LECT1 (214-333) Human

What is the structural composition of LECT1 (214-333) human protein?

LECT1 (214-333) represents a specific fragment of the human Leukocyte Cell-Derived Chemotaxin-1 protein, corresponding to amino acids 214-333 of the full-length protein. This fragment is part of the C-terminal region that contains the 25 kDa mature protein known as chondromodulin-1. The sequence contains important structural elements including cysteine residues that participate in disulfide bonding, which is critical for the protein's biological activity. When examining the amino acid sequence (e.g., MGSSHHHHHH SSGLVPRGSH MGSREVVRKI VPTTTKRPHS GPRSNPGAGR LNNETRPSVQ EDSQAFNPDN PYHQEGESMT FDPRLDHEGI CCIECRRSYT HCQKICEPLG GYYPWPYNYQ GCRSACRVIM PCSWWVARIL GMV for the His-tagged version), researchers should note the characteristic domains that contribute to its functional properties in cartilage development .

How do different expression systems affect the production of recombinant LECT1 (214-333)?

The choice of expression system significantly impacts the structural and functional properties of recombinant LECT1 (214-333). Two primary systems have been documented in the literature:

For studies requiring properly glycosylated protein with native conformation, mammalian expression in HEK293 cells is preferable despite lower yields. When investigating structure-function relationships where glycosylation is not critical, E. coli-expressed protein may be sufficient, though refolding protocols should be carefully optimized to ensure proper disulfide bond formation .

What are the optimal storage conditions for maintaining LECT1 (214-333) stability?

Maintaining stability of purified LECT1 (214-333) requires careful consideration of storage conditions. Lyophilized preparations demonstrate significantly greater long-term stability compared to solutions. For optimal results, store lyophilized LECT1 (214-333) at -20°C to -80°C, where it remains stable for up to 12 months. When working with reconstituted protein, short-term storage (2-7 days) at 4-8°C is acceptable, but for periods exceeding one week, the protein should be aliquoted and stored at -20°C or colder to prevent freeze-thaw cycles that accelerate degradation. The addition of stabilizing agents such as 10% glycerol can improve protein stability by preventing aggregation during freeze-thaw cycles. When preparing working solutions, appropriate buffer systems (typically sterile PBS at pH 7.4) should be used, and the reconstituted protein should be handled with care to minimize contamination .

How can researchers effectively investigate LECT1 (214-333)'s anti-angiogenic properties?

Investigating the anti-angiogenic properties of LECT1 (214-333) requires methodological approaches that assess both direct and indirect effects on vascular cells. A comprehensive experimental design should include:

• Endothelial cell assays: Conduct tube formation assays using human umbilical vein endothelial cells (HUVECs) on Matrigel with varying concentrations of LECT1 (214-333) (typically 1-100 ng/mL). Quantify tube formation by measuring branch points, total tube length, and mesh formation at 6, 12, and 24-hour timepoints.

• Migration/invasion studies: Perform transwell migration assays with a gradient of angiogenic factors (VEGF, bFGF) in the presence/absence of LECT1 (214-333) to assess the protein's ability to inhibit endothelial cell chemotaxis.

• Ex vivo models: Utilize mouse aortic ring assays where vessel sprouting can be visualized and quantified in the presence of LECT1, providing a more physiologically relevant model than cell lines alone.

• In vivo models: Consider chick chorioallantoic membrane (CAM) assays or mouse Matrigel plug assays to evaluate anti-angiogenic activity in complete biological systems.

The experimental design should include appropriate controls, including both positive (known anti-angiogenic compounds) and negative controls, as well as comparison with other LECT1 domains to determine region-specific effects .

What methodologies are most effective for studying LECT1 (214-333)'s role in chondrocyte development?

To investigate LECT1 (214-333)'s role in chondrocyte development, researchers should employ a multi-faceted approach that combines in vitro culture systems with molecular and cellular analyses:

• Primary chondrocyte cultures: Isolate primary chondrocytes from growth plate cartilage and treat with recombinant LECT1 (214-333) at various developmental stages. Monitor proliferation rates using BrdU incorporation assays and assess differentiation markers through quantitative PCR for genes including Sox9, Col2a1, and Aggrecan.

• Micromass cultures: Establish high-density micromass cultures from mesenchymal cells to recapitulate early cartilage formation. Supplement with LECT1 (214-333) (5-50 ng/mL) and analyze changes in nodule formation, glycosaminoglycan production (Alcian blue staining), and expression of chondrogenic markers.

• Explant cultures: Use cartilage explants from developing long bones to maintain tissue architecture and cell-matrix interactions. Culture in the presence of LECT1 (214-333) and analyze histologically for changes in chondrocyte organization, proliferation zones, and hypertrophic differentiation.

• Signal transduction analysis: Investigate the molecular pathways activated by LECT1 (214-333) through phosphorylation studies of relevant signaling molecules (Smads, MAPKs) and use of pathway inhibitors to determine mechanism of action.

These methodologies should be adapted based on specific research questions, with careful consideration of time points that correspond to critical developmental transitions in chondrocyte maturation .

How does tag selection affect the biological activity of recombinant LECT1 (214-333)?

The choice of fusion tag significantly impacts both the purification efficiency and biological activity of recombinant LECT1 (214-333). Researchers must carefully consider these effects when designing experiments:

For studies requiring native-like activity, tag removal using specific proteases (TEV, thrombin, etc.) should be considered, though this introduces additional purification steps and potential for activity loss. When comparing results across studies, researchers should account for the potential confounding effects of different tags. Validation experiments comparing tagged and untagged versions are recommended to establish the impact of the tag on specific biological activities being investigated .

How can researchers address contradictory findings regarding LECT1's role in endochondral ossification?

Contradictory findings regarding LECT1's role in endochondral ossification likely stem from context-dependent functions of the protein. To address these discrepancies, researchers should implement the following methodological approaches:

• Developmental timing studies: Carefully control the developmental stage at which LECT1 (214-333) is introduced or inhibited, as its effects may vary between early chondrogenesis and terminal differentiation phases. Utilize inducible expression systems or timed administration of recombinant protein in developmental models.

• Concentration-dependent studies: Perform detailed dose-response experiments across a wide concentration range (0.1-1000 ng/mL) to identify potential biphasic effects, where low and high concentrations may have opposing actions.

• Microenvironmental considerations: Systematically vary culture conditions (oxygen tension, matrix composition, co-cultured cell types) to identify factors that may switch LECT1's activity from pro-chondrogenic to anti-angiogenic or vice versa.

• Comparative analysis of fragments: Compare the activity of LECT1 (214-333) with other domains of the protein and the full-length molecule to determine if contradictory findings result from domain-specific functions.

• Species-specific differences: Evaluate potential differences between human and other mammalian LECT1 orthologs, particularly in regions with lower sequence conservation.

By systematically controlling these variables and directly comparing results under standardized conditions, researchers can begin to reconcile apparently contradictory findings and develop a more nuanced understanding of LECT1's context-dependent functions in cartilage development and bone formation .

What techniques can effectively distinguish between the direct effects of LECT1 (214-333) on chondrocytes versus indirect effects via angiogenesis inhibition?

Distinguishing between direct effects on chondrocytes and indirect effects via angiogenesis inhibition requires experimental designs that effectively isolate these pathways:

• Co-culture systems with barriers: Establish transwell or microfluidic co-culture systems where chondrocytes and endothelial cells are physically separated but share media. Apply LECT1 (214-333) treatment and analyze cell-type-specific responses independently.

• Conditioned media experiments: Treat endothelial cells with LECT1 (214-333), collect conditioned media, and apply to chondrocyte cultures to determine if endothelial-derived factors mediate observed effects on chondrocytes.

• Selective receptor blocking: Identify and selectively block receptors specific to either chondrocytes or endothelial cells to determine which cell type's response to LECT1 (214-333) is primary.

• Temporal analysis of molecular events: Perform time-course studies measuring phosphorylation of signaling molecules and gene expression changes in both cell types to establish the sequence of events following LECT1 (214-333) exposure.

• In vivo models with cell-type-specific reporters: Utilize transgenic models with fluorescent reporters in either chondrocytes or endothelial cells to visualize and quantify cell-type-specific responses to LECT1 (214-333) in developing cartilage.

These approaches, when used in combination, can help delineate the primary targets of LECT1 (214-333) and the downstream consequences that may affect other cell types indirectly .

What are the most sensitive techniques for quantifying LECT1 (214-333) binding to cellular receptors?

Quantifying LECT1 (214-333) binding to cellular receptors requires sophisticated biophysical techniques that can detect both high and low-affinity interactions:

• Surface Plasmon Resonance (SPR): Immobilize purified receptor candidates on sensor chips and flow LECT1 (214-333) at various concentrations to determine association and dissociation rates (kon and koff). This provides real-time binding kinetics and allows calculation of equilibrium dissociation constants (KD) typically in the range of 10⁻⁹ to 10⁻⁶ M for biologically relevant interactions.

• Bio-Layer Interferometry (BLI): Similar to SPR but allowing for higher throughput screening of potential receptor interactions. Particularly useful for initial screening of multiple candidate receptors.

• Isothermal Titration Calorimetry (ITC): Measures heat changes during binding to provide thermodynamic parameters (ΔH, ΔS, ΔG) in addition to binding constants, offering insights into the nature of the interaction.

• Fluorescence-based techniques:

  • Microscale Thermophoresis (MST): Detects changes in the movement of fluorescently-labeled molecules in temperature gradients upon binding

  • Fluorescence Polarization (FP): Measures changes in the rotational diffusion of labeled molecules upon binding

  • Förster Resonance Energy Transfer (FRET): Detects proximity between donor and acceptor fluorophores attached to LECT1 and potential receptors

• Cell-based binding assays: For contexts where purified receptors are unavailable, develop competitive binding assays using radiolabeled or fluorescently-labeled LECT1 (214-333) on cells expressing candidate receptors.

When interpreting binding data, researchers should consider the potential impact of protein tags on binding kinetics and perform control experiments with tag-only constructs to distinguish specific from non-specific interactions .

How can researchers effectively analyze the impact of post-translational modifications on LECT1 (214-333) function?

Post-translational modifications (PTMs) can significantly alter LECT1 (214-333) function, requiring systematic analytical approaches:

• Mass spectrometry-based PTM mapping: Employ high-resolution LC-MS/MS with multiple fragmentation techniques (CID, ETD, HCD) to comprehensively identify and localize PTMs on LECT1 (214-333). Targeted methods such as Multiple Reaction Monitoring (MRM) can quantify specific modifications across different sample conditions.

• Site-directed mutagenesis: Create point mutations at potential modification sites (changing serine/threonine phosphorylation sites to alanine or tyrosine glycosylation sites to phenylalanine) and assess the impact on biological activity through functional assays.

• Enzymatic deglycosylation/dephosphorylation: Treat recombinant LECT1 (214-333) with specific enzymes (PNGase F, Endo H for N-glycans; alkaline phosphatase for phosphorylation) before functional assays to determine the necessity of these modifications for activity.

• Expression in systems with differing modification capabilities: Compare LECT1 (214-333) produced in E. coli (minimal PTMs), insect cells (intermediate glycosylation complexity), and mammalian cells (complex glycosylation) to determine the impact of glycoform heterogeneity.

• Metabolic labeling for glycan engineering: Use metabolic glycan labeling with modified sugars followed by bio-orthogonal chemistry to create LECT1 variants with defined glycosylation patterns for structure-function studies.

Researchers should systematically document all identified PTMs and their stoichiometry, as variability in modification patterns between preparations may explain inconsistent functional results across different studies .

What emerging technologies might enhance our understanding of LECT1 (214-333) protein interactions in tissue-specific contexts?

Several cutting-edge technologies show promise for advancing our understanding of LECT1 (214-333) protein interactions in tissue-specific contexts:

• Spatial transcriptomics and proteomics: These techniques allow visualization of gene and protein expression with spatial resolution within tissues, enabling the mapping of LECT1-responsive cell populations within the complex architecture of developing cartilage. Particularly, Visium spatial transcriptomics or imaging mass cytometry could reveal how LECT1 signaling varies across different zones of growth plate cartilage.

• Organoid models: Cartilage organoids derived from human iPSCs provide more physiologically relevant 3D models than traditional cell cultures. These systems can recapitulate developmental processes and tissue architecture, allowing for the study of LECT1 (214-333) function in a controlled yet complex environment.

• CRISPR-based technologies:

  • CRISPR activation/interference systems for temporal control of LECT1 expression

  • Base editing or prime editing for precise modification of specific residues within the 214-333 domain

  • CRISPR screens to identify unknown interaction partners or downstream effectors

• Protein engineering approaches: Computational design and directed evolution techniques can generate LECT1 (214-333) variants with enhanced stability, altered specificity, or novel functions for structure-function studies and potential therapeutic applications.

• Advanced imaging techniques: Super-resolution microscopy combined with proximity labeling methods (BioID, APEX) can visualize LECT1 interactions at the nanoscale within cellular compartments and at cell-matrix interfaces.

Integration of these technologies with traditional biochemical and cell biological approaches will provide a more comprehensive understanding of LECT1's role in cartilage development and potential applications in regenerative medicine .