Recombinant Mouse C-type lectin domain family 18 member A (Clec18a)

What is the structural organization of mouse Clec18a and how does it compare to human CLEC18A?

Mouse Clec18a belongs to the C-type lectin family and shares significant structural homology with human CLEC18A. The CLEC18 family in humans includes three closely related paralogs (CLEC18A, CLEC18B, and CLEC18C) that have nearly identical amino acid sequences but differ in specific residues located in the C-type lectin-like domain (CTLD) and the sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain . The mouse genome contains only a single Clec18 gene (Clec18a), unlike the human triplication, making it an interesting model for evolutionary and functional studies . Both human and mouse variants encode N-linked glycoproteins with a C-type lectin domain at the C-terminus.

What are the known ligands of mouse Clec18a and how do they differ from human CLEC18A binding patterns?

Mouse Clec18a primarily binds to sulfonated glycosaminoglycans (GAGs) of proteoglycans, making it unique among C-type lectins . Human CLEC18A also demonstrates this binding specificity, with polysaccharide binding occurring in a calcium-independent manner, which is unusual for C-type lectins that typically require calcium for carbohydrate recognition . Previous glycoarray analyses have shown weak but detectable binding to GlcAβ1-6Galβ structures, which are building blocks in glycosaminoglycans found in proteoglycans . The specific amino acid residues Ser/Arg 339 and Asp/Asn 421 in the CTLD domain contribute significantly to differential binding abilities to polysaccharides isolated from sources like Ganoderma lucidum .

How is the expression of mouse Clec18a regulated at the transcriptional level?

Transcriptional regulation of mouse Clec18a appears tissue-specific, with notable expression in the proximal tubule of the kidney and the medial habenula of the brain . In humans, expression of CLEC18A can be induced by pathogen exposure, as demonstrated by hepatitis C virus (HCV) infection or activation of Toll-like receptors 3/7/8 in hepatic cells . The upregulation mechanism involves pattern recognition receptor signaling, suggesting that Clec18a transcription may be similarly regulated in mice through pathogen-associated molecular pattern (PAMP) recognition pathways. Further research is needed to fully characterize the transcriptional control elements in the mouse Clec18a promoter region.

What is the tissue distribution pattern of Clec18a in mice?

Mouse Clec18a shows a restricted tissue distribution pattern, being predominantly expressed in the proximal tubule of the kidney and the medial habenula of the brain . This contrasts with human CLEC18A, which is more widely expressed in various tissues including abundant expression in hepatocytes and phagocytes . Understanding this species-specific expression pattern is crucial when translating findings between mouse models and human studies. Researchers should consider these differences when designing experiments and interpreting results across species.

What subcellular compartments contain Clec18a and how can this localization be experimentally determined?

Based on human CLEC18A studies, which likely parallel mouse Clec18a, this protein is localized in the endoplasmic reticulum, Golgi apparatus, and endosomes . This subcellular distribution suggests roles in protein processing, glycan recognition, and vesicular trafficking. To experimentally determine subcellular localization, researchers commonly employ:

• Immunofluorescence staining with organelle-specific markers

• Subcellular fractionation followed by Western blotting

• Live-cell imaging with fluorescently tagged Clec18a constructs

For immunofluorescence, cells should be permeabilized with 0.1% saponin before staining with anti-Clec18a antibodies, followed by co-staining with markers for ER (e.g., calnexin), Golgi (e.g., GM130), or endosomes (e.g., EEA1, Rab5, Rab7) .

How does the expression of Clec18a change during cellular differentiation or activation?

While specific data for mouse Clec18a is limited, human studies show that CLEC18 expression increases significantly when monocytes differentiate into macrophages and dendritic cells . This suggests that Clec18a expression in mice might similarly be upregulated during myeloid cell differentiation and activation. Researchers investigating this question should consider time-course experiments measuring Clec18a mRNA and protein levels during differentiation processes, using techniques such as qRT-PCR, Western blotting, and flow cytometry.

What proteins interact with Clec18a and how do these interactions affect cellular function?

Mouse Clec18a, like its human counterpart, interacts with small GTPases, particularly Rab5 and Rab7, which are key regulators of endosomal trafficking . These interactions affect multiple cellular processes:

• Vesicular trafficking: Clec18a can modulate endosome maturation by influencing Rab5 (early endosome) and Rab7 (late endosome) function

• Autophagy: By reducing Rab7 recruitment to autophagosomes, Clec18a can retard autophagosome maturation and impair autophagosome-lysosome fusion

• Interferon signaling: In humans, CLEC18A enhances type I/III interferon production through mechanisms that may involve these Rab protein interactions

To study these interactions in experimental settings, co-immunoprecipitation followed by Western blotting or mass spectrometry can be employed, as previously demonstrated with anti-FLAG M2 affinity gel pulldowns of tagged CLEC18A constructs .

How does Clec18a modulate immune cell function, particularly in phagocytes?

When overexpressed, CLEC18A has been shown to suppress phagocytic activity in phagocytes . This occurs through inhibition of Fc gamma receptor IIA (FcγRIIA) expression in a dose-dependent manner, mediated by the production of NOX-2-dependent reactive oxygen species . This ultimately impairs the uptake of immune complexes.

In mouse models of clear cell renal cell carcinoma (ccRCC), deletion of Clec18a led to increased tumor infiltration by Ly6G+ neutrophils, suggesting a role in regulating myeloid cell recruitment or function in the tumor microenvironment . The table below summarizes these immunomodulatory effects:

What is the role of Clec18a in cancer progression, particularly in renal cell carcinoma?

Recent research has identified a significant role for Clec18a in clear cell renal cell carcinoma (ccRCC) progression . Key findings include:

• High expression of CLEC18A correlates with improved survival in ccRCC patients

• Genetic deletion of Clec18a in a mouse ccRCC cell line (RAG) promotes tumor growth following subcutaneous injection into immunodeficient mice

• Clec18a appears to function through a lymphocyte-independent mechanism, as its tumor-suppressive effects were observed in Rag2−/−Il2rg−/− mice lacking T, B, and NK cells

• Loss of Clec18a leads to significantly increased tumor infiltration by Ly6G+ neutrophils, though no apparent difference in F4/80+ macrophage infiltration was observed

These findings suggest that Clec18a may function as a tumor suppressor in renal cancer, potentially by modulating the tumor microenvironment or through direct effects on tumor cell biology.

How does Clec18a contribute to viral infection responses?

While direct data on mouse Clec18a in viral infections is limited, human CLEC18A has been extensively studied in the context of hepatitis C virus (HCV) infection. CLEC18A expression is induced by HCV infection or activation of Toll-like receptors 3/7/8 . Once upregulated, CLEC18A:

• Interacts with Rab5 and Rab7 to enhance type I/III interferon production

• Inhibits HCV replication in hepatocytes

• May contribute to autoimmunity in chronic infection by impairing phagocytosis through FcγRIIA downregulation

These findings suggest that mouse Clec18a might play similar roles in antiviral immunity and could potentially contribute to immunopathology in chronic viral infections. Researchers studying viral infections in mouse models should consider investigating Clec18a expression and function.

What is the relationship between Clec18a, autophagy, and disease pathogenesis?

Clec18a appears to modulate autophagy, a critical cellular process involved in various diseases. Overexpressed CLEC18A does not affect autophagosome formation but reduces the recruitment of Rab7 to autophagosomes . This retards autophagosome maturation and affects autophagosome-lysosome fusion.

In disease contexts, this modulation of autophagy may:

• Influence viral clearance in infections

• Affect cancer cell survival and metabolism

• Contribute to immune dysregulation through altered processing of self and foreign antigens

Researchers investigating autophagy in disease models should consider monitoring Clec18a expression and its effects on the autophagy pathway, particularly in kidney and brain tissues where mouse Clec18a is predominantly expressed .

What are the recommended methods for detecting and quantifying mouse Clec18a expression?

Based on established protocols for human CLEC18A, researchers can employ several techniques to detect and quantify mouse Clec18a:

RNA detection:

• Real-time RT-PCR using specific primers targeting mouse Clec18a

• The PCR program should include denaturation at 95°C for 10s, annealing at approximately 66°C for 10s, and extension at 72°C for 10s

Protein detection:

• Western blotting using anti-Clec18a antibodies

• Flow cytometry analysis: Cells should be permeabilized with 0.1% saponin and stained with fluorochrome-conjugated anti-Clec18a antibodies

• Immunohistochemistry or immunofluorescence for tissue sections

• ELISA for detection in serum or culture supernatants

When developing or adapting these methods for mouse Clec18a, researchers should validate antibody specificity using appropriate controls, including Clec18a knockout cells as demonstrated in recent studies .

How can functional Clec18a knockout or overexpression models be generated for experimental studies?

Recent research has successfully employed several approaches to manipulate Clec18a expression in experimental systems:

Knockout strategies:

• CRISPR-Cas9-mediated deletion in cell lines: This has been successfully implemented in the RAG murine ccRCC cell line, which exhibits high baseline expression of Clec18a

• Conditional knockout mouse models: While not specifically mentioned in the search results, tissue-specific Cre-loxP systems would be appropriate for studying Clec18a function in specific organs like kidney or brain

Overexpression strategies:

• Lentiviral or plasmid-based expression systems for introducing Clec18a into cell lines with low or no baseline expression, as demonstrated with the E0771 murine mammary cancer cell line

• Transgenic overexpression in mice

The function of these models can be verified through:

• RT-PCR and Western blotting to confirm expression changes

• Functional assays specific to the research question (e.g., phagocytosis assays, autophagy monitoring)

• In vivo tumor growth models for cancer-related studies

What glycan-binding assays are appropriate for characterizing Clec18a interactions with potential ligands?

To characterize Clec18a interactions with glycans, particularly sulfonated GAGs of proteoglycans, researchers can employ several approaches:

• Glycan array screening: While previous glycoarray analysis with 611 common N-linked and O-linked glycostructures did not yield significant interaction partners for human CLEC18A, arrays specifically enriched for glycosaminoglycans and proteoglycans would be more appropriate for Clec18a

• Surface plasmon resonance (SPR): To determine binding kinetics and affinities between recombinant Clec18a and potential GAG ligands

• Pull-down assays: Using immobilized potential ligands to capture Clec18a from cell lysates or recombinant sources

• Competitive binding assays: To evaluate relative binding preferences among different GAG structures

• Calcium dependency tests: To confirm the unusual Ca²⁺-independent binding mode observed with human CLEC18 proteins

When designing these experiments, researchers should consider that specific amino acid residues in the CTLD domain (corresponding to positions 339 and 421 in human CLEC18) may significantly influence binding specificity .

How can recombinant mouse Clec18a be utilized to study tumor microenvironment interactions?

Recombinant mouse Clec18a offers several approaches to investigate tumor microenvironment interactions:

• Exogenous administration: Purified recombinant Clec18a can be introduced into tumor models to assess effects on:

  • Tumor growth kinetics

  • Immune cell infiltration patterns, particularly neutrophils which show differential recruitment in Clec18a-deficient tumors

  • Cytokine/chemokine profiles within the tumor microenvironment

• Mechanistic studies: Recombinant Clec18a with mutations in key binding domains can help determine:

  • Which structural features are essential for its tumor-suppressive functions

  • How glycan recognition contributes to its effects on immune cells

  • Whether direct tumor cell binding or immune cell modulation drives its effects

• Imaging applications: Fluorescently labeled recombinant Clec18a can track:

  • Distribution within tumor tissues

  • Cellular binding partners in situ

  • Dynamic interactions with GAGs in the tumor microenvironment

Given that Clec18a deletion promoted tumor growth of ccRCC cells in mouse models , therapeutic applications using recombinant Clec18a as a potential tumor suppressor merit further investigation.

What is the relationship between Clec18a-mediated autophagy regulation and its roles in cancer and infection?

The dual role of Clec18a in both autophagy regulation and disease processes presents intriguing research questions:

In cancer contexts, Clec18a's interaction with Rab proteins, particularly Rab7, affects autophagosome maturation and autophagosome-lysosome fusion . This may:

• Influence tumor cell metabolism and survival under stress conditions

• Affect antigen presentation and immune surveillance

• Modulate the degradation of signaling molecules involved in proliferation and apoptosis

In infectious disease contexts, particularly viral infections:

• Clec18a-mediated autophagy modulation may affect viral clearance mechanisms

• Enhanced type I/III interferon production through Clec18a upregulation could promote antiviral state

• Impaired phagocytosis through FcγRIIA downregulation might contribute to persistence of immune complexes and autoimmunity

Future research should explore how these seemingly contradictory functions (antiviral vs. immunosuppressive) are balanced in different cellular contexts and disease states. Co-immunoprecipitation studies combined with functional autophagy assays could help delineate these complex relationships.

How might genetic variations in mouse Clec18a impact experimental outcomes across different mouse strains?

Genetic variations in mouse Clec18a across different strains represent an important consideration for experimental design and interpretation:

• Expression level variations: Different mouse strains may have baseline differences in Clec18a expression, potentially affecting:

  • Susceptibility to renal tumors

  • Immune responses to viral infections

  • Autophagy regulation in relevant tissues

• Functional polymorphisms: Amino acid variations corresponding to the critical residues identified in human CLEC18 proteins (positions 339 and 421) might exist between mouse strains, potentially altering:

  • Binding specificity to glycosaminoglycans

  • Interactions with Rab proteins

  • Downstream signaling effects

• Experimental considerations: Researchers should:

  • Document the mouse strain background in all Clec18a studies

  • Consider backcrossing Clec18a genetic models to multiple strain backgrounds for robust phenotypic assessment

  • Sequence the Clec18a gene in experimental strains to identify potential functional variations

While limited data exists on strain-specific Clec18a variations, the lessons from human CLEC18 polymorphism studies suggest this could be an important area for future investigation in mouse models.