Recombinant Mouse Opalin (Opalin)

What is Opalin and where is it expressed?

Opalin, also called Tmem10 or oligodendrocytic paranodal loop protein, is a transmembrane protein detected specifically in mammalian oligodendrocytes. Its expression is predominantly localized to white matter-rich regions of the central nervous system (CNS) but is absent in the peripheral nervous system (PNS). Importantly, Opalin homologs are found exclusively in mammals and not in non-mammalian species with myelinated axons, suggesting a mammal-specific evolutionary role .

When is Opalin expressed during development?

Opalin expression is significantly upregulated during postnatal developmental stages, coinciding with active myelination. The protein's levels increase drastically at the postnatal myelination stage, suggesting its importance in the maturation of the myelin sheath. This temporal expression pattern correlates with oligodendrocyte differentiation and maturation processes .

What are the structural characteristics of recombinant mouse Opalin?

Recombinant mouse Opalin is a transmembrane sialylglycoprotein. The protein contains specific domains critical for its function, particularly the N-terminal domain which is essential for binding to LGI1. The functional recombinant protein maintains the native binding properties and can be used to study Opalin-LGI1 interactions in experimental settings .

What is the functional significance of Opalin?

Opalin functions as a receptor for LGI1, a secretory protein produced mainly by neurons that plays a critical role in myelination. Through this interaction, Opalin mediates oligodendrocyte differentiation. Conditional knockout of OPALIN in the oligodendrocyte lineage causes hypomyelination and white matter abnormalities (WMAs), similar to LGI1 deficiency, demonstrating its critical role in myelin formation .

How does the LGI1-Opalin signaling axis regulate oligodendrocyte differentiation?

The LGI1-Opalin interaction triggers downstream signaling that regulates transcription factors critical for oligodendrocyte differentiation, notably Sox10 and Olig2. Biochemical analysis of Opalin-deficient mice revealed downregulation of these transcription factors, providing strong evidence that the LGI1-Opalin signaling axis is crucial for proper oligodendrocyte maturation. This pathway represents a potential target for therapeutic interventions in demyelinating diseases .

What are the specific binding properties between recombinant Opalin and LGI1?

The N-terminal domain of Opalin is critical for LGI1 binding. Mutational studies have identified key residues in this interaction, particularly at positions K23 and D26. The OPALIN_K23A/D26A mutant fails to bind LGI1 and cannot rescue the hypomyelination phenotype in Opalin knockout mice when reintroduced through viral vectors. This suggests these specific amino acid residues are essential for the functional interaction between Opalin and LGI1 .

How does conditional knockout of Opalin affect oligodendrocyte lineage cells?

Conditional knockout (cKO) of Opalin specifically in the oligodendrocyte lineage results in hypomyelination and white matter abnormalities, similar to LGI1 deficiency. The phenotype can be rescued by virus-mediated re-expression of wild-type Opalin but not by the LGI1-unbound OPALIN_K23A/D26A mutant. This confirms that Opalin's function in oligodendrocytes is dependent on its ability to bind LGI1 and that this interaction is critical for proper myelination in the CNS .

What are optimal protocols for producing and purifying recombinant mouse Opalin?

For producing recombinant mouse Opalin, mammalian expression systems are recommended to ensure proper post-translational modifications, particularly sialylation which may be important for function. The recombinant protein can be designed with a signal sequence and appropriate tags for purification. For purification, affinity chromatography using anti-Opalin antibodies or tag-specific resins followed by size-exclusion chromatography has proven effective. Care should be taken to maintain the native conformation, especially of the N-terminal domain critical for LGI1 binding .

How should recombinant Opalin be stored and handled for optimal activity?

Recombinant Opalin, like many transmembrane proteins, requires careful handling to maintain activity. Based on similar proteins, lyophilization in the presence of stabilizers may be appropriate. For reconstitution, gentler buffers that maintain protein conformation are recommended. After reconstitution, the protein should be stored in small aliquots at -20°C to -80°C to avoid freeze-thaw cycles. Do not freeze when still lyophilized; store at +4°C in this state .

What experimental systems are most suitable for studying Opalin function?

Several experimental systems have proven effective for studying Opalin function:

• In vitro oligodendrocyte cultures: Primary cultures or oligodendrocyte cell lines can be treated with recombinant Opalin or LGI1 to assess effects on differentiation.

• Knockout mouse models: Both conventional and conditional knockout approaches have provided valuable insights into Opalin function.

• Slice cultures: Ex vivo brain slice cultures allow for the study of Opalin in a more intact system with preserved cell-cell interactions.

• Binding assays: Surface plasmon resonance or co-immunoprecipitation can be used to study the interaction between Opalin and LGI1 or to screen for other potential binding partners .

What controls and validation steps are critical when working with recombinant Opalin?

When working with recombinant Opalin, several validation steps are essential:

Additionally, including both positive controls (wild-type Opalin) and negative controls (non-binding mutants like OPALIN_K23A/D26A) in functional assays is critical for result interpretation .

How can recombinant Opalin be used to study demyelinating diseases?

Recombinant Opalin can serve as a valuable tool for studying demyelinating diseases through several approaches:

• Therapeutic screening: The protein can be used to screen for compounds that enhance LGI1-Opalin interaction or downstream signaling.

• Biomarker development: Detecting alterations in Opalin expression or its fragments in patient samples may serve as biomarkers for myelin integrity.

• Disease modeling: Recombinant Opalin can be used in cellular models of demyelinating diseases to assess its potential protective or restorative effects.

• Mechanistic studies: The protein allows for detailed investigation of molecular pathways disrupted in demyelinating conditions .

What insights can Opalin provide into mammalian-specific aspects of myelination?

Since Opalin is exclusive to mammals and absent in non-mammalian vertebrates with myelinated axons, it offers unique insights into mammalian-specific aspects of myelination. Studying recombinant Opalin can help elucidate:

• The evolutionary adaptations in mammalian myelin that may contribute to advanced cognitive functions

• Specialized paranodal structures unique to mammalian CNS

• Differential regulation of oligodendrocyte maturation in mammals versus non-mammals

• Potential therapeutic targets specific to human demyelinating diseases

How can recombinant Opalin be used to investigate neuron-oligodendrocyte communication?

Recombinant Opalin provides a powerful tool for studying neuron-oligodendrocyte communication, particularly through the LGI1-Opalin axis. This protein can be used to:

• Map the spatial and temporal dynamics of LGI1-Opalin signaling during development

• Identify additional components of this signaling pathway through protein interaction studies

• Develop co-culture systems where neuronal LGI1 and oligodendrocyte Opalin interactions can be manipulated

• Investigate how electrical activity in neurons might regulate Opalin expression and function in oligodendrocytes

What are promising avenues for therapeutic applications targeting the Opalin-LGI1 axis?

Given Opalin's role as an LGI1 receptor that promotes oligodendrocyte differentiation, several therapeutic strategies could be developed:

• Small molecule enhancers: Compounds that enhance LGI1-Opalin binding might promote remyelination.

• Gene therapy approaches: Viral delivery of Opalin to demyelinated regions could potentially restore proper myelination.

• Peptide mimetics: Designed peptides that mimic the active domains of Opalin might activate downstream signaling pathways.

• Combined approaches: Therapies targeting both neuronal LGI1 secretion and oligodendrocyte Opalin expression might provide synergistic benefits in treating demyelinating diseases .

What technological developments would advance Opalin research?

Several technological advancements would significantly benefit Opalin research:

• Improved recombinant production systems: Development of expression systems that better maintain the native structural properties of Opalin.

• Advanced imaging techniques: Methods to visualize Opalin-LGI1 interactions in live tissue would provide important temporal and spatial information.

• Single-cell analysis tools: Techniques to assess Opalin function in individual oligodendrocytes at different maturation stages.

• In vivo sensors: Development of biosensors to monitor Opalin activity in real-time in animal models .