Recombinant Chicken Nicalin (NCLN), partial

Basic Research Questions

• What is chicken Nicalin and what is its biological function?

  Chicken Nicalin (NCLN) is a transmembrane protein containing an aminopeptidase (AP) domain similar to that found in Nicastrin. It forms a complex with its binding partner Nomo (Nodal modulator), which is distinct from the γ-secretase complex. This Nicalin/Nomo complex plays a crucial role in modulating Nodal signaling during embryonic development .

  Methodologically, the function of Nicalin can be studied through:

  • Co-immunoprecipitation experiments to identify binding partners

  • Blue Native-PAGE analysis to determine complex formation (typically 500-550 kDa)

  • In vitro cell-based assays measuring Nodal-dependent signaling inhibition

  • In vivo functional studies in model organisms like zebrafish to observe mesendodermal patterning effects

• How is recombinant chicken Nicalin typically produced in laboratory settings?

  Recombinant chicken Nicalin can be produced using several expression systems:

  E. coli expression system:

  • Advantages: High yield, cost-effective, rapid production

  • Limitations: Lacks post-translational modifications, potential for inclusion body formation

  • Protocol typically involves:

    1. Cloning the chicken Nicalin gene into an appropriate expression vector with His-tag

    2. Transformation into competent E. coli cells

    3. Induction of protein expression with IPTG

    4. Purification via Ni-chelate chromatography

  Eukaryotic expression systems (preferred for functional studies):

  • Mammalian cells (HEK293, CHO) for proper folding and post-translational modifications

  • Baculovirus-insect cell system for higher yields with eukaryotic modifications

  • Yeast expression systems for glycosylated proteins

• What is the tissue distribution pattern of Nicalin in chickens?

  Chicken Nicalin shows a tissue-specific expression pattern similar to its binding partner Nomo, with:

  • Highest mRNA levels detected in pancreas, skeletal muscle, and heart

  • Moderate expression in other tissues including liver

  • Three different Nicalin mRNA transcripts (4.6, 4.0 and 2.3 kb) have been detected, which differ in the length of their 3′ untranslated regions and are generated through alternative polyadenylation

  Methodological approach: Tissue distribution can be studied using:

  • Northern blot analysis using labeled Nicalin-specific probes

  • Quantitative real-time PCR with primer pairs targeting conserved regions

  • In situ hybridization for localization studies

  • Immunohistochemistry with anti-Nicalin antibodies

• How can researchers differentiate between Nicalin and other AP domain-containing proteins?

  Differentiating Nicalin from other AP domain proteins requires:

  Sequence analysis:

  • Multiple sequence alignment to identify conserved and unique regions

  • Phylogenetic analysis to determine evolutionary relationships

  Structural characterization:

  • Domain organization analysis shows Nicalin contains an ∼200-amino-acid region with an AP-like fold

  • Unlike active aminopeptidases, Nicalin may lack catalytic residues

  Functional assays:

  • Specific inhibitors of aminopeptidases can be used to distinguish enzymatic activity

  • Co-immunoprecipitation with Nomo (its specific binding partner)

  • Nodal signaling modulation assays, as this is a specific function of the Nicalin/Nomo complex

Advanced Research Questions

• What are the optimal purification strategies for recombinant chicken Nicalin?

  Multi-step purification protocol:

  1. Initial preparation:

     • For membrane proteins like Nicalin, solubilization with appropriate detergents (e.g., DDM as used for Nicalin complexes) is critical

     • Centrifugation to separate soluble fraction (11,400 × g, 30 min, 4°C)

  2. Chromatography sequence:

     • Affinity chromatography: For His-tagged Nicalin, Ni-chelate chromatography

     • Ion exchange: DEAE-Sepharose CL-6B column using gradient elution with NaCl (0-0.1 M)

     • Size exclusion: Toyopearl HW65F column for final polishing

  3. Quality assessment:

     • SDS-PAGE with Coomassie staining for purity evaluation

     • Western blot with anti-Nicalin antibodies for identity confirmation

     • Blue Native-PAGE to verify intact complex formation (500-550 kDa)

  4. Maintaining complex integrity:

     • Avoid Triton X-100 which disrupts the Nicalin complex

     • Use DDM for complex preservation during co-immunoprecipitation studies

• How can next-generation sequencing approaches be used to study Nicalin expression?

  RNA-Seq methodology for Nicalin expression analysis:

  1. Experimental design:

     • Tissue collection from various chicken organs (following the pattern of highest expression in pancreas, skeletal muscle, and heart)

     • RNA extraction using FastPure Cell/Tissue Total RNA Isolation Kit

     • Library preparation and sequencing (Illumina platform)

  2. Bioinformatic analysis pipeline:

     • Quality control with FastQC and data processing with Trimmomatic

     • Genome alignment using HISAT2 followed by statistical alignment with RSeQC

     • Transcript assembly with StringTie RABT

     • Expression quantification using TPM (Transcripts Per Million) values

  3. Differential expression analysis:

     • Use DESeq2 for identifying significant differential expression (q-value ≤ 0.05)

     • STEM (Short Time-Series Expression Miner) for clustering co-expressed genes

  4. Functional annotation:

     • GO analysis using DAVID software and KEGG pathway analysis using KOBAS

     • Guilt-by-association principle to predict function based on co-expression patterns

• What are the considerations for designing functional assays for recombinant chicken Nicalin?

  Functional assay design protocol:

  1. Complex formation assays:

     • Co-immunoprecipitation with anti-Nicalin antibodies to detect Nomo interaction

     • Blue Native-PAGE (BN-PAGE) to analyze the intact Nicalin/Nomo complex (~500-550 kDa)

     • Size exclusion chromatography coupled with multi-angle light scattering for complex stoichiometry

  2. Nodal signaling inhibition assays:

     • Reporter gene assays using Nodal-responsive elements

     • Cell-based assays comparing control vs. Nicalin-overexpressing cells

     • Monitoring downstream effectors of Nodal signaling (e.g., Smad phosphorylation)

  3. Developmental biology assays:

     • Zebrafish embryo model to study mesendodermal patterning

     • Analysis of Lefty function modulation by the Nicalin/Nomo complex

     • Assessment of potential interaction with Activin-dependent signaling

  4. Controls and validation:

     • Use of wild-type vs. mutant Nicalin constructs

     • Dose-dependent response curves

     • Positive controls (known Nodal inhibitors) and negative controls

• How can genetic modification of chickens enhance production of recombinant Nicalin?

  Genetic engineering strategy for improved Nicalin production:

  1. CRISPR/Cas9 gene editing approaches:

     • Design of guide RNAs targeting specific loci for optimal expression

     • Integration of Nicalin expression cassettes into chicken genome

     • Potential targeting of the ovalbumin locus for egg white expression

  2. Promoter selection for tissue-specific expression:

     • Ovarian-specific promoters for production in egg white

     • Cytomegalovirus (CMV) promoter for high-level expression

     • Tissue-specific promoters based on Nicalin's natural expression pattern

  3. Optimizing post-translational modifications:

     • Engineering glycosylation patterns for enhanced activity

     • Considering that chicken-derived glycoproteins lack α1,3-Gal, which reduces immunogenicity

     • Enhancing sialylation by targeting expression in egg yolk rather than egg white, as yolk-derived proteins show higher sialylation levels

  4. Production and purification:

     • Eggs as a bioreactor system offering advantages including:

       • Short production cycle

       • High production efficiency

       • Lower research costs

       • Expression products closer to natural state

     • Purification from egg white or yolk using affinity chromatography

• What are the analytical methods for characterizing post-translational modifications of recombinant chicken Nicalin?

  Comprehensive PTM analysis workflow:

  1. Glycosylation analysis:

     • PNGase F treatment to remove N-linked glycans

     • UPLC-MS/MS analysis of released glycans

     • Lectin blot to determine sialic acid linkages (e.g., α2,6-sialic acid)

     • Quantification of different glycoforms as percentages of total glycan content

  2. Mass spectrometry-based approaches:

     • Tryptic digestion followed by LC-MS/MS for peptide mapping

     • Electron transfer dissociation (ETD) for intact glycopeptide analysis

     • MALDI analysis for peptide mass fingerprinting

  3. Site-specific modification analysis:

     • Site-directed mutagenesis of potential glycosylation sites

     • Comparison of mobility shifts before and after deglycosylation

     • Enrichment of glycopeptides prior to MS analysis

  4. Comparison of expression systems:

     • Analysis of glycosylation differences between E. coli (non-glycosylated), yeast, and avian expression systems

     • Evaluation of sialylation levels in egg yolk (higher) versus egg white (lower)

     • Assessment of fucosylation status, as chicken-derived proteins tend to be afucosylated

• How can RNA editing affect the expression and function of chicken Nicalin?

  RNA editing investigation methodology:

  1. Detection of RNA editing:

     • Deep sequencing of genomic DNA and mRNA to identify A-to-I or C-to-U substitutions

     • Analysis of conserved editing sites in Nicalin transcripts

     • RT-PCR and Sanger sequencing of individual clones to verify editing events

  2. Frequency analysis:

     • Quantification of edited versus non-edited transcripts

     • Investigation of tissue-specific editing patterns

     • Effects of editing on mRNA stability and translation efficiency

  3. Functional consequences:

     • Generation of recombinant constructs with edited sequences

     • Comparison of protein folding, complex formation, and signaling activity

     • Analysis drawing from studies of other proteins where RNA editing affects function

  4. Evolutionary analysis:

     • Comparison of RNA editing sites across species

     • Assessment of conservation of editing mechanisms in avian species

     • Investigation of potential compensatory mutations that affect editing frequency

• What are the approaches for studying Nicalin-Nomo protein-protein interactions?

  Protein interaction analysis protocol:

  1. Co-immunoprecipitation approaches:

     • Use of detergents that preserve complex integrity (DDM recommended)

     • Immunoprecipitation with anti-Nicalin antibodies followed by immunoblotting for Nomo

     • Reciprocal IP with anti-Nomo antibodies to confirm specificity

  2. Structural analysis methods:

     • X-ray crystallography of the purified complex

     • Cryo-EM for 3D structure determination

     • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  3. Biophysical characterization:

     • Surface plasmon resonance (SPR) for binding kinetics

     • Isothermal titration calorimetry (ITC) for thermodynamic parameters

     • Microscale thermophoresis for affinity measurements

  4. Computational approaches:

     • Molecular docking simulations

     • Molecular dynamics to study complex stability

     • Alanine scanning mutagenesis to identify critical residues for interaction

• How can chicken-specific culture systems be optimized for functional studies of recombinant Nicalin?

  Chicken cell culture optimization strategy:

  1. Cell line selection:

     • DF-1 chicken fibroblast cell line for stable expression

     • Chicken embryonic fibroblasts (CEFs) for primary culture

     • HD11 macrophage cell line for immune response studies

  2. Culture conditions:

     • Optimal temperature: 41°C for chicken cells versus 37°C for mammalian cells

     • Media composition: RPMI1640 supplemented with appropriate serum

     • Growth factors specific for avian cells

  3. Transfection methods:

     • Lipofection protocols optimized for chicken cells

     • Viral vector systems including RCAS (Replication-Competent ALV LTR with a Splice acceptor)

     • Electroporation parameters specific for chicken cell types

  4. Functional assays in chicken cells:

     • Stimulation protocols using chicken-specific cytokines and growth factors

     • RT-PCR using chicken-specific primers for quantifying gene expression

     • Flow cytometry with appropriate antibodies for chicken cell surface markers

Table 1: Comparison of Expression Systems for Recombinant Chicken Proteins

Table 2: Tissue Distribution of Nicalin and Binding Partner Nomo

Table 3: N-Glycan Profiles of Recombinant Proteins in Chicken Expression Systems