CLEC10A Human

What is CLEC10A and what are its alternative names in scientific literature?

CLEC10A, also known as macrophage galactose/N-acetyl-galactosamine (GalNAc) specific lectin (MGL), CD301, DC-ASGPR, and HML, is a 40 kDa type II transmembrane glycoprotein belonging to the C-type lectin family . In scientific databases, you may find it referenced under accession number Q8IUN9-2, with additional synonyms including CLECSF13, CLECSF14, HML2, and MGL . Unlike mice, which have two homologs (CD301a/MGL1 and CD301b/MGL2), humans possess a single CLEC10A gene .

What is the molecular structure of human CLEC10A?

Human CLEC10A comprises three distinct domains: a 39 amino acid cytoplasmic region, a 21 amino acid transmembrane segment, and a 256 amino acid extracellular domain (ECD) . The ECD features one carbohydrate recognition domain (CRD) and a neck region . The protein has a predicted molecular weight of 27.3 kDa, but due to post-translational glycosylation, it typically migrates to 30-40 kDa when analyzed by Bis-Tris PAGE . Alternative splicing generates multiple isoforms with 27 aa, 3 aa, and/or 4 aa deletions within the ECD .

Which cell types express CLEC10A in humans?

CLEC10A shows a highly specific expression pattern in the human immune system. It is primarily expressed on:

• CD1c+ dendritic cells (DCs) in blood and lymphoid tissues (including spleen and thymus)

• Immature myeloid dendritic cells

• Alternatively activated (tolerogenic) macrophages

• Immature monocyte-derived DCs (moDCs), with reduced expression on mature moDCs

Notably, CLEC10A is not expressed or is expressed at very low levels on CD141+ DCs, plasmacytoid DCs (pDCs), and monocytes, making it an excellent marker for distinguishing CD1c+ DCs from other myeloid lineages .

What are the primary ligands of CLEC10A and how does it function?

CLEC10A selectively binds and internalizes terminal nonsialylated alpha- or beta-linked GalNAc moieties on O-linked carbohydrates, including the Tn carcinoma antigen . The carbohydrate recognition domain specifically recognizes galactose/N-acetylgalactosamine (Tn antigen) .

When studying CLEC10A-ligand interactions, researchers often use synthetic MUC-1-(Tn)2 peptides (Tn-glycosylated MUC-1) as experimental ligands . Upon binding its ligands, CLEC10A rapidly internalizes into CD1c+ DCs, making it an attractive target for antigen delivery in immunotherapeutic approaches .

How does CLEC10A expression change during dendritic cell maturation?

CLEC10A expression is dynamically regulated during dendritic cell maturation. In monocyte-derived DCs (moDCs), CLEC10A is expressed at low to intermediate levels in immature cells . Upon maturation with inflammatory stimuli (IL-1β, IL-6, TNFα, and prostaglandin E2), CLEC10A expression is significantly reduced, though not completely absent .

Similarly, in primary CD1c+ DCs, Toll-like receptor (TLR) stimulation with R848 (TLR7/8 ligand) or pIC (TLR3 ligand) leads to reduced surface expression of CLEC10A, with TLR7/8 stimulation causing a more pronounced reduction than TLR3 stimulation . This downregulation pattern is consistent with other type I C-type lectin receptors such as DCIR, DC-Sign, and CLEC9A .

What role does CLEC10A play in immunomodulation?

CLEC10A plays a vital role in modulating both innate and adaptive immunity . When CD1c+ DCs are stimulated with the CLEC10A ligand (Tn-glycosylated MUC-1 peptide) in combination with the TLR7/8 agonist R848, they exhibit enhanced secretion of cytokines including IL-8, IL-10, and TNFα . This suggests that CLEC10A signaling can modulate TLR-induced inflammatory responses.

CLEC10A expressed on tolerogenic dendritic cells binds carbohydrate determinants on the CD45 molecule (specifically RA, RB, and RC but not RO isoforms) expressed by T, NK, and B cells, potentially mediating interactions between these immune cell types .

What techniques are most effective for detecting CLEC10A expression in human cells?

For comprehensive CLEC10A expression analysis, researchers should employ a multi-modal approach:

• Flow cytometry: Using anti-CLEC10A antibodies (such as clone H037G3) for surface protein detection . For optimal results, compare with appropriate isotype controls (e.g., mouse IgG2a, clone MOPC-173).

• Transcriptomic analysis: mRNA expression can be assessed through RNA sequencing or microarray analysis, with subsequent bioinformatic processing including differential gene expression analysis (DEGs), principal component analysis (PCA), and hierarchical clustering .

• Ligand binding assays: Using fluorescently-labeled natural ligands such as FITC-βAla-GVTSAPDTRPAPGSTAPPAH(GalNAc)GVTSAPDTRPAPGSTAPPAH(GalNAc), a Tn-glycosylated MUC-1 peptide, compared against non-glycosylated control peptides .

How can researchers effectively study CLEC10A internalization in dendritic cells?

To study CLEC10A internalization, the following protocol has been validated:

• Stain cells with PE-coupled anti-CLEC10A antibody (or appropriate isotype control) for 15 minutes on ice

• Wash and resuspend cells in PBS containing 2% human sera

• Incubate at 37°C for various time points (0, 5, 15, 30, and 60 minutes) to allow internalization

• Detect remaining surface CLEC10A by secondary staining with anti-PE antibody followed by fluorescently-labeled tertiary antibody

• Analyze by flow cytometry, measuring the decrease in surface signal over time

This approach allows quantification of internalization kinetics, which is critical for evaluating CLEC10A's potential as an antigen delivery receptor.

What are the considerations for recombinant CLEC10A protein production?

When producing recombinant CLEC10A for research purposes:

• Expression system: HEK293 cells are commonly used to ensure proper mammalian glycosylation

• Protein region: Most successful constructs include the extracellular domain (Gln61-His292 or Gln61-His316)

• Purification tags: N-terminal 6-His tags facilitate purification without interfering with the carbohydrate recognition domain

• Quality control: Assess purity (>95%) using Bis-Tris PAGE and confirm low endotoxin levels (<1EU per μg) using the LAL method

• Storage: Lyophilize from filtered PBS (pH 7.4) with 8% trehalose as a protectant

How can CLEC10A be utilized for targeted antigen delivery to human dendritic cells?

CLEC10A represents a promising target for antigen delivery to CD1c+ DCs based on several characteristics:

• Cell-type specificity: CLEC10A is selectively expressed on CD1c+ DCs, allowing targeted delivery to this DC subset

• Rapid internalization: Upon binding ligands or antibodies, CLEC10A efficiently internalizes into DCs

• Antigen processing potential: CLEC10A has been demonstrated to traffic to HLA I and II rich compartments in immature moDCs, suggesting potential for both MHC class I and II presentation

For antigen targeting applications, researchers can use:

• Anti-CLEC10A antibodies conjugated to antigens of interest

• Synthetic glycopeptides containing GalNAc moieties (such as Tn-MUC1) linked to antigenic epitopes

• Nanoparticles decorated with CLEC10A ligands for payload delivery

What is the potential of CLEC10A as an immunotherapy target for cancer?

CLEC10A has shown promise as an immunotherapy target for cancers, particularly for strategies targeting dendritic cells . Several factors make it attractive for cancer immunotherapy research:

• It facilitates uptake of tumor-associated carbohydrate antigens like the Tn antigen, which is commonly expressed on various carcinomas

• Its selective expression on CD1c+ DCs enables targeted delivery of cancer antigens to professional antigen-presenting cells

• The combination of CLEC10A targeting with TLR ligands (such as R848) enhances cytokine production by DCs, potentially boosting anti-tumor immune responses

Research into CLEC10A-based cancer immunotherapies is still emerging, with particular interest in lung adenocarcinoma (LUAD), though functional research in this area remains limited .

How does CLEC10A trafficking influence antigen presentation in dendritic cells?

The intracellular trafficking of CLEC10A is critical for understanding its role in antigen presentation. Upon internalization:

• CLEC10A has been reported to traffic to HLA I and II rich compartments in human immature moDCs

• This suggests that antigens targeted via CLEC10A could be presented on both MHC class I and II molecules, facilitating activation of both CD8+ and CD4+ T cells

• This dual presentation capability could be particularly valuable for cancer immunotherapy approaches requiring both helper and cytotoxic T cell responses

Unlike some other C-type lectin receptors that may deliver signals upon binding, CLEC10A appears to separate internalization from signaling events . Further research is needed to fully characterize the precise compartmentalization of CLEC10A and its influence on cross-presentation efficiency.

How does human CLEC10A differ from mouse CD301a/MGL1 and CD301b/MGL2?

Understanding the differences between human CLEC10A and mouse homologs is critical for translational research:

This differential expression pattern between species suggests that researchers should be cautious when extrapolating findings from mouse models to human biology .

What are the challenges in developing CLEC10A-targeted therapies from preclinical to clinical applications?

Translating CLEC10A-targeted therapies from laboratory studies to clinical applications faces several challenges:

• Species differences: The divergence between human CLEC10A and mouse homologs complicates preclinical testing in mouse models

• DC subset heterogeneity: CD1c+ DCs themselves are heterogeneous, and targeting efficiency may vary among subpopulations

• Expression regulation: CLEC10A expression is downregulated during DC maturation, potentially limiting the window for therapeutic targeting

• Glycan complexity: The natural glycan ligands of CLEC10A are diverse and complex, requiring sophisticated chemistry for synthetic targeting ligands

• Functional outcomes: While CLEC10A efficiently internalizes cargo, the immunological outcome (immunity vs. tolerance) may depend on additional signals

Addressing these challenges requires comprehensive preclinical studies using humanized mouse models or human ex vivo systems before clinical translation.

What are the emerging research questions about CLEC10A in human immunology?

Several important questions remain to be fully addressed regarding CLEC10A:

• What is the complete repertoire of natural ligands for CLEC10A in various tissues and disease states?

• How does CLEC10A signaling integrate with other pattern recognition receptor pathways?

• What is the role of CLEC10A in maintaining tolerance versus immunity in different contexts?

• How might CLEC10A function be altered in autoimmune diseases, infectious diseases, or cancer?

• Can CLEC10A be effectively targeted for vaccination or immunotherapy without causing off-target effects?

What methodological advances could accelerate CLEC10A research?

Future CLEC10A research would benefit from:

• Development of improved monoclonal antibodies against different epitopes of CLEC10A

• Generation of reporter cell lines for high-throughput screening of CLEC10A ligands

• Advanced imaging techniques to track CLEC10A trafficking in real-time

• Conditional knockout systems in human dendritic cells to assess CLEC10A function

• Glycan array technologies to comprehensively characterize CLEC10A binding preferences