Recombinant Human Vacuolar protein sorting-associated protein 13B (VPS13B), partial

What is the structure and function of VPS13B protein?

VPS13B (also known as COH1) is a 4022-amino-acid transmembrane protein located on chromosome 8 (8q22.2). It functions as a Golgi-associated peripheral membrane protein involved in Golgi integrity, homeostasis, and membrane transport . The protein contains ten transmembrane domains, a potential vacuolar targeting motif, an endoplasmic reticulum retention signal on the C-terminus, and two peroxisomal matrix protein targeting signal 2 (PTS2) consensus sequences on both N- and C-termini . VPS13B belongs to the VPS13 protein family, which is highly conserved in eukaryotic cells and plays critical roles in intracellular protein transport and vesicle-mediated sorting . The protein is widely expressed in brain, blood, small intestine, muscles, placenta, heart, retina, kidney, and lung tissues .

How can I express recombinant VPS13B in mammalian cell lines?

Methodological approach:

• Clone the VPS13B gene into a mammalian expression vector (e.g., pcDNA3.1_VPS13B)

• Verify the sequence integrity through Sanger sequencing

• Transfect standard cell cultures (e.g., HeLa cells) using appropriate transfection reagents

• Optimize transfection conditions (DNA concentration, cell density, incubation time)

• Confirm expression through western blotting or immunofluorescence microscopy

The expression of complete VPS13B is challenging due to its large size (4022 amino acids). Therefore, working with partial constructs containing specific functional domains may be more practical for certain experiments. When using partial constructs, ensure they contain the domains of interest, such as transmembrane regions or Golgi-targeting sequences .

What are the basic methods to detect VPS13B protein in cell cultures?

Methodological approach:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% normal goat serum

  • Incubate with anti-VPS13B primary antibody

  • Use appropriate fluorescent secondary antibodies

  • Co-stain with Golgi markers (e.g., GM130) to assess localization

• Western blotting:

  • Prepare cell lysates in appropriate lysis buffer

  • Separate proteins on SDS-PAGE (note: use low percentage gels for full-length VPS13B)

  • Transfer to PVDF membrane

  • Probe with anti-VPS13B antibody

  • Visualize using chemiluminescence or fluorescent detection systems

• Quantification methods:

  • For localization studies, quantify Golgi enrichment by measuring the percentage of Golgi-associated VPS13B fluorescence compared to total VPS13B cell fluorescence intensity using ImageJ or similar software

How can I functionally characterize VPS13B missense variants using recombinant protein expression?

Methodological approach:

• Generate VPS13B constructs containing the missense variants of interest using site-directed mutagenesis

• Express wild-type and mutant VPS13B proteins in appropriate cell lines

• Assess subcellular localization through immunofluorescence microscopy:

  • Define two regions of interest (ROIs) for each cell:
    a. Total cell ROI (outlines the cell border and measures total VPS13B immunofluorescence)
    b. Golgi ROI (outlines the GM130-positive Golgi structure and measures Golgi-associated VPS13B immunofluorescence)

  • Calculate the percentage of Golgi-associated VPS13B fluorescence compared to total VPS13B cell fluorescence

  • Compare Golgi enrichment between wild-type and mutant proteins

• Include appropriate controls:

  • Positive control: A known pathogenic variant (e.g., p.Gly2704Arg) showing reduced Golgi localization

  • Negative control: A benign variant (e.g., p.Ala590Thr) with normal subcellular distribution

• Perform statistical analysis to determine significance of differences in localization patterns

This approach has been validated for characterizing VPS13B missense variants and can provide functional evidence for pathogenicity classification according to ACMG guidelines .

What are the challenges in working with recombinant full-length VPS13B and how can they be addressed?

Methodological solutions:

• Size limitations:

  • The full-length VPS13B cDNA (approximately 12 kb) is challenging to manipulate

  • Solution: Work with partial constructs containing specific functional domains

  • Validate that partial constructs retain relevant biological activities

• Expression efficiency:

  • Large proteins often express poorly in heterologous systems

  • Solutions:
    a. Optimize codon usage for the host system
    b. Use strong promoters (e.g., CMV for mammalian cells)
    c. Consider inducible expression systems to minimize toxicity
    d. Test different cell lines for optimal expression

• Protein stability:

  • Large proteins may be subject to increased degradation

  • Solutions:
    a. Include proteasome inhibitors during protein extraction
    b. Optimize extraction buffers with appropriate protease inhibitors
    c. Perform experiments at shorter time points after transfection

• Functional analysis:

  • Develop specific assays to measure VPS13B activity:
    a. Golgi morphology analysis
    b. Vesicle trafficking assays
    c. Protein-protein interaction studies (co-immunoprecipitation, proximity labeling)

How can I assess the impact of VPS13B mutations on Golgi structure and function?

Methodological approach:

• Express wild-type or mutant VPS13B in cell culture systems

• Analyze Golgi morphology:

  • Immunostain for Golgi markers (GM130, TGN46)

  • Quantify Golgi area, fragmentation, and distribution

  • Use high-resolution microscopy techniques (confocal, super-resolution)

• Assess Golgi function:

  • Protein glycosylation assays

  • Vesicle trafficking analyses

  • Golgi stress response measurements

• Quantitative analysis:

  • Measure the area covered by the Golgi complex

  • Assess Golgi fragmentation index

  • Evaluate cisternal stacking through electron microscopy

• Recovery experiments:

  • Rescue experiments by co-expressing wild-type VPS13B in cells with mutant variants

  • Assess whether Golgi abnormalities can be reversed

These approaches allow for comprehensive characterization of how VPS13B mutations impact Golgi structure and function, providing insights into pathogenic mechanisms .

What are the best experimental models to study recombinant VPS13B function?

Methodological comparison:

For initial characterization of recombinant VPS13B variants, HeLa cells provide an accessible and well-characterized system, particularly for subcellular localization studies . For more disease-relevant contexts, neuronal models or patient-derived cells may be more appropriate, depending on the specific research questions.

How can I optimize the expression and purification of recombinant VPS13B protein fragments for structural studies?

Methodological approach:

• Domain identification and construct design:

  • Analyze VPS13B sequence using bioinformatics tools to identify stable domains

  • Design expression constructs with appropriate boundaries to enhance solubility

  • Include affinity tags (e.g., His, GST, MBP) to facilitate purification

• Expression optimization:

  • Test multiple expression systems:
    a. Bacterial systems (E. coli): Fast but may not provide proper folding
    b. Insect cells (Sf9, High Five): Better for complex eukaryotic proteins
    c. Mammalian cells (HEK293, CHO): Best for post-translational modifications

  • Optimize temperature, induction conditions, and expression time

• Solubility enhancement:

  • Consider fusion partners that enhance solubility (MBP, SUMO, thioredoxin)

  • Test different lysis buffers with varying salt concentrations, pH, and detergents

  • For membrane-associated domains, include appropriate detergents (DDM, CHAPS)

• Purification strategy:

  • Multi-step purification combining:
    a. Affinity chromatography (based on chosen tag)
    b. Ion exchange chromatography
    c. Size exclusion chromatography

  • Assess protein purity by SDS-PAGE and protein quality by dynamic light scattering

• Structural validation:

  • Circular dichroism to confirm secondary structure

  • Limited proteolysis to identify stable domains

  • Thermal shift assays to assess stability

  • Initial characterization by negative-stain electron microscopy

How should I analyze the functional impact of VPS13B variants compared to wild-type protein?

Methodological approach:

• Quantitative analysis of subcellular localization:

  • Define clear metrics for Golgi enrichment

  • Calculate the percentage of Golgi-associated VPS13B fluorescence compared to total VPS13B cell fluorescence

  • Use appropriate statistical tests (t-test, ANOVA) to compare variants

• Data normalization and controls:

  • Include positive controls (known pathogenic variants) and negative controls (benign variants)

  • Normalize data relative to wild-type VPS13B to account for experiment-to-experiment variation

  • Assess multiple parameters to create a comprehensive functional profile

• Integration with pathogenicity prediction:

  • Combine functional data with in silico prediction tools

  • Apply ACMG classification guidelines to interpret variants

  • Correlate functional deficits with clinical phenotypes when possible

• Presentation of results:

  • Use clear graphical representations showing percent Golgi enrichment with error bars

  • Include representative images for each variant

  • Present data from multiple experiments to demonstrate reproducibility

What are the common pitfalls in interpreting VPS13B variant functional data and how can they be avoided?

Methodological considerations:

• Expression level variations:

  • Problem: Different expression levels can affect localization patterns

  • Solution: Analyze cells with comparable expression levels; normalize data appropriately

• Cell-to-cell variability:

  • Problem: Heterogeneous response in cell populations

  • Solution: Analyze sufficient cell numbers (>30 cells per condition); present distribution of results

• Partial loss of function:

  • Problem: Subtle functional defects may be missed

  • Solution: Use quantitative methods with appropriate statistical power; compare to variants with known effects

• Overexpression artifacts:

  • Problem: Overexpression can lead to mislocalization unrelated to variant effects

  • Solution: Include wild-type controls at similar expression levels; consider inducible systems

• Context dependence:

  • Problem: Effects may vary in different cell types

  • Solution: Validate key findings in multiple cell types, including disease-relevant cells when possible

• Interpretation guidelines:

  • Problem: Translating functional data to clinical significance

  • Solution: Use established frameworks (e.g., ACMG guidelines) and integrate multiple lines of evidence

How can recombinant VPS13B be used to study Cohen Syndrome pathophysiology?

Methodological applications:

• Structure-function analyses:

  • Express recombinant VPS13B constructs containing patient-specific mutations

  • Assess effects on protein localization, stability, and function

  • Compare cellular phenotypes with clinical presentations

• Disease modeling:

  • Introduce VPS13B mutations in cellular models using CRISPR/Cas9

  • Analyze effects on Golgi structure, protein trafficking, and cellular homeostasis

  • Develop phenotypic assays relevant to Cohen Syndrome

• Protein interaction studies:

  • Identify VPS13B binding partners using techniques such as:
    a. Co-immunoprecipitation with recombinant VPS13B
    b. Proximity labeling (BioID, APEX)
    c. Yeast two-hybrid screening with VPS13B domains

  • Determine how disease-causing mutations affect these interactions

• Therapeutic development:

  • Use recombinant VPS13B systems to screen for compounds that:
    a. Stabilize mutant VPS13B proteins
    b. Enhance their correct localization
    c. Bypass the functional defects caused by mutations

• Biomarker identification:

  • Assess downstream effects of VPS13B dysfunction

  • Identify potential biomarkers for disease progression and treatment response

What are the emerging techniques for studying VPS13B protein dynamics and interactions?

Methodological innovations:

• Live-cell imaging approaches:

  • Express VPS13B fused to fluorescent proteins (e.g., GFP, mCherry)

  • Track protein dynamics and localization in real-time

  • Utilize FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

• Proximity labeling techniques:

  • Fuse VPS13B to promiscuous biotin ligases (BioID, TurboID) or peroxidases (APEX)

  • Identify proximal proteins through biotinylation followed by streptavidin pulldown and mass spectrometry

  • Map the spatial organization of VPS13B in specific cellular compartments

• Structural biology approaches:

  • Cryo-electron microscopy of VPS13B protein domains

  • X-ray crystallography of stable VPS13B fragments

  • Molecular dynamics simulations to understand conformational changes

• Protein-lipid interaction assays:

  • Liposome binding assays to assess interaction with membrane lipids

  • Identify lipid binding domains within VPS13B

  • Determine how mutations affect lipid interactions

• Systems biology integration:

  • Transcriptomics and proteomics to identify pathways affected by VPS13B dysfunction

  • Network analysis to place VPS13B in cellular signaling contexts

  • Multi-omics approaches to understand global effects of VPS13B mutations