Recombinant Mouse Integral membrane protein 2C (Itm2c)

What is the structural composition of Mouse Itm2c protein?

Mouse Integral membrane protein 2C (Itm2c) is a type II integral transmembrane protein with its N-terminus located intracellularly and C-terminus positioned either extracellularly or within a luminal organelle domain. The protein consists of 269 amino acids with a calculated molecular weight of 30,482 Da and a theoretical isoelectric point (pI) of 8.83 . The full amino acid sequence is:

MVKISFQPAVAGIKADKADKAAASGPASASAPAAEILLTPAREERPPRHRSRKGGSVGGVCYLSMGMVVLLMGLVFASVYIYRYFFLAQLARDNFFHCGVLYEDSLSSQIRTRLELEEDVKIYLEENYERINVPVPQFGGGDPADIIHDFQRGLTAYHDISLDKCYVIELNTTIVLPPRNFWELLMNVKRGTYLPQTYIIQEEMVVTEHVRDKEALGSFIYHLCNGKDTYRLRRRSTRRRRINKRGGKNCNAIRHFENTFVVETLICGVV

When conducting structural analysis, researchers should note that the protein contains a single putative N-glycosylation site at amino acid position 171 (Asn), which is conserved across all three members of the Itm2 family in both mice and humans .

How does Itm2c expression vary across tissue types?

Mouse Itm2c exhibits a distinctive tissue-specific expression pattern that differs from other members of the Itm2 family. Northern blot analysis has detected a single ~2.1 kb transcript at varying levels across different tissues. Notably, Itm2c is highly expressed in both adult and postimplantation embryonic brain tissues, with lower expression levels detected in other adult tissues .

RT-PCR analyses of various adult tissues confirm that Itm2c expression is highest in the adult brain, suggesting a potential specialized neurological function. This brain-specific expression pattern distinguishes Itm2c from other members of the Itm2 family, indicating possible unique roles in neuronal development and function . Researchers investigating Itm2c should consider this tissue distribution pattern when designing experiments and interpreting results.

What are the recommended storage and handling protocols for recombinant Itm2c?

For optimal stability and experimental reproducibility, recombinant Itm2c protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple uses. Repeated freeze-thaw cycles should be strictly avoided as they can compromise protein integrity and function .

For reconstitution, follow this methodological approach:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) to the solution

• Aliquot for long-term storage at -20°C/-80°C (50% is the recommended final glycerol concentration)

• Thaw aliquots on ice before use

When working with the protein, store working aliquots at 4°C for up to one week to minimize degradation. The protein is typically supplied in Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain stability .

What biological functions have been attributed to Itm2c?

Itm2c serves important regulatory functions in several neurological pathways:

These diverse functions position Itm2c at the intersection of neurodegeneration, development, and cell survival pathways, making it a compelling target for neuroscience and neurological disease research.

How does Itm2c relate to other members of the Itm2 family?

The Itm2 family consists of three main members: Itm2a, Itm2b, and Itm2c. Sequence analysis reveals that mouse Itm2c shares 41% amino acid identity with Itm2a and 49% with Itm2b, indicating moderate conservation within this protein family .

• Itm2c: Primarily expressed in adult brain tissues

• Itm2a/Itm2b: More widely distributed expression patterns

ITM2B is considered an important paralog of ITM2C, and mutations in ITM2B are associated with Cerebral Amyloid Angiopathy . Despite their similarities, each family member likely serves specialized functions, with Itm2c's brain-specific expression pattern suggesting a unique role in neuronal biology compared to its paralogs.

What experimental applications is recombinant Itm2c suitable for?

Recombinant Mouse Itm2c protein is suitable for multiple experimental applications in basic and translational research:

• SDS-PAGE analysis: The protein can be used as a standard for molecular weight comparison and identification .

• Antibody production: High-purity recombinant Itm2c serves as an excellent antigen for generating specific antibodies against the protein .

• Protein-protein interaction studies: Tagged versions of the protein facilitate pull-down assays, co-immunoprecipitation, and other interaction studies to identify binding partners.

• Functional assays: The protein can be employed in enzymatic and binding assays to study its interaction with amyloid-beta and other potential substrates or binding partners .

• Structural studies: Highly purified protein can be used for crystallography or other structural determination methods.

When selecting recombinant Itm2c for these applications, researchers should consider the expression system (E. coli vs. mammalian cells) based on their specific experimental requirements, particularly regarding post-translational modifications.

What methodological approaches can optimize recombinant Itm2c expression and purification?

For researchers seeking to express and purify recombinant Itm2c, several methodological considerations can enhance yield and quality:

Expression Systems:

• E. coli: Suitable for basic structural studies and applications not requiring mammalian post-translational modifications. The protein with His-tag has been successfully expressed in E. coli systems .

• HEK-293 Cells: Preferable for studies requiring mammalian glycosylation patterns and proper protein folding, particularly important given the N-glycosylation site at position 171 .

• Cell-free protein synthesis (CFPS): An alternative approach that can yield functional protein with advantages in scaling and modification options .

Purification Strategy:

• Employ affinity chromatography using the appropriate tag (His, Strep, or Myc-DYKDDDDK)

• Include protease inhibitors during extraction to prevent degradation

• Optimize buffer conditions (pH 8.0 appears suitable based on storage buffer composition)

• Consider size exclusion chromatography for higher purity applications

• Validate protein quality using multiple methods including SDS-PAGE, Western blotting, and analytical SEC (HPLC)

For transmembrane proteins like Itm2c, the addition of mild detergents during extraction and purification may improve solubility and native conformation retention. Purity levels greater than 90% are achievable with optimized protocols, as demonstrated in commercial preparations .

How can researchers investigate Itm2c's role in neurodegenerative pathways?

To explore Itm2c's involvement in neurodegenerative processes, particularly its interaction with amyloid-beta pathways, researchers can implement these experimental approaches:

• Cell-based APP processing assays:

  • Transfect neuronal cell lines with Itm2c expression vectors

  • Measure APP processing products (sAPPα, sAPPβ, Aβ peptides) by ELISA or Western blot

  • Use dose-response studies to assess concentration-dependent effects

  • Compare wild-type Itm2c with mutated versions to identify functional domains

• Secretase accessibility assays:

  • Develop co-immunoprecipitation protocols to assess Itm2c binding to APP

  • Investigate whether Itm2c physically blocks alpha- or beta-secretase access to APP

  • Use proximity ligation assays to visualize protein interactions in situ

• Transgenic mouse models:

  • Generate conditional Itm2c knockout or overexpression mouse models

  • Assess neuronal morphology, particularly dendritic complexity given Itm2c's role in neuron projection development

  • Analyze amyloid pathology in aged mice or when crossed with AD model mice

  • Perform behavioral testing to correlate molecular findings with cognitive outcomes

• Comparative analysis with Itm2b:

  • Since Itm2b mutations are linked to Cerebral Amyloid Angiopathy , comparative functional studies between Itm2c and Itm2b could reveal shared mechanisms

  • Develop domain-swapping experiments to identify functional differences between family members

These approaches should be complemented with careful controls and validation using both in vitro and in vivo systems to establish physiologically relevant findings.

What techniques are appropriate for studying Itm2c's N-glycosylation and its functional significance?

The conserved N-glycosylation site at position 171 (Asn) in Itm2c warrants specific investigation approaches:

• Site-directed mutagenesis:

  • Generate Asn171→Gln mutant versions of Itm2c

  • Compare subcellular localization of wild-type and glycosylation-deficient mutants using fluorescent protein tags

  • Assess functional differences in amyloid-beta binding and APP processing

• Glycosylation analysis:

  • Employ endoglycosidases (PNGase F, Endo H) to remove N-linked glycans

  • Analyze mobility shifts by Western blotting to confirm glycosylation status

  • Use lectin affinity chromatography to isolate glycosylated forms

  • Apply mass spectrometry for detailed glycan composition analysis

• Trafficking studies:

  • Track protein movement through secretory pathways using pulse-chase experiments

  • Compare wild-type versus glycosylation-deficient mutants

  • Assess co-localization with Golgi, lysosomal, and plasma membrane markers

• Protein stability assessment:

  • Determine half-life differences between glycosylated and non-glycosylated forms

  • Use cycloheximide chase assays to monitor degradation rates

  • Investigate whether glycosylation affects proteolytic processing

This multifaceted approach can reveal whether N-glycosylation primarily affects protein stability, trafficking, binding interactions, or enzymatic function, providing insights into this conserved post-translational modification.

How might Itm2c serve as a potential biomarker in disease contexts?

Recent research suggests potential biomarker applications for Itm2c, particularly in colorectal cancer (CRC):

Analysis of The Cancer Genome Atlas (TCGA) colorectal cancer dataset identified ITM2C as part of a gene signature with significant diagnostic potential. When combined with CA7 and CA2 genes, ITM2C showed remarkable classification accuracy:

ROC curve analysis indicated an impressive AUC value of 0.995 for ITM2C in CRC detection .

To investigate Itm2c's biomarker potential, researchers should consider:

• Expression profiling across disease stages and subtypes

• Correlation analysis with established disease markers and patient outcomes

• Functional validation through knockdown/overexpression studies

• Development of detection methods in accessible clinical samples

While current evidence points to potential in CRC detection, similar approaches could be explored for neurological conditions given Itm2c's prominent brain expression and involvement in neurodegenerative pathways .

What control strategies should be implemented when studying Itm2c function?

Robust experimental design for Itm2c research requires carefully selected controls:

• Protein expression controls:

  • Use empty vector transfections alongside Itm2c expression constructs

  • Include related family members (Itm2a, Itm2b) as specificity controls

  • For tagged versions, create constructs with the tag alone to control for tag-mediated effects

• Tissue-specific considerations:

  • Given Itm2c's brain-predominant expression , include non-neuronal tissues as negative controls

  • When studying neuronal effects, compare multiple brain regions with varying Itm2c expression levels

• Functional redundancy assessment:

  • In knockout/knockdown studies, measure expression of Itm2a and Itm2b to detect compensatory upregulation

  • Consider double or triple knockdowns to address functional redundancy

• Species comparisons:

  • Where appropriate, compare mouse and human Itm2c to validate conserved functions

  • Note that mouse Itm2c has 269 amino acids versus 267 in human , which may affect certain interaction studies

• Glycosylation controls:

  • Include enzymatically deglycosylated protein preparations

  • Utilize glycosylation-site mutants alongside wild-type protein

These control strategies will strengthen data interpretation and minimize confounding factors in Itm2c research.

How can researchers effectively investigate Itm2c interactions with amyloid-beta pathways?

To elucidate the mechanisms through which Itm2c regulates amyloid-beta pathways, researchers should implement these specialized techniques:

• Direct binding assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics between purified Itm2c and various amyloid-beta species

  • ELISA-based binding assays with immobilized Itm2c or amyloid-beta

  • Employ proper controls including heat-denatured proteins and competitive binding assays

• Cellular APP processing models:

  • Establish dose-dependent effects of Itm2c expression on APP processing

  • Quantify all APP processing products (sAPPα, sAPPβ, CTFs, Aβ peptides)

  • Use pharmacological secretase inhibitors to determine which processing step is affected

• Secretase enzyme activity assays:

  • Determine whether Itm2c directly inhibits secretase enzymatic activity using fluorogenic substrates

  • Compare with known secretase inhibitors to quantify inhibition efficiency

• Domain mapping:

  • Generate truncated versions of Itm2c to identify which domains are essential for APP processing effects

  • Create chimeric proteins with domains from Itm2a or Itm2b to determine family-specific functions

• Live-cell imaging:

  • Use fluorescently tagged Itm2c and APP to visualize interactions in real time

  • Employ FRET (Förster Resonance Energy Transfer) to confirm protein proximity

These approaches will help establish whether Itm2c acts through direct binding to APP, affects secretase access, directly inhibits secretase activity, or functions through alternative mechanisms .

What approaches can resolve contradictory findings in Itm2c research?

When facing contradictory results in Itm2c studies, researchers should systematically address potential sources of discrepancy:

This structured approach can help resolve contradictions and advance the field toward consensus on Itm2c functions and mechanisms.

What emerging technologies could advance understanding of Itm2c biology?

Several cutting-edge technologies hold promise for deepening our understanding of Itm2c:

• Cryo-electron microscopy (Cryo-EM):

  • Determine high-resolution structure of Itm2c alone and in complex with interaction partners

  • Reveal conformational changes associated with binding events

  • Visualize membrane topology and protein dynamics

• CRISPR-based approaches:

  • Generate precise knockin mutations to model disease-relevant variants

  • Create reporter cell lines with endogenous tagging of Itm2c

  • Implement CRISPR activation/interference for dose-dependent functional studies

• Single-cell transcriptomics:

  • Map Itm2c expression across brain cell types at single-cell resolution

  • Identify co-expressed gene networks to reveal functional associations

  • Track expression changes during development and in disease states

• Proteomics technologies:

  • Apply proximity labeling (BioID, APEX) to identify Itm2c's proximal interactome

  • Use quantitative interaction proteomics to map dynamic protein complexes

  • Employ phosphoproteomics to reveal signaling pathways affected by Itm2c

• Organoid and iPSC-based models:

  • Study Itm2c function in human brain organoids

  • Compare effects in organoids derived from healthy versus disease-affected individuals

  • Model developmental roles using differentiation protocols

These technologies will help address current knowledge gaps and potentially reveal unexpected functions and interactions of Itm2c in neuronal biology and disease contexts.

How might understanding Itm2c biology contribute to therapeutic development?

The unique characteristics and functions of Itm2c suggest several therapeutic avenues worth exploring:

• Amyloid modulation strategies:

  • Develop peptide mimetics based on Itm2c domains that inhibit APP processing

  • Design small molecules that enhance Itm2c's native amyloid-regulatory function

  • Create antibodies that stabilize Itm2c-APP interactions

• Neuronal development applications:

  • Exploit Itm2c's role in neuronal differentiation for stem cell therapies

  • Develop modulators of Itm2c function to promote neuronal repair after injury

  • Target downstream pathways for neurodevelopmental disorders

• Biomarker implementation:

  • Leverage Itm2c's diagnostic potential in colorectal cancer

  • Explore similar applications in neurological conditions

  • Develop sensitive detection methods for clinical samples

• Combination approaches:

  • Identify synergistic effects with existing amyloid-targeting therapies

  • Develop dual-targeting strategies addressing multiple disease mechanisms

These therapeutic directions should be pursued with careful attention to Itm2c's brain-specific expression pattern and potential for both on-target and off-target effects in various tissues.