Recombinant Morus nigra Agglutinin beta-2 chain isoform 2

What is Morus nigra Agglutinin and how does it relate to other plant lectins?

Morus nigra Agglutinin refers to lectins isolated from black mulberry (Morus nigra) that belong to the jacalin-related lectin (JRL) family. These lectins adopt the typical β-prism motif found in other JRLs and form tetrameric assemblies similar to jacalin . Two main variants have been characterized: MornigaM (mannose-specific) and MornigaG (galactose-specific), with the beta chains likely responsible for carbohydrate binding specificity . This follows a pattern similar to type-2 ribosome-inactivating proteins (RIPs) like mistletoe lectins, where the B-chain accounts for carbohydrate binding and immunomodulatory activities while the A-chain provides enzymatic functions .

How does the structure of the beta-2 chain isoform 2 determine its functional properties?

The beta-2 chain isoform 2, as part of the tetrameric assembly, likely contributes significantly to the carbohydrate-binding pocket of the Morus nigra Agglutinin. Based on structural studies of MornigaM, we know that the carbohydrate-binding cavity readily binds mannose without requiring major structural rearrangements upon binding . The specific amino acid composition and three-dimensional arrangement of the binding site determine both binding specificity and affinity. By analogy with other plant lectins, subtle variations in the binding site architecture between different isoforms can significantly alter carbohydrate recognition patterns, explaining the existence of multiple isoforms that may recognize different glycan targets or bind with varying affinities.

What high-resolution techniques are most informative for analyzing the carbohydrate-binding site?

X-ray crystallography has already proven valuable for studying Morus nigra lectins, with structures of MornigaM and the MornigaM-mannose complex determined at 1.8 Å and 2.0 Å resolution, respectively . This approach remains the gold standard for visualizing atomic-level details of the binding site architecture and interactions with carbohydrates. For the beta-2 chain isoform 2, co-crystallization with various glycan structures would provide comprehensive binding site characterization. Complementary techniques include NMR spectroscopy for examining solution dynamics, hydrogen-deuterium exchange mass spectrometry for identifying flexible regions, and computational approaches like molecular dynamics simulations to model conformational changes upon glycan binding.

How do mutations in the binding site affect carbohydrate specificity?

While specific data for the beta-2 chain isoform 2 is not available in the search results, structure-function relationships can be inferred from other lectins. In related lectins, even single amino acid substitutions in the binding pocket can dramatically alter carbohydrate specificity. For instance, the binding specificity difference between mannose-specific and galactose-specific lectins often hinges on key residues that form hydrogen bonds with the axial (mannose) or equatorial (galactose) hydroxyl groups at the C2 position. Systematic site-directed mutagenesis studies targeting residues in the binding site would be invaluable for mapping the structural determinants of specificity in the beta-2 chain isoform 2 and potentially engineering variants with altered binding profiles.

What computational approaches are useful for predicting glycan binding interactions?

Molecular docking simulations can predict binding modes and energetics for different glycans with the beta-2 chain. Molecular dynamics simulations can further refine these predictions by accounting for protein flexibility and solvent effects. More advanced approaches include quantum mechanics/molecular mechanics (QM/MM) calculations for more accurate modeling of hydrogen bonding networks and CH-π interactions that are common in protein-carbohydrate recognition. Machine learning approaches trained on glycan array data from related lectins could also help predict binding specificities. These computational methods are particularly valuable for designing experiments, interpreting structural data, and developing hypotheses about binding specificity differences between isoforms.

How can the carbohydrate binding specificity be comprehensively profiled?

The most comprehensive approach to characterizing binding specificity is glycan array screening, which can test binding against hundreds of structurally diverse glycans simultaneously. This should be complemented with quantitative affinity measurements using techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) for key glycan structures. Cell-based binding assays using flow cytometry can confirm the ability to recognize relevant glycans in their native cellular context. For the beta-2 chain isoform 2, comparing its glycan binding profile with other Morus nigra lectin isoforms would be particularly informative. The results should be analyzed in the context of what is known about related lectins, such as MornigaM's mannose specificity and the preference of mistletoe lectins for terminally α2-6-sialylated neolacto-series gangliosides .

What immunomodulatory activities has Morus nigra Agglutinin demonstrated?

While specific immunomodulatory activities of the beta-2 chain isoform 2 are not detailed in the search results, extracts from Morus nigra have demonstrated significant anti-inflammatory properties. For example, Morus nigra leaf extract inhibited carrageenan-induced paw edema with an IC50 of 15.2 mg/kg and reduced granulomatous tissue formation with an IC50 of 71.1 mg/kg . Compounds isolated from the stem bark (mornigrol D and norartocarpetin) showed potent anti-inflammatory activity, inhibiting β-glucuronidase release from rat polymorphonuclear leukocytes by 65.9% and 67.7%, respectively, at 10^-5 M concentration . By analogy with mistletoe lectins, where the B-chain accounts for immunomodulatory activities through mechanisms like augmented IL-12 production and increased NK cell activation , the beta-2 chain may contribute to similar pathways.

How does the beta-2 chain interact with cell surface receptors?

The specific cell surface interactions of the beta-2 chain isoform 2 would need experimental determination, but insights can be drawn from related lectins. Mistletoe lectins recognize CD75s, which has glycans with terminal α2-6-sialyl-lactosamine and is expressed on activated B-cells, T-cells, and immature dendritic cells . The Korean mistletoe lectin interacts with TLR-4 molecules, leading to macrophage activation and cytokine production . Similar receptor interactions might be expected for the beta-2 chain of Morus nigra Agglutinin. To identify relevant receptors, techniques such as glycoproteomic analysis of pull-down fractions, flow cytometry with receptor-specific antibodies, and cell-based binding assays with receptor knockout lines would be valuable experimental approaches.

What are the most reliable activity assays for the recombinant beta-2 chain?

A multi-faceted approach to activity assessment is recommended:

• Glycan binding assays:

  • Enzyme-linked lectin assays (ELLA)

  • Glycan array screening

  • Isothermal titration calorimetry

  • Surface plasmon resonance

• Cell-based assays:

  • Flow cytometry to measure binding to cell surface glycans

  • Cell agglutination assays if the recombinant protein forms multimers

• Functional immunological assays:

  • Cytokine induction in peripheral blood mononuclear cells (PBMCs)

  • NK cell activation assays

  • Dendritic cell maturation assays

Each assay provides complementary information, with glycan binding assays confirming molecular recognition properties while cell-based and immunological assays validate functional relevance. Appropriate controls including heat-inactivated protein and competitive inhibition with specific sugars should be included to confirm specificity.

How should researchers design experiments to investigate immunomodulatory effects?

Investigating immunomodulatory effects requires a systematic approach:

For in vitro studies, dose-response relationships and time-course analyses are essential. When investigating anti-inflammatory properties, researchers should consider both acute models (like carrageenan-induced paw edema) and chronic models (like cotton pellet-induced granuloma), similar to studies with Morus nigra leaf extracts . Mechanistic studies should explore whether the beta-2 chain affects the NF-κB pathway and nitric oxide production, which are linked to the anti-inflammatory effects observed with Morus nigra flavonoids .

What approaches can distinguish the activities of the beta-2 chain from other isoforms?

Distinguishing the specific activities of the beta-2 chain isoform 2 requires:

• Comparative recombinant expression: Produce multiple isoforms under identical conditions to enable direct comparison.

• Isoform-specific antibodies: Develop antibodies that can specifically recognize the beta-2 chain isoform 2 for immunolocalization and neutralization studies.

• Chimeric constructs: Create chimeric proteins by swapping domains between isoforms to map functional regions.

• Comparative glycan array profiling: Identify unique glycan recognition patterns that differentiate this isoform.

• Competitive binding studies: Determine whether different isoforms compete for the same cellular receptors.

• Domain-specific mutagenesis: Target mutations to regions that differ between isoforms to identify functional hotspots.

These approaches, particularly when used in combination, can help establish the unique functional profile of the beta-2 chain isoform 2 relative to other Morus nigra lectin variants.

How can the beta-2 chain be utilized in glycobiology research?

The beta-2 chain has several valuable applications in glycobiology research:

• Glycan structure detection: As a reagent for detecting specific glycan structures in tissues, cells, or on purified proteins.

• Affinity purification: For isolating glycoproteins bearing its target glycans from complex mixtures.

• Cell type identification: For characterizing cells based on their surface glycosylation patterns.

• Glycodynamics studies: For tracking changes in glycosylation during development, differentiation, or disease progression.

• Comparative glycomics: For comparing glycosylation patterns across species or between normal and pathological states.

The specificity of the beta-2 chain for particular glycan structures makes it a valuable tool for these applications, complementing other analytical techniques like mass spectrometry and enabling visualization of glycans in their native cellular context.

What is the potential of the beta-2 chain in immunology research?

Based on the immunomodulatory properties of related lectins, the beta-2 chain has significant potential in immunology research:

• Immune cell activation studies: For investigating mechanisms of innate immune activation, particularly if it interacts with pattern recognition receptors like TLR-4, as observed with Korean mistletoe lectin .

• Cytokine modulation research: For studying how glycan recognition influences cytokine production patterns, similar to how Morus nigra flavonoids reduce IL-6 and increase IL-10 .

• Anti-inflammatory mechanism investigation: For exploring novel anti-inflammatory pathways, building on the observed anti-inflammatory effects of Morus nigra compounds .

• Cancer immunology: For studying how lectin-glycan interactions might enhance anti-tumor immune responses, similar to mistletoe lectins that stimulate Th1 cells which may mediate anti-tumor T-cell responses .

• Tolerance and autoimmunity research: For investigating how glycan recognition might influence self/non-self discrimination, similar to how ricin B-chain-proinsulin fusion protein suppressed autoimmune insulitis in NOD mice .

These applications could provide insights into novel immunomodulatory mechanisms and potentially inform therapeutic approaches.

What are the challenges in translating in vitro findings to in vivo models?

Several important challenges must be addressed when translating findings from in vitro to in vivo systems:

• Bioavailability and biodistribution: Understanding how the recombinant protein is distributed in vivo and whether it reaches relevant target tissues.

• Stability and half-life: Determining protein stability in physiological conditions and its circulation time.

• Immune recognition: Assessing whether the recombinant protein itself triggers immune responses that could confound interpretation.

• Dose translation: Establishing appropriate dosing regimens that correlate with effective concentrations from in vitro studies.

• Species-specific glycan differences: Accounting for differences in glycosylation patterns between humans and model organisms.

• Complex microenvironments: Considering how the tissue microenvironment might affect protein activity compared to simplified in vitro systems.

• Multiple cell type interactions: Assessing effects on complex cellular networks rather than isolated cell populations.

Careful experimental design, including appropriate controls and dose-response studies, is essential for meaningful translation between in vitro and in vivo findings.

How do post-translational modifications affect the activity of recombinant versus native beta-2 chain?

The impact of post-translational modifications on the beta-2 chain represents an important research consideration. While specific data for this protein is not available in the search results, lessons from other plant lectins suggest several key points:

• Glycosylation status: If the native beta-2 chain is glycosylated, recombinant versions produced in bacterial systems will lack these modifications, potentially affecting folding, stability, or activity.

• Disulfide bond formation: Proper disulfide bond formation is critical for many lectins' structural integrity and is dependent on the expression system's oxidizing environment.

• Proteolytic processing: Some plant lectins undergo specific proteolytic processing that may be important for activity and might not occur correctly in heterologous expression systems.

• Comparative analysis approaches: Researchers should compare recombinant and native proteins using multiple methods including circular dichroism spectroscopy (for secondary structure), thermal stability assays, and side-by-side functional comparisons.

Understanding these differences is crucial for interpreting results obtained with recombinant proteins and may explain discrepancies between studies using proteins from different sources.

How can researchers address conflicting data on immunomodulatory activities?

When confronted with conflicting data regarding immunomodulatory activities, researchers should:

• Standardize protein preparations: Ensure consistent protein quality, purity, and endotoxin levels, as contamination can significantly confound immunological studies.

• Account for isoform differences: Clearly identify which specific isoform (e.g., beta-2 chain isoform 2 versus other variants) was used in each study.

• Consider experimental context: Variations in cell types, culture conditions, and assay readouts can lead to apparently contradictory results.

• Evaluate dose-response relationships: Immunomodulatory proteins often show bell-shaped dose-response curves, with different activities at different concentrations.

• Examine temporal dynamics: The timing of measurements can significantly affect results, particularly for transient responses.

• Perform multi-laboratory validation: Collaborative studies using identical protocols and reagents can help resolve discrepancies.

• Integrate multiple readouts: Combining measures of different aspects of immune function provides a more comprehensive picture than single readouts.

This systematic approach can help reconcile apparently conflicting findings and build a more coherent understanding of the protein's activities.

What novel techniques might advance understanding of structure-function relationships?

Several cutting-edge approaches could provide new insights into structure-function relationships:

• Cryo-electron microscopy: For visualizing protein-glycan complexes without crystallization, potentially capturing more natural conformations.

• Single-molecule techniques: Including atomic force microscopy and optical tweezers to study binding dynamics at the individual molecule level.

• Hydrogen-deuterium exchange mass spectrometry: For mapping conformational changes and binding interfaces with high resolution.

• Native mass spectrometry: For analyzing intact protein-glycan complexes and determining stoichiometry.

• CRISPR-based glycoengineering: For creating cell lines with specific glycosylation patterns to test binding specificity in cellular contexts.

• AI-powered structural prediction: Using tools like AlphaFold2 to predict structures of variant isoforms and their complexes with glycans.

• In-cell NMR: For studying lectin-glycan interactions in the cellular environment.

• Glycan and protein microarrays: For high-throughput analysis of binding specificity across multiple protein variants and glycan structures.

These advanced techniques, when combined with traditional approaches, can provide unprecedented resolution of the molecular mechanisms underlying the beta-2 chain's functional properties.