Recombinant Human C-type lectin domain family 2 member L (CLEC2L)

What is Recombinant Human C-type lectin domain family 2 member L (CLEC2L)?

Recombinant Human C-type lectin domain family 2 member L (CLEC2L) is a type II transmembrane protein belonging to the CLEC2 family of proteins, which includes other members such as CD69, LLT1, AICL, and KACL. Unlike other CLEC2 family members that show broad tissue expression patterns, CLEC2L is predominantly expressed in the brain, earning it the alternative designation as brain-associated C-type lectin (BACL). CLEC2L functions as a homodimeric cell surface receptor, suggesting important roles in cell-cell recognition or immune function in neural tissues .

The mature human CLEC2L protein has a specific structural composition that includes a 68 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 125 amino acid extracellular domain. This structure is characteristic of type II transmembrane proteins, with the N-terminus located intracellularly and the C-terminus extending into the extracellular space .

How does CLEC2L differ from other members of the CLEC2 protein family?

CLEC2L distinguishes itself from other CLEC2 family members primarily through its tissue-specific expression pattern. While other family members like LLT1, AICL, and KACL are expressed on B cells, monocytes, and keratinocytes respectively, CLEC2L shows predominant expression in brain tissue . This brain-specific expression pattern suggests specialized functions in neural environments that may differ significantly from the roles of other CLEC2 family proteins in immune cell recognition and interaction.

The structural differences between CLEC2L and other family members, particularly in the extracellular domain, likely contribute to distinct binding partners and signaling pathways. Understanding these differences is crucial for researchers investigating the specific functions of CLEC2L in comparison to other C-type lectins .

What techniques are available for studying CLEC2L gene expression?

Researchers have multiple options for studying CLEC2L gene expression, with lentiviral activation systems representing a particularly powerful approach. The CRISPR Activation Plasmid products enable specific upregulation of CLEC2L gene expression by utilizing a deactivated Cas9 (dCas9) nuclease fused to a VP64 activation domain, in conjunction with sgRNA (MS2) engineered to bind the MS2-P65-HSF1 fusion protein .

This synergistic activation mediator (SAM) transcription activation system provides a robust method to maximize endogenous CLEC2L expression. The system works by targeting the CLEC2L gene locus (which maps to 6 B1 in mouse) and inducing transcriptional activation without altering the gene sequence itself . For monitoring activation efficacy, researchers can employ antibodies like CLEC-2L (D-5): sc-390424 to validate expression levels through techniques such as Western blotting before and after activation.

How can recombinant CLEC2L protein be characterized biochemically?

Recombinant CLEC2L protein characterization typically involves several biochemical approaches:

• SDS-PAGE analysis: When resolved under reducing and non-reducing conditions, Recombinant Human CLEC2L Fc Chimera shows bands at 39-44 kDa and 80-90 kDa, respectively, as visualized by Coomassie Blue staining. This difference in molecular weight indicates the presence of disulfide bonds that maintain the protein's dimeric structure under native conditions .

• Binding assays: Functional characterization can be performed through binding studies. For example, when Recombinant Human CLEC2L Fc Chimera is immobilized at 5 μg/mL (100 μL/well), Recombinant Human Galectin-3 binds with an ED50 of 1.5-12 μg/mL, demonstrating a specific interaction between these proteins .

The following table summarizes key biochemical properties of Recombinant Human CLEC2L:

How can CLEC2L expression be artificially upregulated in experimental models?

Artificial upregulation of CLEC2L expression can be achieved through several state-of-the-art approaches, with CRISPR-based methods showing particular promise. The CRISPR Activation system, utilizing a deactivated Cas9 (dCas9) fused to activation domains, provides a targeted approach for enhancing gene expression without altering the underlying genetic sequence .

For CLEC2L specifically, researchers can employ Lentiviral Activation Particles designed for the mouse Clec2l gene. These particles deliver the necessary components of the Synergistic Activation Mediator (SAM) transcription activation system, including:

• dCas9-VP64: A nuclease-deficient Cas9 (with D10A and N863A mutations) fused to the VP64 transcriptional activation domain

• sgRNA(MS2): A guide RNA targeting the CLEC2L promoter region, engineered with MS2 loop structures

• MS2-P65-HSF1 fusion protein: A transcriptional activator that binds to the MS2 loops in the sgRNA

This system allows for robust activation of endogenous CLEC2L expression, particularly valuable for studying the functional consequences of increased CLEC2L levels in neuronal cells or brain tissue models .

What is the significance of CLEC2L's brain-specific expression pattern?

The predominant expression of CLEC2L in brain tissue, earning it the designation "brain-associated C-type lectin" (BACL), suggests specialized functions in the central nervous system that distinguish it from other CLEC2 family members . This brain-specific expression pattern raises several important research questions:

First, CLEC2L may play roles in neuronal cell recognition, adhesion, or communication, potentially contributing to synaptic formation or maintenance. Second, as a C-type lectin, CLEC2L might recognize specific carbohydrate structures on neural cells, potentially mediating interactions between different cell types in the brain. Third, the protein could have immunomodulatory functions within the brain's unique immune environment, possibly interacting with microglia or infiltrating immune cells during neuroinflammatory processes .

Researchers investigating CLEC2L should consider designing experiments that specifically address its function within neural contexts, potentially using primary neuronal cultures, brain organoids, or animal models with manipulated CLEC2L expression to elucidate its physiological relevance.

How do experimental findings with CLEC2L compare to studies of CLEC-2?

While CLEC2L and CLEC-2 share nomenclature similarities, they represent distinct proteins with different expression patterns and functions. CLEC-2 (C-type lectin-like receptor 2) is primarily expressed on platelets and serves as a receptor for podoplanin (PDPN), mediating platelet activation and potentially contributing to cancer metastasis. In contrast, CLEC2L shows predominant expression in brain tissue .

Experimental studies with CLEC-2 have demonstrated:

• CLEC-2 expression decreases upon platelet activation with agonists like ADP and PDPN, as measured by flow cytometry

• Contrary to previous hypotheses, soluble CLEC-2 (sCLEC-2) levels in plasma decrease rather than increase following platelet activation

• Patients with certain cancer types (colorectal carcinoma, melanoma, and breast cancer) show lower sCLEC-2 plasma levels compared to healthy controls

These findings contrast with the limited available data on CLEC2L function, highlighting the importance of not confusing these distinct proteins despite their similar nomenclature. Researchers working with CLEC2L should be careful to distinguish relevant literature and experimental approaches from those developed for CLEC-2 .

What are the optimal conditions for working with Recombinant Human CLEC2L Fc Chimera protein?

When working with Recombinant Human CLEC2L Fc Chimera protein, researchers should consider several factors to maintain protein stability and functionality:

For immobilization in binding assays, a concentration of 5 μg/mL (100 μL/well) has been validated for interaction studies with binding partners such as Recombinant Human Galectin-3 . This concentration provides sufficient protein density for reliable detection of specific interactions while minimizing non-specific binding.

For SDS-PAGE analysis, both reducing and non-reducing conditions should be examined to properly assess the protein's structural integrity. Under reducing conditions, CLEC2L Fc Chimera appears at 39-44 kDa, while under non-reducing conditions, it shows bands at 80-90 kDa, reflecting its native dimeric state .

Proper reconstitution of lyophilized protein is crucial for maintaining activity. Manufacturers typically provide specific reconstitution instructions that should be followed precisely to ensure optimal protein conformation and function .

How can researchers differentiate between CLEC2L and other CLEC family members in experimental systems?

Differentiating between CLEC2L and other CLEC family members requires careful selection of detection methods and experimental controls:

• Antibody selection: Use highly specific antibodies validated for CLEC2L detection, such as CLEC-2L (D-5): sc-390424, which have been confirmed not to cross-react with other CLEC family proteins .

• Expression analysis: Take advantage of CLEC2L's predominant brain expression pattern. When working with non-neural tissues, detection of CLEC2L should be confirmed through multiple methods, as expression levels are expected to be significantly lower than in brain tissue .

• Molecular weight verification: The specific molecular weight pattern of CLEC2L (39-44 kDa under reducing conditions, 80-90 kDa under non-reducing conditions for the Fc chimera version) can help distinguish it from other CLEC family proteins .

• Functional assays: CLEC2L's specific binding to partners like Galectin-3 can be used to confirm identity through functional interaction studies .

• Genetic approaches: For definitive identification, genetic techniques such as specific siRNA knockdown or CRISPR-based targeting can confirm the identity of the protein being studied through loss-of-function effects.

How should researchers interpret contradictory findings regarding CLEC family proteins in disease contexts?

When interpreting contradictory findings regarding CLEC family proteins, researchers should carefully consider several factors that may contribute to discrepancies:

First, clear distinction between different CLEC proteins is essential. For example, while increased soluble CLEC-2 levels were previously hypothesized to serve as a biomarker of platelet activation and tumor progression, newer research demonstrates that platelet activation actually leads to decreased CLEC-2 expression and reduced soluble CLEC-2 levels in plasma. This contradicts earlier assumptions about CLEC-2 release mechanisms .

The table below illustrates this contradiction with data from platelet stimulation experiments:

This data clearly shows that contrary to previous hypotheses, platelet activation (confirmed by increased CD62P expression) coincides with decreased CLEC-2 expression and decreased soluble CLEC-2 in plasma, while total CLEC-2 protein levels remain unchanged .

When extending these considerations to CLEC2L research, investigators should maintain skepticism toward established paradigms and verify mechanisms through multiple experimental approaches. Tissue-specific effects, differences in experimental systems, and variations in detection methodologies may all contribute to apparently contradictory findings.

What are the future research directions for CLEC2L in neurological disorders?

Given CLEC2L's predominant expression in brain tissue, several promising research directions emerge for investigating its potential roles in neurological disorders:

• Neurodegenerative diseases: Researchers should investigate whether CLEC2L expression or function is altered in conditions like Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis. As a cell surface receptor, CLEC2L might influence neuroinflammatory processes or neuron-glia interactions relevant to neurodegeneration .

• Neuroimmune interactions: The C-type lectin domain structure suggests potential roles in recognition of specific carbohydrate moieties. Exploration of CLEC2L's interaction with microglia or infiltrating immune cells during neuroinflammation could reveal important regulatory mechanisms .

• Synaptic plasticity: Investigation of CLEC2L's potential involvement in synaptic formation, maintenance, or plasticity would be valuable, particularly through knockout or overexpression studies in neural culture systems or animal models.

• Biomarker development: While studies with CLEC-2 suggest caution in interpreting soluble lectin levels as disease biomarkers, carefully designed studies might evaluate whether cerebrospinal fluid levels of soluble CLEC2L correlate with specific neurological conditions .

• Therapeutic targeting: Development of tools to modulate CLEC2L expression or function, such as the CRISPR activation systems described in the literature, could enable evaluation of CLEC2L as a potential therapeutic target for neurological disorders .