Recombinant Pongo abelii N-terminal EF-hand calcium-binding protein 1 (NECAB1)

What is NECAB1 and what are its structural characteristics?

NECAB1 (N-terminal EF-hand calcium-binding protein 1), also known as EFCBP1, is a member of the NECAB family of neuronal calcium-binding proteins . The full-length human NECAB1 cDNA spans 5229 bp and encodes a 351 amino acid protein containing two EF-hand calcium-binding motifs and a C-terminal antibiotic biosynthesis monooxygenase (ABM) domain . This protein shares 49.9% and 56.8% global homology with human NECAB2 and NECAB3, respectively . The EF-hand domains are responsible for calcium binding, while the ABM domain is functionally less characterized but is evolutionarily ancient and similar to atypical heme oxygenases in prokaryotes .

What is the evolutionary conservation of NECAB1 across species?

NECAB1 shows significant conservation across mammalian species, though with some species-specific expression patterns. While the gene is present in various primates including Pongo abelii (Sumatran orangutan) , its distribution pattern in brain tissues shows some variability between species. For example, when comparing mice and European moles, NECAB1 stains a subset of granule cells in moles but is absent from dentate granule cells in mice . Despite these differences, the core structure and major expression patterns are relatively conserved, suggesting evolutionary importance of this protein's function .

What is the primary tissue distribution of NECAB1?

NECAB1 is predominantly expressed in the central nervous system . Northern hybridization has revealed that NECAB1 is specifically expressed in brain tissues with varying abundance across different brain regions . The highest expression levels are found in:

• Temporal lobe (3.4-fold higher than average cerebral cortex expression)

• Frontal lobe (1.9-fold higher)

• Occipital pole (1.5-fold higher)

Equal expression levels are observed in the putamen and cerebral cortex, while no hybridization signal is detected in the spinal cord . Within the brain, NECAB1 has been identified in the cerebral cortex, hippocampus, basolateral amygdala, and striatum .

How does NECAB1 expression compare to other calcium-binding proteins in neurons?

Unlike classical neuronal calcium-binding proteins (parvalbumin, calbindin, and calretinin) that are expressed in specific, limited neuronal populations, NECAB1 is expressed in a much larger proportion of neurons . In dorsal root ganglia (DRG), NECAB1 is found in approximately 65% of all neuron profiles, predominantly in small and medium-sized neurons . Colocalization studies have shown that:

This distribution pattern suggests that NECAB1 serves distinct physiological functions from classical calcium-binding proteins .

What are the optimal conditions for expressing recombinant Pongo abelii NECAB1?

For expression of recombinant Pongo abelii NECAB1, baculovirus expression systems have proven effective . The full-length protein (amino acids 1-275) can be produced with optimal results when expressed with appropriate tags (determined during the manufacturing process) . For storage and stability:

• Use deionized sterile water for reconstitution to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Store at -20°C/-80°C for up to 12 months (lyophilized form) or 6 months (liquid form)

Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided as it may compromise protein integrity .

What antibodies and detection methods are most effective for NECAB1 research?

Several validated antibodies have been developed for NECAB1 detection:

• Rabbit polyclonal antibodies (e.g., ab224450) that are suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) and Western blotting (WB)

• For optimal IHC-P results, a dilution of 1/1000 has been validated for detecting NECAB1 in human pancreas, colon, cerebral cortex, and liver tissues, as well as mouse parietal association cortex and hippocampus

For tissue staining and cellular localization studies, both standard immunohistochemistry and advanced techniques like CLARITY have been successfully employed . The CLARITY method provides particular advantages for visualizing the NECAB1 system in three dimensions, revealing features such as the bilateral connection of NECAB1-positive neurons with dendrites that embrace the dorsal columns .

How can I differentiate between NECAB1, NECAB2, and NECAB3 in experimental settings?

Differentiation between the three NECAB family members requires careful selection of detection methods:

• Antibody selection: Use antibodies that have been validated for specificity against each NECAB family member. Early studies using antisera were cross-reactive between NECAB proteins, leading to misinterpretation of expression patterns .

• Subcellular localization: Each NECAB protein has a distinct subcellular distribution pattern that can aid in identification:

  • NECAB1: Primarily in the nucleus and cytosol

  • NECAB2: Predominantly in endosomes and plasma membrane

  • NECAB3: Mainly in the endoplasmic reticulum and Golgi apparatus

• Expression patterns: In the spinal cord, NECAB1 and NECAB2 show complementary, non-overlapping distribution patterns, which can be used as a distinguishing feature . In the hippocampus, NECAB1 is expressed throughout cell-sparse layers of Ammon's horn and the hilus of the dentate gyrus, while NECAB2 is enriched in pyramidal cells of the CA2 region .

• PCR primers: Design region-specific primers that target unique sequences in each NECAB gene for quantitative PCR differentiation.

How does NECAB1 contribute to neuronal calcium signaling and what methodologies best capture this function?

NECAB1 plays a significant role in neuronal calcium signaling, and several methodologies can be employed to investigate this function:

• Calcium imaging techniques: Using fluorescent calcium indicators (e.g., Fura-2, Fluo-4) in NECAB1-expressing neurons to measure real-time changes in intracellular calcium levels in response to various stimuli.

• Electrophysiology: Patch-clamp recordings combined with calcium imaging can correlate electrical activity with calcium dynamics in neurons expressing NECAB1.

• CRISPR-Cas9 gene editing: Knockout or mutation of NECAB1 in neuronal cultures or animal models followed by functional calcium signaling assays can reveal its specific contributions.

• Protein-protein interaction studies: Since NECAB1 associates with synaptotagmin-1, a critical protein involved in membrane trafficking and synaptic vesicle exocytosis , co-immunoprecipitation or proximity ligation assays can elucidate how this interaction affects calcium-dependent neurotransmitter release.

In research settings, it's important to note that NECAB1's functional impact on calcium signaling may differ between neuronal subtypes. For example, in CB1 cannabinoid receptor/cholecystokinin (CB1/CCK)-positive interneurons, NECAB1 and NECAB2 show subcellular nanoscale differences that suggest a functional division of labor between these calcium-binding proteins .

What role does NECAB1 play in pain pathways and how can this be investigated?

NECAB1 has significant expression in pain-related neural circuits, particularly in dorsal root ganglia (DRG) neurons and spinal cord pathways associated with nociception . To investigate its role in pain:

• Phenotypic characterization: In DRGs, approximately 65% of neurons express NECAB1, with the following phenotypic distribution:

  Neuronal Marker	% of NECAB1+ neurons
  CGRP (peptidergic)	~40%
  IB4 (nonpeptidergic)	42.5 ± 2.5%
  NF200 (myelinated)	25-30%

  This broad distribution suggests involvement in multiple aspects of somatosensory processing .

• Pain model studies: Examine changes in NECAB1 expression following peripheral nerve injury in animal models of neuropathic pain. Unlike NECAB2, which is downregulated after peripheral nerve injury, NECAB1 expression remains stable .

• Functional manipulation: Use intrathecal administration of NECAB1-targeting siRNA or viral vectors expressing NECAB1 shRNA to assess the behavioral consequences of NECAB1 knockdown in pain models.

• Anatomical characterization: NECAB1 labels interneurons and a plexus of processes in superficial layers of the spinal dorsal horn, as well as commissural neurons in the intermediate area, suggesting involvement in both local and long-range pain processing circuits .

How does NECAB1 expression relate to pathological conditions and what research approaches can explore these connections?

NECAB1 has been implicated in several pathological conditions, which can be investigated through various research approaches:

• Developmental language disorders and autism spectrum disorders:

  • Missense mutations in NECAB1 have been linked to developmental language disorders, a common co-occurring condition in individuals with autism spectrum disorder

  • Research approach: Genotype-phenotype correlation studies in patient cohorts, functional characterization of identified mutations using cell culture models

• Schizophrenia:

  • Expression analysis suggests NECAB1 could serve as a biomarker for schizophrenia

  • Research approach: Transcriptomic analysis of post-mortem brain samples, correlation with clinical phenotypes, animal models of schizophrenia-like symptoms

• Diabetes mellitus:

  • NECAB1 is induced in pancreatic β cell lines upon exposure to cortisol or corticosterone and in pancreatic islets of obese db/db mice

  • NECAB1 may negatively regulate glucose-induced insulin secretion in pancreatic β-cells

  • Research approach: In vitro models using pancreatic β-cell lines, ex vivo islet studies, genetic manipulation in diabetes animal models

• Pulmonary fibrosis and cardiovascular events:

  • Correlatory evidence suggests NECAB1 could be a potential diagnostic biomarker for immune infiltration in idiopathic pulmonary fibrosis and for major adverse cardiovascular events in patients with acute coronary syndromes

  • Research approach: Biomarker validation studies, mechanistic investigations in relevant tissue samples

What is the current understanding of the antibiotic biosynthesis monooxygenase (ABM) domain in NECAB1 and how can its function be investigated?

The C-terminal antibiotic biosynthesis monooxygenase (ABM) domain in NECAB1 is functionally less well characterized but evolutionarily ancient . Current understanding and investigation approaches include:

• Evolutionary analysis: The ABM domain in NECAB1 is similar to atypical heme oxygenases in prokaryotes where these domains typically form dimers . Comparative genomics and phylogenetic analyses can provide insights into the evolutionary origin and conservation of this domain.

• Structural biology approaches: X-ray crystallography or cryo-EM studies of the isolated ABM domain or the full-length NECAB1 protein can reveal structural features that might suggest function.

• Biochemical assays: Since prokaryotic ABM domains often have enzymatic activity, in vitro assays to test potential substrates could reveal if the ABM domain in NECAB1 retains any enzymatic function.

• Domain interaction studies: Calcium binding to the EF-hand domains might induce conformational changes that affect the ABM domain. FRET-based approaches or limited proteolysis experiments can test if calcium binding alters the conformation or accessibility of the ABM domain.

• Hypothesized mechanisms: Current hypotheses suggest that calcium-mediated conformational changes might:

  • Induce ABM domain dimerization, potentially activating enzymatic properties

  • Influence protein interactions by controlling the availability of protein-protein interaction domains situated between the EF hands and the ABM domain

  • Affect subcellular localization of NECAB proteins

What are the major discrepancies in NECAB1 research findings and how can they be reconciled?

Several discrepancies exist in the NECAB1 literature that require careful consideration:

• Expression pattern contradictions:

  • Early studies described NECAB1 expression primarily in the striatum and layer 4 of the rat cerebral cortex, but later studies showed different patterns including interneurons in the hippocampus, lumbar dorsal root ganglia, spinal cord neurons, and principal neurons in the somatosensory cortex and basolateral amygdala

  • Resolution approach: The antisera used in early studies were cross-reactive to NECAB2 and NECAB3 . Using highly specific antibodies or complementary techniques like in situ hybridization can help resolve these discrepancies.

• Species differences in expression:

  • NECAB1 stains a subset of granule cells in moles while it is absent from dentate granule cells in mice

  • Resolution approach: Systematic comparative studies across species using identical methodologies to determine genuine species differences versus technical variations.

• Functional roles:

  • While NECAB1 is proposed as a marker for CB1/CCK-containing GABAergic interneurons , its functional significance in these cells remains poorly understood

  • Resolution approach: Cell-type specific knockout or knockdown studies combined with functional assays.

What novel technologies or approaches could advance NECAB1 research?

Several emerging technologies could significantly advance our understanding of NECAB1:

• Single-cell multi-omics: Combining single-cell RNA sequencing with proteomics and epigenomics to comprehensively characterize NECAB1-expressing cells across different brain regions and physiological states.

• CRISPR-based screening: Genome-wide or targeted CRISPR screens to identify genes that functionally interact with NECAB1.

• Optogenetic and chemogenetic tools: Development of cell-type specific tools to manipulate NECAB1-expressing neurons in vivo to understand their circuit functions.

• Advanced calcium imaging: Development of genetically encoded calcium indicators that specifically report on calcium binding to NECAB1 rather than global calcium changes.

• In situ protein interaction mapping: Techniques like proximity labeling combined with mass spectrometry to map the interactome of NECAB1 in its native cellular context.

• Cryo-electron tomography: To visualize NECAB1 in its native cellular environment at near-atomic resolution.

• Biosensors: Development of FRET-based biosensors to monitor NECAB1 conformational changes in response to calcium binding in real-time.

How can researchers address the challenges in studying the functional effects of NECAB1 mutations associated with neurological disorders?

Addressing the challenges in studying NECAB1 mutations requires a multi-faceted approach:

• Patient-derived models: Generate induced pluripotent stem cells (iPSCs) from patients with NECAB1 mutations and differentiate them into relevant neuronal subtypes to study cellular phenotypes.

• Precision gene editing: Create isogenic cell lines that differ only in the NECAB1 mutation of interest to control for genetic background variability.

• Animal models: Develop knock-in mouse models carrying human NECAB1 mutations to study effects at circuit and behavioral levels.

• Domain-specific functional assays: Develop assays that specifically test how mutations affect:

  • Calcium binding affinity and kinetics

  • Protein-protein interactions, particularly with synaptotagmin-1

  • Subcellular localization

  • Potential enzymatic activity of the ABM domain

• Translational biomarkers: Identify measurable correlates of NECAB1 dysfunction that could be used in clinical studies.

• High-throughput drug screening: Develop cellular assays suitable for screening compounds that might rescue phenotypes associated with NECAB1 mutations.

How does NECAB1 research intersect with the broader field of calcium signaling in cellular physiology?

NECAB1 research offers important insights into calcium signaling that extend beyond neuroscience:

• Calcium buffering diversity: NECAB1 represents a calcium-binding protein with expression patterns distinct from classical calcium buffers (parvalbumin, calbindin, calretinin), suggesting specialized roles in calcium homeostasis .

• Cell-type specific calcium handling: The predominant expression of NECAB1 in CB1/CCK-positive interneurons correlates with their unique calcium transient properties compared to other interneuron types , highlighting how different cell types evolved specialized calcium handling mechanisms.

• Calcium-dependent protein interactions: NECAB1 associates with synaptotagmin-1 , suggesting roles in calcium-dependent regulation of protein complex formation and function.

• Non-neuronal calcium signaling: Recent findings of NECAB1 involvement in pancreatic β-cell function demonstrate how neuronal calcium-binding proteins can have important roles in other tissues .

• Calcium domains and microdomains: The distinct subcellular localization of NECAB proteins points to their potential roles in regulating calcium signaling within specific cellular compartments.

What implications does NECAB1 research have for understanding and treating metabolic disorders?

Recent research has revealed unexpected connections between NECAB1 and metabolic regulation:

• Pancreatic β-cell function: NECAB1 is induced in pancreatic β cell lines upon exposure to glucocorticoids (cortisol, corticosterone) and in the pancreatic islets of obese db/db mice . This induction suppresses insulin secretion by reducing intracellular calcium levels .

• Glucocorticoid receptor pathway: NECAB1 upregulation is dependent on glucocorticoid receptor (GR) activation, with binding of the GR to upstream regions of the NECAB1 gene being essential for this effect . This finding connects stress hormone signaling to β-cell dysfunction through NECAB1.

• Adipoinsular axis: NECAB1 may play a novel role in the adipoinsular axis and could be involved in the pathophysiology of obesity-related diabetes mellitus . This suggests therapeutic potential in targeting NECAB1 for metabolic disorders.

• Research implications:

  • Developing NECAB1 inhibitors could potentially enhance insulin secretion in type 2 diabetes

  • Measuring NECAB1 levels might serve as a biomarker for stress-induced β-cell dysfunction

  • Investigating the relationship between NECAB1 and other calcium-dependent processes in metabolic tissues could reveal new regulatory mechanisms

How can computational and structural biology approaches enhance our understanding of NECAB1 function?

Computational and structural biology approaches offer powerful tools for understanding NECAB1:

• Protein structure prediction: The advent of AI-based structure prediction tools like AlphaFold2 can generate high-confidence models of NECAB1 structure, particularly for regions that have been challenging to crystallize.

• Molecular dynamics simulations: Can provide insights into how calcium binding affects the conformation of NECAB1 and potentially the function of its ABM domain.

• Protein-protein interaction prediction: Computational methods can predict potential interaction partners of NECAB1 based on structural complementarity and sequence features.

• Systems biology approaches: Network analysis integrating transcriptomic, proteomic, and functional data can place NECAB1 within broader signaling networks and predict its functional associations.

• Evolutionary analysis: Detailed phylogenetic analysis of the NECAB family can reveal patterns of conservation and divergence that might correspond to functional specialization.

• Virtual screening: Computational screening of compound libraries can identify potential small molecule modulators of NECAB1 function for experimental validation.

• Integration with experimental data: Computational methods are most powerful when integrated with experimental approaches such as cross-linking mass spectrometry, hydrogen-deuterium exchange, or electron paramagnetic resonance to validate structural predictions and dynamics.