative disorders, diabetes, and inflammatory conditions, might benefit from Stbd1-targeted approaches

• Enhancing Stbd1 function could potentially promote cell survival under ER stress conditions

Metabolic disorders:

• Stbd1's involvement in glycogen metabolism suggests potential applications in metabolic disorders characterized by dysregulated energy homeostasis

• Tissue-specific modulation of Stbd1 activity might offer approaches to regulate glycogen storage and utilization

Potential therapeutic strategies:

• Small molecule modulators of Stbd1 activity

• Gene therapy approaches using AAV vectors to deliver functional Stbd1

• Peptide-based therapies targeting specific Stbd1 interactions

• Combined therapies addressing both Stbd1 and complementary pathways

These potential applications highlight the need for continued research to fully characterize Stbd1's physiological roles and resolve existing contradictions in the literature.

What are the key unanswered questions about Stbd1 function that require further investigation?

Despite significant progress in understanding Stbd1, several crucial questions remain unanswered and warrant further investigation:

Mechanism of glycogen trafficking:

• What is the precise mechanism by which Stbd1 facilitates glycogen transport to lysosomes?

• How is this process regulated under different physiological conditions?

• Why does Stbd1 knockdown in skeletal muscle not affect lysosomal glycogen accumulation in Pompe disease models?

Tissue-specific functions:

• Does Stbd1 play different roles in different tissues?

• What factors determine the tissue-specific effects of Stbd1 deficiency?

• Are there tissue-specific interaction partners that modify Stbd1 function?

Relationship with the ubiquitin-proteasome system:

• What is the significance of Stbd1 ubiquitination that leads to proteasomal degradation?

• How is Stbd1 stability regulated, and how does this impact its function?

• What is the interplay between autophagy and proteasomal degradation in Stbd1 regulation?

ER stress response:

• What molecular mechanisms underlie Stbd1 upregulation during ER stress?

• How exactly does Stbd1-mediated glycogen clustering protect against ER stress-induced apoptosis?

• Is the protective effect of Stbd1 during ER stress conserved across different cell types and tissues?

Developmental and physiological regulation:

• How is Stbd1 expression and function regulated during development?

• What is the physiological significance of Stbd1 in normal glycogen metabolism?

• How does Stbd1 function change in response to different nutritional states or exercise?

Addressing these questions will require integrated approaches combining advanced genetic models, cell biology techniques, and physiological studies. The answers will significantly enhance our understanding of glycogen metabolism, autophagy, and ER stress responses.

What emerging technologies might advance Stbd1 research in the coming years?

Several cutting-edge technologies hold promise for advancing Stbd1 research and resolving key questions about its function:

Advanced imaging techniques:

• Super-resolution microscopy (STORM, PALM, SIM) to visualize Stbd1-glycogen interactions at nanoscale resolution

• Live-cell imaging with optogenetic tools to monitor dynamic changes in Stbd1 localization and function

• Correlative light and electron microscopy (CLEM) to connect fluorescence observations with ultrastructural details

• Volume electron microscopy to generate 3D reconstructions of glycogen-containing structures

CRISPR-based technologies:

• CRISPR activation (CRISPRa) and interference (CRISPRi) for precise temporal control of Stbd1 expression

• Base editing and prime editing for introducing specific point mutations in Stbd1 domains

• CRISPR screens to identify novel regulators and interaction partners of Stbd1

• Tissue-specific and inducible CRISPR systems for spatiotemporal control of Stbd1 expression in vivo

Proteomics and interaction mapping:

• Proximity labeling approaches (BioID, APEX) to comprehensively map the Stbd1 interactome

• Quantitative phosphoproteomics to elucidate Stbd1 regulation by phosphorylation

• Crosslinking mass spectrometry to capture transient interactions

• Thermal proteome profiling to identify direct binding partners and effects of small molecules

Single-cell technologies:

• Single-cell RNA-seq to explore cell-to-cell variability in Stbd1 expression

• Single-cell proteomics to examine variation in Stbd1 protein levels and modifications

• Spatial transcriptomics to map Stbd1 expression patterns within tissues

Advanced animal models:

• Human iPSC-derived organoids to study Stbd1 in human tissue contexts

• Conditional and inducible knockout models with tissue-specific and temporal control

• Humanized mouse models expressing human Stbd1 variants

• Non-human primate models for translational studies of Stbd1 function

These emerging technologies, particularly when applied in combination, have the potential to resolve current contradictions in the literature and advance our understanding of Stbd1's complex roles in glycogen metabolism, ER stress responses, and disease pathogenesis.