Gly m 4.0101

What is Gly m 4.0101 and what is its biological significance?

Gly m 4.0101 is a soybean protein isolated from Glycine max that belongs to the Bet v 1 homologue family. It is also known as starvation-associated message 22 (SAM 22) or stress-induced protein SAM22, as its expression increases under stress conditions such as starvation . Biologically, it serves as a pathogenesis-related protein involved in plant defense mechanisms. As a food allergen, Gly m 4 (including its variant Gly m 4.0101) is one of the most clinically significant allergens from soybeans, alongside other major allergens like Gly m 8 . Unlike many other food Bet v 1 homologues that typically cause mild local symptoms, Gly m 4 can induce severe systemic allergic reactions, making it a marker allergen for severe food-allergic reactions to soy .

How does Gly m 4.0101 relate to birch pollen allergies?

Gly m 4.0101 is a homologue of the birch pollen allergen Bet v 1, which is a primary sensitizer responsible for the development of pollen and food allergic cross-reactions . The structural similarities between these proteins explain the clinical observation of cross-reactivity, where individuals sensitized to birch pollen may experience allergic reactions when consuming soy products. This phenomenon is part of the broader birch-fruit-vegetable-nut-legume syndrome. Research indicates that while most Bet v 1 homologues in foods cause only mild oral allergy syndrome in Bet v 1-sensitized individuals, Gly m 4 has the potential to trigger severe systemic reactions . Understanding this relationship is crucial for comprehending the immunological mechanisms underlying these cross-reactive allergic responses.

What are the key structural features of Gly m 4.0101?

Gly m 4.0101, like other Bet v 1 homologues, possesses distinct structural characteristics that contribute to its allergenicity:

• It contains a large internal hydrophobic cavity capable of accommodating various ligands .

• The protein maintains a conserved fold characteristic of the PR-10 protein family (pathogenesis-related proteins).

• The tertiary structure includes a seven-stranded anti-parallel β-sheet and three α-helices.

These structural features are critical for its biological function and allergenic properties. The hydrophobic cavity, in particular, plays a significant role in ligand binding, which may affect stability, proteolytic resistance, and immunogenicity of the protein . Researchers should note that structural modifications, either through ligand binding or processing-induced changes, might alter the allergenicity profile of Gly m 4.0101.

How can I investigate ligand binding to Gly m 4.0101?

The investigation of ligand binding to Gly m 4.0101 can be approached using multiple complementary techniques:

• Fluorescence spectroscopy: Employ fluorescent probes like TNS (6-(p-toluidino)-2-naphthalenesulfonic acid) to study ligand binding. The research methodology involves:

  • Measuring base-line fluorescence of TNS (typically at 4 μM) in phosphate buffer

  • Recording emission spectra from 330 to 550 nm after excitation at 320 nm

  • Titrating the ligand of interest (e.g., quercetin-3,4′-diglucoside) into the protein solution

  • Subtracting contributions of buffer, protein, and ligand to isolate binding-specific signals

• Molecular docking: In silico approaches provide valuable insights into binding mechanisms:

  • Prepare protein and ligand structures using tools like UCSF Chimera's DockPrep

  • Perform blind docking using algorithms such as AutoDock Vina

  • Analyze binding poses and energetics to identify potential interaction sites

  • Visualize complex structures with programs like Discovery Studio Visualizer

• Functional assays: Assess how ligand binding affects protein properties:

  • Evaluate changes in proteolytic susceptibility through digestion assays

  • Examine alterations in epithelial transport rates in the presence of ligands

  • Investigate modifications in immunological responses in cell culture models

These methodologies provide comprehensive insights into the binding mechanisms and the functional consequences of Gly m 4.0101-ligand interactions.

What expression systems are most effective for producing recombinant Gly m 4.0101?

For producing recombinant Gly m 4.0101, E. coli expression systems have proven effective. The methodology involves:

• Vector design: Construct a recombinant plasmid (e.g., pET-His8-TrxL-Gly m 4) containing:

  • T7 promoter

  • Ribosome binding site

  • lac-operator

  • A fusion tag sequence (e.g., octahistidine tag)

  • A carrier protein like TrxL to enhance solubility

  • The Gly m 4.0101 coding sequence

• Expression conditions: Optimize parameters such as:

  • IPTG concentration for induction

  • Temperature during induction (often lower temperatures like 16-25°C improve soluble protein yield)

  • Duration of induction

  • Media composition

• Purification strategy: Implement a multi-step purification process:

  • Initial capture using immobilized metal affinity chromatography (IMAC) via the His-tag

  • Optional carrier protein removal by protease cleavage

  • Polishing steps using size exclusion or ion exchange chromatography

  • Quality control through SDS-PAGE, mass spectrometry, and circular dichroism

These approaches enable the production of high-quality recombinant Gly m 4.0101 suitable for structural, functional, and immunological studies.

How can I prepare fluorescently labeled Gly m 4.0101 for transport studies?

Preparation of fluorescently labeled Gly m 4.0101 for transport studies requires careful conjugation chemistry to maintain protein functionality. A recommended protocol based on research practices is:

• Labeling procedure:

  • Reconstitute 1.5 mg of purified recombinant Gly m 4.0101 in 50 μL of DMSO

  • Add to 300 μL of coupling buffer (0.1 M sodium carbonate, 0.1 M sodium bicarbonate, pH 9.6)

  • Add 2.9 mg of fluorescein isothiocyanate isomer I (FITC) in 100 μL of DMSO

  • Conduct the coupling reaction for 2 hours at 20°C in the dark

• Purification of labeled protein:

  • Load the reaction mixture onto a PD10 gel-filtration column pre-equilibrated with distilled water

  • Collect fractions and assess protein content and labeling efficiency

  • Confirm labeling through spectrophotometric analysis and calculate the dye-to-protein ratio

• Validation of labeled protein functionality:

  • Verify that labeling does not significantly alter protein structure using circular dichroism

  • Confirm that allergenic epitopes remain intact through immunoassays

  • Test for maintained ligand-binding capacity if relevant to the study

This labeled protein can then be used in epithelial transport studies, allowing for sensitive detection of protein movement across cellular barriers.

What methodologies are recommended for studying Gly m 4.0101 transport across intestinal epithelium?

The investigation of Gly m 4.0101 transport across intestinal epithelium typically employs Caco-2 cell monolayers as a model system. The recommended methodology includes:

• Cell culture preparation:

  • Grow Caco-2 cells on permeable supports (e.g., Transwell inserts) for 21 days to allow differentiation

  • Monitor transepithelial electrical resistance (TEER) to confirm monolayer integrity (values >250 Ω·cm² indicate a well-formed barrier)

  • Use inserts with appropriate pore size (typically 0.4 μm) and membrane material

• Transport studies:

  • For apical-to-basolateral transport: Add 0.4 mL of 2 μM FITC-labeled Gly m 4.0101 (with or without ligands) to the apical compartment and 0.7 mL of transport buffer to the basolateral side

  • For basolateral-to-apical transport: Add 0.7 mL of 2 μM FITC-labeled Gly m 4.0101 to the basolateral compartment and 0.4 mL of transport buffer to the apical side

  • Conduct transport experiments at 37°C for 90 minutes

  • Collect samples from the receiver compartment at defined intervals

• Data analysis:

  • Calculate the apparent permeability coefficient (Papp) using the equation:
    Papp = (V/(A × Ci)) × ΔC/Δt
    where V is the volume of the acceptor chamber, A is the area of the membrane insert, Ci is the initial concentration of Gly m 4.0101, and ΔC/Δt is the solute flux across the barrier

  • Determine uptake ratios (UR = Papp(A→B)/Papp(B→A)) and efflux ratios (ER = Papp(B→A)/Papp(A→B))

  • Verify monolayer integrity by measuring TEER before and after the experiment

These methodologies provide quantitative assessment of Gly m 4.0101 transport rates and mechanisms, essential for understanding how this allergen crosses intestinal barriers.

How can I evaluate the immunological responses to Gly m 4.0101 in cell culture models?

Evaluating immunological responses to Gly m 4.0101 requires sophisticated cell culture models that replicate aspects of the intestinal immune system. The following methodological approach is recommended:

• Co-culture system establishment:

  • Develop a Caco-2/immune cell co-culture model incorporating relevant immune cell types (dendritic cells, T cells, etc.)

  • Use Transwell systems to separate epithelial cells from immune cells, or more advanced 3D culture systems

  • Ensure physiologically relevant cell ratios and culture conditions

• Stimulation protocol:

  • Expose the apical side of the epithelial layer to Gly m 4.0101 at various concentrations

  • Include appropriate controls (untreated, LPS positive control, etc.)

  • For transport and immune response studies, consider using Gly m 4.0101 with and without potential ligands

  • Examine the effects of intact Gly m 4.0101 versus its proteolytic fragments

• Response assessment:

  • Measure cytokine production using multiplex technology (e.g., xMAP) to quantify multiple cytokines simultaneously

  • Focus on Th2-polarizing cytokines (IL-4, IL-5, IL-10, IL-13) relevant to allergic responses

  • Assess dendritic cell maturation markers and T cell activation

  • Evaluate epithelial barrier function in response to allergen exposure

This approach provides comprehensive insights into how Gly m 4.0101 interacts with the intestinal immune system, potentially leading to sensitization or allergic responses.

What protocols should be used to investigate Gly m 4.0101 susceptibility to gastrointestinal digestion?

Investigation of Gly m 4.0101 digestibility requires simulation of gastrointestinal conditions through a sequential in vitro digestion protocol:

• Gastric phase simulation:

  • Prepare Gly m 4.0101 at a final concentration of 0.05 mM in 0.05 M HCl, pH 2.0

  • Add pepsin at a ratio of 50 ng (0.1 U) per 1 μg of protein

  • Incubate the mixture at 37°C for 2 hours with gentle agitation

  • Take aliquots at different time points (0, 15, 30, 60, 120 min) to monitor digestion kinetics

• Intestinal phase simulation:

  • Adjust the pH of the gastric digestion mixture to 8.0 using ammonium bicarbonate

  • Add trypsin (2.5 ng, 0.03 U per 1 μg of substrate) and α-chymotrypsin (10 ng, 0.4 × 10⁻³ U per 1 μg of substrate)

  • Incubate at 37°C for an additional 2 hours

  • Sample at various timepoints throughout the intestinal phase

• Analysis of digestion products:

  • Monitor proteolysis using SDS-PAGE to visualize protein fragmentation patterns

  • Identify specific peptide fragments using mass spectrometry (LC-MS/MS)

  • Assess the effect of ligand binding on proteolytic susceptibility by preincubating Gly m 4.0101 with ligands (e.g., quercetin-3,4′-diglucoside at a 1:4 protein-to-ligand molar ratio)

  • Evaluate the transport and immunological properties of identified resistant peptides

This methodological approach provides insights into the digestive stability of Gly m 4.0101 and helps identify potentially allergenic peptide fragments that might survive digestion and contribute to sensitization or allergic reactions.

How do processing methods affect Gly m 4.0101 allergenicity in soy products?

The effects of processing methods on Gly m 4.0101 allergenicity require systematic evaluation using the following research approach:

• Processing simulation:

  • Apply various processing techniques to soy materials:

    • Thermal processing (boiling, roasting, autoclaving)

    • Fermentation (traditional and controlled)

    • High-pressure processing

    • Extrusion

    • Enzymatic hydrolysis

  • Process under controlled conditions with precise documentation of parameters

• Allergen quantification:

  • Extract proteins from processed samples using appropriate buffers

  • Implement LC-MS/MS quantification methods using signature peptides (e.g., NPFLFGSNR for Gly m 5.0101)

  • Develop and validate similar quantification methods for Gly m 4.0101

  • Compare allergen content across differently processed samples

• Functional allergenicity assessment:

  • Evaluate IgE-binding capacity of processed samples using sera from allergic patients

  • Assess structural modifications through spectroscopic techniques

  • Examine digestibility changes using the in vitro digestion protocol

  • Test immunological responses in cell culture models

This comprehensive approach helps determine which processing methods might reduce Gly m 4.0101 allergenicity while maintaining the nutritional quality of soy products, providing valuable information for both research and potential clinical applications.

What methodologies are recommended for predicting cross-reactivity between Gly m 4.0101 and other allergens?

Prediction of cross-reactivity between Gly m 4.0101 and other allergens requires a multi-faceted approach combining computational and experimental methods:

• Sequence-based analysis:

  • Perform multiple sequence alignment of Gly m 4.0101 with potential cross-reactive allergens

  • Calculate sequence identity and similarity percentages

  • Apply the A-RISC (Allergen-Relative Identity, Similarity and Cross-reactivity) index methodology:

    • A-RISC = (SI + (SS-SI)/2)/100
      where SI is sequence identity and SS is sequence similarity

  • Generate similarity matrices and visualization maps to identify potential cross-reactivity clusters

• Structural analysis:

  • Compare 3D structures using structural alignment tools

  • Map similar/identical residues onto molecular surfaces

  • Calculate solvent-accessible surface areas of conserved regions

  • Identify conserved epitopes using computational epitope prediction tools

• Epitope-focused approaches:

  • Identify potential B-cell and T-cell epitopes

  • Assess epitope conservation across allergens

  • Evaluate surface exposure of conserved epitopes

  • Consider the spatial distribution of conserved residues

The A-RISC methodology has been shown to be particularly useful, as it provides a quantitative index that correlates well with observed patterns of cross-reactivity. Values above 0.75 suggest a high risk of cross-reactivity, 0.5-0.75 indicate medium-high risk, 0.25-0.5 suggest medium-low risk, and values below 0.25 represent low risk .

How can I experimentally validate predicted cross-reactivity between Gly m 4.0101 and other allergens?

Experimental validation of predicted cross-reactivity requires multiple complementary approaches:

• Immunological methods:

  • Conduct inhibition ELISA experiments:

    • Pre-incubate patient sera with varying concentrations of soluble Gly m 4.0101

    • Test the inhibited sera against immobilized potentially cross-reactive allergens

    • Calculate IC50 values to quantify cross-reactivity

  • Perform IgE immunoblotting with sera from patients with known allergies

  • Use basophil activation tests (BAT) to assess functional cross-reactivity

• Epitope mapping:

  • Generate overlapping peptides spanning the sequence of Gly m 4.0101

  • Test IgE binding to these peptides

  • Compare binding patterns with peptides derived from potentially cross-reactive allergens

  • Consider using phage display or peptide microarrays for high-throughput analysis

• Advanced structural studies:

  • Use X-ray crystallography or NMR to determine structures in complex with antibodies

  • Apply hydrogen-deuterium exchange mass spectrometry to identify binding regions

  • Employ molecular dynamics simulations to investigate structural dynamics and epitope accessibility

• In vivo models:

  • Develop animal models sensitized to Gly m 4.0101

  • Challenge with potentially cross-reactive allergens

  • Assess immune responses and clinical manifestations

  • Consider humanized mouse models for more relevant results

These experimental approaches provide robust validation of computational predictions and advance our understanding of the molecular basis for cross-reactivity between Gly m 4.0101 and other allergens, especially within the broader Bet v 1 homologue family.

What are the optimal approaches for quantifying Gly m 4.0101 in soybean samples?

Accurate quantification of Gly m 4.0101 in soybean samples can be achieved using mass spectrometry-based approaches. The following methodology is recommended, adapted from successful approaches used for other soy allergens:

• Sample preparation:

  • Grind soybean samples to a fine powder

  • Defat samples using hexane or similar solvent

  • Extract proteins using an appropriate buffer (e.g., phosphate buffer with detergents)

  • Filter and centrifuge to obtain a clear protein extract

• Protein digestion:

  • Implement on-filter digestion using trypsin:

    • Apply protein extract to filter units

    • Wash with appropriate buffers

    • Add trypsin (typically 1:50 enzyme:protein ratio)

    • Incubate for 16-18 hours at 37°C

    • Collect peptides by centrifugation

• LC-MS/MS analysis:

  • Develop a multiple reaction monitoring (MRM) method targeting specific peptides unique to Gly m 4.0101

  • Select reliable signature peptides showing good chromatographic behavior and reproducible fragmentation

  • Use synthetic peptide standards for absolute quantification

  • Establish a calibration curve using the synthetic peptide standards

• Method validation:

  • Determine the limit of detection (LOD) and limit of quantification (LOQ)

  • Establish linearity range (e.g., 1.6-500 ng/mL as demonstrated for Gly m 5.0101)

  • Validate precision (intra- and inter-day)

  • Assess accuracy through spike-recovery experiments

  • Ensure specificity by analyzing samples with known allergen content

This LC-MS/MS approach provides sensitive and specific quantification of Gly m 4.0101 in various soybean samples and products, enabling comparative studies across varieties and processing conditions.

How can I analyze Gly m 4.0101 levels in processed food products?

Analysis of Gly m 4.0101 in processed food products presents unique challenges due to matrix effects and protein modifications. The following comprehensive approach is recommended:

• Optimized extraction protocols:

  • Develop matrix-specific extraction buffers (considering fat content, pH, etc.)

  • Include denaturants (urea, SDS) to enhance extraction of modified proteins

  • Consider extraction additives that prevent protein-matrix interactions

  • Implement sequential extraction procedures for complex matrices

• Sample clean-up strategies:

  • Apply solid-phase extraction (SPE) to remove interfering compounds

  • Consider immunoaffinity clean-up using anti-Gly m 4 antibodies

  • Implement protein precipitation or ultrafiltration steps as needed

  • Use two-dimensional clean-up approaches for highly complex matrices

• LC-MS/MS quantification:

  • Adapt the MRM method developed for soybean samples

  • Include matrix-matched calibration curves

  • Add isotopically labeled peptide standards as internal references

  • Monitor multiple peptides per protein to account for processing-induced modifications

• Method performance assessment:

  • Validate method in different food matrices (dairy, baked goods, meat products, etc.)

  • Determine matrix-specific detection limits

  • Assess recovery rates in processed foods

  • Compare results with immunological methods to identify processing-induced epitope modifications

This comprehensive approach enables reliable quantification of Gly m 4.0101 in diverse food products, supporting allergen risk assessment and management in food production.

What are the key unresolved questions in Gly m 4.0101 research?

Several critical questions remain unresolved in Gly m 4.0101 research that warrant further investigation:

• Sensitization mechanisms:

  • What are the precise mechanisms by which Gly m 4.0101 acts as a sensitizer rather than just an elicitor?

  • How do intact protein versus digestion fragments contribute to sensitization?

  • What is the role of the microbiome in modulating responses to Gly m 4.0101?

• Structure-function relationships:

  • How do natural ligands of Gly m 4.0101 modify its allergenic properties?

  • What structural elements distinguish it from less allergenic Bet v 1 homologues?

  • How do post-translational modifications affect allergenicity?

• Cross-reactivity thresholds:

  • What are the minimal structural requirements for cross-reactivity with Bet v 1?

  • Can quantitative thresholds for sequence and structural similarity predict clinical cross-reactivity?

  • How do cross-reactive epitopes evolve in different plant species?

• Population-specific responses:

  • Are there genetic factors predisposing certain individuals to Gly m 4.0101 sensitization?

  • How do environmental factors influence sensitivity to this allergen?

  • Are there geographic or demographic variations in response patterns?

Addressing these questions will require integrative approaches combining structural biology, immunology, genetics, and clinical research to advance our understanding of this important food allergen.

What novel methodologies show promise for advancing Gly m 4.0101 research?

Several emerging methodologies hold significant promise for advancing Gly m 4.0101 research:

• Single-cell technologies:

  • Single-cell RNA sequencing to identify specific immune cell populations responding to Gly m 4.0101

  • CyTOF or spectral flow cytometry for high-dimensional phenotyping of allergic responses

  • Single-cell proteomics to characterize cell-specific signaling pathways

• Advanced structural biology approaches:

  • Cryo-electron microscopy for visualizing allergen-antibody complexes

  • Hydrogen-deuterium exchange mass spectrometry for mapping conformational epitopes

  • AlphaFold and other AI-driven structure prediction tools for modeling variant structures

• Organoid and advanced tissue models:

  • Intestinal organoids incorporating epithelial and immune components

  • Organ-on-chip technologies reproducing mucosal barrier functions

  • 3D bioprinted tissues mimicking the intestinal immune system

• Computational immunology tools:

  • Improved epitope prediction algorithms incorporating T cell receptor recognition patterns

  • Network analysis approaches to understand allergen cross-reactivity

  • Systems biology models of allergic sensitization pathways

• CRISPR-based approaches:

  • Gene editing of soybean to create hypoallergenic variants with modified Gly m 4.0101

  • Cellular models with specific receptor knockouts to elucidate mechanism

  • In vivo models with humanized immune components

These emerging methodologies promise to overcome current technical limitations and provide deeper insights into the molecular and cellular mechanisms underlying Gly m 4.0101 allergenicity and cross-reactivity.