CLEC1B Human

What is the structural composition of human CLEC1B?

Human CLEC1B (also known as CLEC-2) is a 32 kDa type II transmembrane glycoprotein belonging to the C-type lectin-like family of receptors. Its structure consists of three distinct domains: a 33 amino acid cytoplasmic domain, a 21 amino acid transmembrane region, and a 175 amino acid extracellular domain. The cytoplasmic domain contains multiple threonine and serine residues that serve as potential phosphorylation sites, along with a critical YXXL (Tyr-Xaa-Xaa-Leu) motif that is essential for signaling functions. The protein's extracellular domain contains the C-type lectin-like domain (CTLD) that facilitates ligand recognition and binding .

What are the primary biological functions of CLEC1B in normal human physiology?

CLEC1B serves multiple critical physiological functions in humans. It acts as a platelet-related molecule and functions as an activating receptor for snake venom toxin rhodocytin as well as the endogenous ligand podoplanin. It plays essential roles in several biological processes including:

• Platelet activation and aggregation

• Lymphatic/blood vessel separation

• Immune response regulation

• Tumor cell-induced platelet aggregation

Research indicates that CLEC1B has inhibitory effects on platelet aggregation and tumor metastasis in various cancer types, including colon carcinoma. The receptor's binding to ligands triggers Src kinase-dependent tyrosine phosphorylation of the YXXL sequence, activating the tyrosine kinase Syk and initiating a signaling cascade that ultimately leads to phospholipase C gamma 2 activation .

How can researchers detect CLEC1B expression in clinical samples?

Multiple methodological approaches can be employed to detect CLEC1B expression in clinical samples:

• Flow Cytometry: Human whole blood samples can be stained with fluorescently-conjugated anti-CLEC1B antibodies (such as Mouse Anti-Human CLEC-2 APC-conjugated Monoclonal Antibody) along with other markers like CD41. This technique allows for specific detection of CLEC1B in platelet populations and other cell types .

• Immunohistochemistry (IHC): For tissue samples, IHC can be performed using anti-CLEC1B antibodies. The staining intensity and percentage of positively stained cells are evaluated on a semi-quantitative scoring system:

  • 0-1: Negative

  • 2-3: Weak

  • 4-5: Moderate

  • 6-8: Strong

  Assessment should be performed by two independent pathologists to ensure reliability .

• Tissue Microarray (TMA): This high-throughput method allows simultaneous analysis of CLEC1B expression across multiple tissue samples, facilitating correlation studies with clinical parameters and other markers like CD4 and CD8 .

• Transcriptional Analysis: RNA sequencing or qPCR can be used to analyze CLEC1B mRNA expression levels in tissue samples, particularly useful when comparing tumor and normal tissues .

What is the role of CLEC1B in tumor immune microenvironment regulation?

CLEC1B plays a significant role in modulating the tumor immune microenvironment (TIME), particularly in hepatocellular carcinoma (HCC). Research findings suggest that:

• CLEC1B expression is significantly associated with immune cell infiltration and function within the tumor microenvironment. Gene Ontology (GO) enrichment analysis reveals that CLEC1B's biological processes are primarily associated with T cell activation and lymphocyte differentiation .

• GSEA analysis of CLEC1B-related genes showed that the top positively regulated hallmark signatures include inflammatory response, interferon gamma response, and TNFα signaling. This suggests that CLEC1B activates immune response signaling in HCC, leading to the secretion of essential pro-inflammatory cytokines that can activate CD8+ T cells .

• CLEC1B expression positively correlates with multiple immunomodulators and is implicated in various immune-related processes. Specifically, low CLEC1B expression in tumors is associated with reduced CD8+ T cell infiltration, suggesting its importance in cytotoxic T cell recruitment or retention within the tumor microenvironment .

• The protein appears to have an immunologic enhancement role in the HCC tumor microenvironment, potentially serving as a novel immunoregulator for HCC therapy .

How does CLEC1B expression correlate with patient outcomes in hepatocellular carcinoma?

CLEC1B expression demonstrates significant prognostic value in HCC patients:

What methodologies are most effective for studying CLEC1B in relation to immune infiltration?

Several complementary methodological approaches are recommended for investigating CLEC1B's relationship with immune infiltration:

• Single-Cell RNA Sequencing: The Tumor Immune Single-cell Hub (TISCH) database can be utilized for single-cell analysis of CLEC1B expression across different immune cell populations within tumors .

• Immune Correlation Analysis: Dividing patients into CLEC1B-high and CLEC1B-low groups based on median expression values allows for correlation analysis between CLEC1B expression and infiltrating immune cells using databases like TISIDB .

• Immunohistochemical Co-staining: Performing IHC for CLEC1B alongside immune cell markers (CD4, CD8, etc.) on tissue microarrays enables direct visualization and quantification of the relationship between CLEC1B expression and immune cell infiltration .

• Gene Set Enrichment Analysis (GSEA): This approach helps identify signaling pathways significantly enriched in relation to CLEC1B expression levels. The analysis can reveal associations with immune-related pathways such as inflammatory response and interferon gamma response .

• Differential Gene Expression Analysis: Constructing volcano plots to elucidate differentially expressed genes between CLEC1B-high and CLEC1B-low groups can identify immune-related genes co-regulated with CLEC1B .

How does CLEC1B influence response to cancer therapeutics?

CLEC1B expression appears to significantly influence treatment responses, particularly to targeted therapies for HCC:

• Sorafenib Response: Research has demonstrated that overexpression of CLEC1B significantly impacts the treatment effects of sorafenib on HCC cells. This suggests that CLEC1B levels may be an important consideration when predicting patient responses to this first-line targeted therapy .

• Immunotherapy Implications: Given CLEC1B's correlation with immune infiltration and association with inflammatory and interferon response pathways, its expression level may potentially predict immunotherapy effectiveness. Low CLEC1B and high PD-L1 have been reported to be a valuable prognostic factor implying worse clinical outcomes in HCC .

• Therapeutic Resistance Mechanisms: GSEA analysis indicates that CLEC1B expression is negatively associated with pathways involved in tumor progression such as DNA repair, E2F targets, and MYC targets. This suggests that low CLEC1B expression may contribute to therapeutic resistance through activation of these proliferative and survival pathways .

What experimental approaches are recommended for validating CLEC1B as a prognostic biomarker?

To validate CLEC1B as a prognostic biomarker for HCC or other cancers, researchers should consider implementing the following experimental approaches:

• Multi-cohort Validation: Analyze CLEC1B expression across independent patient cohorts using databases like TCGA and validate findings in separate clinical cohorts. Statistical approaches should include:

  • Cox proportional hazards regression models

  • Kaplan-Meier survival curves with Log-rank tests

  • Nomogram construction and calibration curve analysis

• Tissue Microarray Analysis: Construct tissue microarrays with paired tumor and non-tumor samples to evaluate CLEC1B protein expression by immunohistochemistry. Semi-quantitative scoring should be performed by multiple pathologists to ensure reliability .

• ROC Curve Analysis: Determine the diagnostic value of CLEC1B by calculating the area under the ROC curve with 95% confidence intervals .

• Multivariate Analysis: Combine CLEC1B expression with established clinicopathological factors to develop comprehensive prognostic models that outperform individual markers .

• Functional Validation: Perform in vitro and in vivo experiments with CLEC1B overexpression or knockdown to validate the causal relationship between CLEC1B levels and cancer progression phenotypes .

What signaling mechanisms are activated by CLEC1B in human cells?

CLEC1B activates specific signaling cascades through well-defined molecular mechanisms:

• Ligand-Induced Activation: When ligands such as podoplanin or rhodocytin bind to CLEC1B, they induce receptor cross-linking on the cell surface .

• YXXL Motif Phosphorylation: This cross-linking triggers Src kinase-dependent tyrosine phosphorylation of the YXXL sequence in the cytoplasmic domain. This phosphorylation event is critical for downstream signal transduction .

• Syk Kinase Activation: Following YXXL phosphorylation, the tyrosine kinase Syk is recruited and activated .

• Downstream Signaling: The activated Syk initiates a signaling pathway that ultimately leads to phospholipase C gamma 2 (PLCγ2) activation. In platelets, this results in calcium mobilization, granule secretion, and integrin activation .

• Immune-Related Pathways: In immune contexts, CLEC1B signaling appears to activate pathways related to T cell receptor signaling, inflammatory response, and interferon gamma response, as revealed by KEGG enrichment and GSEA analyses .

How do genetic variations in CLEC1B affect its function and disease associations?

While the search results don't provide specific information about CLEC1B genetic variations, researchers investigating this area should consider:

• Mutation Analysis: Analyzing CLEC1B mutations across cancer databases to identify potential loss-of-function or gain-of-function variants that might influence disease progression.

• Single Nucleotide Polymorphisms (SNPs): Investigating whether common SNPs in CLEC1B correlate with altered expression levels, protein function, or disease susceptibility.

• Structure-Function Relationships: Examining how variations in key domains (especially the YXXL motif or ligand-binding regions) might affect signaling capacity and downstream pathway activation.

• Expression Quantitative Trait Loci (eQTLs): Identifying genetic variants that affect CLEC1B expression levels and correlating these with disease phenotypes.

This represents an important area for future research given CLEC1B's significant associations with cancer progression and immune function.

What are the most reliable antibodies and detection methods for CLEC1B research?

Based on the search results, researchers should consider these validated approaches for CLEC1B detection:

• Flow Cytometry Applications:

  • Mouse Anti-Human CLEC-2 APC-conjugated Monoclonal Antibody (Clone #219133, Catalog #FAB1718A) has been validated for flow cytometric detection of CLEC1B in human whole blood and platelets .

  • Mouse Anti-Human CD41 FITC-conjugated Monoclonal Antibody or Mouse Anti-Human Integrin alpha 2b/CD41 PE-conjugated Monoclonal Antibody (Catalog #FAB7616P) can be used as co-staining markers to identify platelet populations .

  • Appropriate isotype controls such as Mouse IgG2A Allophycocyanin Isotype Control (Catalog #IC0041A) should be included .

• Immunohistochemistry:

  • Anti-CLEC1B antibody (1:1000, Proteintech) has been validated for IHC applications on tissue microarrays .

  • For co-staining studies, antibodies against CD4 and CD8 from the same manufacturer can be used to maintain consistent protocols .

• Western Blotting:
  While not explicitly mentioned in the search results, researchers typically use the same primary antibodies validated for IHC at appropriate dilutions for Western blot applications.

• Protocol Recommendations:

  • For membrane-associated protein staining, researchers should follow established protocols for staining membrane-associated proteins as referenced in the search results .

  • Optimal dilutions should be determined by each laboratory for each application to ensure optimal signal-to-noise ratios .

How can researchers effectively investigate CLEC1B's role in the tumor-immune microenvironment?

To comprehensively investigate CLEC1B's role in the tumor-immune microenvironment, researchers should implement a multi-faceted approach:

• Bioinformatic Analysis:

  • Use ssGSEA algorithm for studies related to immune infiltration .

  • Perform single-gene differential expression analysis using the DESeq2 method to identify CLEC1B-related genes .

  • Conduct GO, KEGG, and GSEA analyses to reveal CLEC1B-associated immune pathways .

• Tissue-Based Analysis:

  • Construct tissue microarrays including tumor and normal tissues.

  • Perform multiplex immunohistochemistry for CLEC1B along with immune cell markers (CD4, CD8, etc.) .

  • Quantify spatial relationships between CLEC1B-expressing cells and tumor-infiltrating lymphocytes.

• Functional Studies:

  • Develop CLEC1B overexpression and knockdown cell models.

  • Assess immune cell migration, adhesion, and activation in co-culture systems with modified cancer cells.

  • Evaluate changes in cytokine/chemokine production and immune checkpoint molecule expression.

• In Vivo Models:

  • Generate CLEC1B knockout or overexpression mouse models.

  • Analyze immune infiltration in spontaneous or implanted tumors.

  • Test immunotherapy efficacy in the context of different CLEC1B expression levels.

• Clinical Correlation:

  • Divide patient cohorts into CLEC1B-high and CLEC1B-low groups to analyze differences in immune infiltration and response to therapy .

  • Correlate CLEC1B expression with markers of immune activation or suppression in patient samples.