CTGF Human, His

What is the molecular structure of human CTGF and how does the His-tag modification affect its properties?

Human CTGF is a 38 kDa secreted protein belonging to the CCN family of matricellular proteins. It consists of four conserved domains: an insulin-like growth factor binding protein domain, a von Willebrand factor type C repeat, a thrombospondin type 1 repeat, and a C-terminal domain containing a cystine knot motif . The His-tag modification, typically added to the N- or C-terminus, consists of 6-10 histidine residues that facilitate protein purification through metal affinity chromatography without significantly altering the protein's biological activity in most applications.

How is CTGF expression regulated in human neural tissues and what techniques can detect these changes?

CTGF expression in neural tissues is regulated through multiple mechanisms including stress responses and growth factor signaling pathways. Research has demonstrated that CTGF expression is significantly increased in the amygdala of individuals with major depressive disorder (MDD) . Specifically, quantitative real-time PCR analysis revealed that CTGF expression was significantly elevated in the accessory basal nucleus (p < .001), amygdalohippocampal nucleus (p < .001), and lateral nucleus (p < .001) in postmortem brain samples from MDD patients compared to controls .

For detection and quantification of CTGF expression in neural tissues, researchers commonly employ:

• Quantitative real-time PCR (qRT-PCR) with primers specific to human CTGF

• In situ hybridization to localize CTGF mRNA expression in specific brain regions

• Laser capture microdissection combined with RNA extraction for nucleus-specific expression analysis

• Microarray analysis for high-throughput screening of expression changes

When studying CTGF in neural tissues, it is critical to use appropriate housekeeping genes (such as GAPDH) for normalization and to employ the Livak method for accurate fold-change calculations .

What are the standard methods for purifying His-tagged human CTGF protein for experimental use?

The purification of His-tagged human CTGF typically follows a multi-step process designed to maximize protein yield while preserving biological activity:

• Expression System Selection: Mammalian expression systems (HEK293 or CHO cells) are preferred for human CTGF expression as they provide appropriate post-translational modifications critical for CTGF function.

• Immobilized Metal Affinity Chromatography (IMAC): The standard first purification step utilizes Ni-NTA or Co-NTA resins that bind the His-tag with high affinity. Elution is typically performed using a gradient or step-wise increase of imidazole concentration (50-300 mM).

• Secondary Purification: Size exclusion chromatography (SEC) or ion-exchange chromatography is recommended to remove protein aggregates and increase purity.

• Quality Control Testing: Purified His-tagged CTGF should be validated by:

  • SDS-PAGE with Coomassie staining for purity assessment

  • Western blotting with anti-CTGF and anti-His antibodies

  • Functional assays to confirm biological activity

When working with CTGF, researchers should be aware that the protein may interact with various extracellular matrix components during purification, potentially affecting yield. Adding low concentrations of detergent (0.05% Tween-20) or adjusting salt concentration can help minimize these interactions without compromising protein structure.

How can researchers effectively use His-tagged CTGF to study its role in depression and what controls are essential?

Investigating CTGF's role in depression requires careful experimental design and appropriate controls. Based on findings showing increased CTGF expression in the amygdala of MDD patients, researchers should consider the following methodological approach:

• In vitro studies:

  • Treat primary neuronal or glial cultures with purified His-tagged CTGF at physiologically relevant concentrations (10-100 ng/ml)

  • Controls must include heat-inactivated CTGF protein and a tag-only control peptide

  • Measure endpoints such as neuronal survival, synapse formation, and gene expression changes

  • Validate findings using CTGF with an alternative tag (e.g., FLAG) to confirm results are not tag-dependent

• In vivo studies:

  • Develop models with localized CTGF expression or administration in relevant brain regions

  • Compare centrally administered His-tagged CTGF with vehicle control using stereotaxic injection techniques

  • Assess depression-like behaviors using validated paradigms such as forced swim test, sucrose preference test, and social interaction tests

  • Include anti-CTGF antibody treatment (e.g., FG-3019) as a blocking control

Research has demonstrated that central administration of CTGF increases depression-like behavior in outbred rats, while antibody treatment with FG-3019 decreases depression-like behavior and reduces CTGF expression while increasing PDGFβ expression in the dentate gyrus . These findings provide a methodological framework for further mechanistic studies.

What are the molecular mechanisms through which CTGF influences neuronal survival, and how can these be experimentally interrogated?

CTGF appears to function as a proapoptotic molecule in neuronal contexts, with studies suggesting several mechanisms that researchers can experimentally interrogate:

• CTGF interaction with cell surface receptors:

  • CTGF interacts with integrins, TrkA, and LRP receptors to activate downstream signaling

  • Methodological approach: Use receptor-blocking antibodies or siRNA knockdown of specific receptors to identify which are essential for CTGF's effects on neuronal survival

  • Key controls: Include scrambled siRNAs and isotype control antibodies

• Modulation of growth factor signaling:

  • CTGF may antagonize survival-promoting growth factors like FGF2

  • Evidence suggests early-life FGF2 administration decreases CTGF expression and anxiety-like behavior in bred low responder rats

  • Experimental approach: Co-administer His-tagged CTGF with various growth factors and assess whether CTGF blocks their pro-survival effects

• Intracellular signaling pathways:

  • Monitor activation of apoptotic signaling molecules (caspases, Bax/Bcl-2 ratio)

  • Assess changes in stress-responsive signaling pathways (MAPK, JNK, NFκB)

  • Utilize pathway-specific inhibitors to determine which signaling cascades are necessary for CTGF's effects

Research has shown that CTGF knockdown rescued apoptosis in the olfactory bulb, suggesting a direct role in cellular survival . For experimental interrogation, researchers should employ both gain-of-function (applying His-tagged CTGF) and loss-of-function (CTGF knockdown or antibody neutralization) approaches to comprehensively understand these mechanisms.

What are the current challenges in translating CTGF research findings from animal models to human clinical applications?

Several significant challenges exist in translating CTGF research from animal models to human applications:

• Species-specific differences in CTGF signaling:

  • Human and rodent CTGF share approximately 91% amino acid identity but may differ in post-translational modifications and binding partners

  • Methodological solution: Conduct comparative studies using both human and rodent CTGF in the same experimental systems

  • Employ humanized animal models where appropriate

• Tissue-specific effects of CTGF:

  • CTGF functions differently across tissues, with research showing distinct roles in the amygdala versus hippocampus

  • Regional specificity must be considered when designing therapeutic approaches

  • Complex interactions with the extracellular environment mean the net effect of CTGF depends on other molecules in the region

• Temporal dynamics of CTGF expression:

  • Acute versus chronic CTGF elevation may have different consequences

  • In animal models, early-life interventions affecting CTGF produced long-lasting effects on behavior

  • Human studies suggest chronic CTGF elevation in MDD, but the temporal dynamics remain poorly understood

• Sex differences in CTGF regulation and function:

  • Human postmortem studies on CTGF in MDD have been limited to male subjects

  • Methodological consideration: Include both sexes in preclinical and clinical studies

  • Analyze data for sex-specific effects of CTGF manipulation

These translational challenges underscore the importance of using multiple complementary approaches and carefully validating findings across experimental systems.

How should researchers design experiments to study the role of CTGF in hematological malignancies?

Based on evidence that CTGF is overexpressed in B-cell acute lymphoblastic leukemia (B-ALL) and associated with poor prognosis , researchers should consider the following experimental design principles:

What experimental controls are crucial when studying the effects of CTGF on neural tissues?

When investigating CTGF effects on neural tissues, researchers must employ rigorous controls to ensure valid and reproducible results:

• Protein-specific controls:

  • Denatured/heat-inactivated CTGF to control for non-specific protein effects

  • Alternative tagged version of CTGF (e.g., FLAG-tag instead of His-tag) to rule out tag-specific effects

  • CTGF with site-directed mutations in functional domains to identify structure-function relationships

  • Dose-response experiments to establish physiologically relevant concentrations

• Tissue and cell type controls:

  • Include multiple brain regions in analyses, as CTGF effects vary by region

  • Utilize cell-type specific markers to determine which neural cells respond to CTGF

  • Compare effects across different neural cell types (neurons, astrocytes, microglia, oligodendrocytes)

• Genetic and pharmacological controls:

  • CTGF neutralizing antibodies (e.g., FG-3019) to confirm specificity of observed effects

  • siRNA or shRNA knockdown of CTGF receptors to validate signaling pathways

  • Pathway-specific inhibitors to confirm downstream mechanisms

• Behavioral controls in animal studies:

  • Include non-stressed control groups when studying stress-induced CTGF expression

  • Control for potential confounding variables such as age, sex, and estrous cycle

  • Use multiple behavioral tests to assess the same construct (e.g., multiple tests of depression-like behavior)

These controls are essential because CTGF interacts with various extracellular matrix proteins and cell surface receptors, and its net effect depends on other molecules in the cellular environment .

What are the most effective analytical methods for detecting and quantifying CTGF-protein interactions in neural tissues?

Several complementary analytical methods can be employed to effectively detect and quantify CTGF-protein interactions in neural tissues:

• Co-immunoprecipitation (Co-IP):

  • Utilize anti-His antibodies to pull down His-tagged CTGF and associated proteins

  • Perform reciprocal Co-IP with antibodies against suspected binding partners

  • Controls should include IgG isotype controls and lysates from tissues not expressing the His-tagged protein

  • Western blot analysis of immunoprecipitated complexes can confirm specific interactions

• Proximity Ligation Assay (PLA):

  • Enables visualization of protein-protein interactions in situ with subcellular resolution

  • Particularly valuable for neural tissues where cellular heterogeneity is high

  • Can detect interactions between CTGF and receptors like integrins, TrkA, and LRP

  • Control experiments should include omission of one primary antibody

• Surface Plasmon Resonance (SPR):

  • Provides quantitative binding kinetics (ka, kd) and affinity (KD) measurements

  • His-tagged CTGF can be immobilized on Ni-NTA sensor chips

  • Requires purified interaction partners and may not reflect the complexity of the neural environment

  • Essential for determining whether interactions are direct or indirect

• Cross-linking Mass Spectrometry (XL-MS):

  • Enables unbiased identification of proteins interacting with CTGF in complex neural tissues

  • Chemical cross-linkers stabilize transient interactions before protein complex isolation

  • Mass spectrometry identifies cross-linked peptides and interaction sites

  • Bioinformatic analysis reveals interaction networks and structural insights

These methods should be used in combination, as each provides complementary information about CTGF interactions. For neural tissues specifically, attention must be paid to preserving the native cellular environment as much as possible, as CTGF functions are highly context-dependent .

How can researchers accurately model the effects of stress-induced CTGF expression changes in experimental systems?

To accurately model stress-induced CTGF expression changes, researchers should consider the following methodological approaches:

• Selection of appropriate stress paradigms:

  • Social defeat stress has been demonstrated to increase CTGF expression in the dentate gyrus of bred low responder rats

  • Protocol: 15 minutes of social defeat daily for 4 days with a resident aggressor, followed by separation by wire mesh cage until the end of the session

  • Tissue collection timing: 4 days after the completion of social defeat testing

  • Controls must include non-stressed animals housed in identical conditions

• Genetic models with differential stress susceptibility:

  • Utilize selectively bred high responder (bHR) and low responder (bLR) rat lines that differ in novelty-seeking, anxiety behavior, and stress reactivity

  • bLRs exhibit greater anxiety-like and depression-like behavior compared to bHRs

  • bLRs show elevated CTGF expression in the dentate gyrus compared to bHRs

  • This model allows for the study of both baseline differences and stress-induced changes

• Temporal dynamics assessment:

  • Implement time-course studies measuring CTGF expression at multiple points after stress exposure

  • Use in situ hybridization to detect rapid changes in CTGF mRNA

  • Complement with protein-level measurements using immunohistochemistry or western blotting

  • Consider both acute and chronic stress paradigms to distinguish between transient and persistent changes

• Validation in human samples:

  • When possible, correlate animal findings with human postmortem tissue analyses

  • Consider using human induced pluripotent stem cell (iPSC)-derived neural cultures exposed to stress hormones

  • Implement careful statistical controls for postmortem variables in human studies

This approach is supported by research showing that social defeat stress significantly increased CTGF expression in the dentate gyrus of bred low responder rats (p < .05) , providing a validated methodological framework.

What strategies can researchers employ to overcome challenges in studying CTGF's role in complex multifactorial diseases?

Studying CTGF in complex multifactorial diseases such as major depressive disorder and hematological malignancies presents unique challenges that can be addressed through several methodological strategies:

• Integrative multi-omics approaches:

  • Combine transcriptomics, proteomics, and metabolomics data to understand CTGF in disease context

  • Example methodology: In MDD studies, correlate CTGF expression with broader gene network changes

  • Unbiased pathway analysis has identified networks related to nervous system development, tissue development, and connective tissue function as significantly altered in relation to CTGF

  • Implement systems biology approaches to model complex interactions

• Cell type-specific analyses:

  • Employ single-cell RNA sequencing to identify cell populations where CTGF expression changes are most pronounced

  • Use laser capture microdissection for region-specific analyses, as demonstrated in amygdala nuclei studies

  • Develop cell type-specific CTGF knockout or overexpression models

  • This granular approach helps overcome the heterogeneity inherent in complex diseases

• Stratification approaches for clinical samples:

  • Categorize patients based on CTGF expression tertiles for survival analyses

  • Incorporate CTGF expression with established biomarkers and clinical parameters

  • In B-ALL studies, this approach revealed that patients with high CTGF expression had significantly worse outcomes (5-year OS: 12% vs. 58% for low expression)

  • Develop composite biomarker panels including CTGF for improved prognostication

• Mechanistic dissection through conditional manipulation:

  • Implement temporally controlled and tissue-specific CTGF manipulation

  • Use inducible expression systems to model disease progression

  • Combine with environmental challenges (e.g., stress exposure) to study gene-environment interactions

  • This approach helps separate causal roles from secondary responses

These strategies collectively enable researchers to address the complexity of CTGF's role in multifactorial diseases while maintaining scientific rigor and translational relevance.

How can researchers address the issue of protein aggregation when working with recombinant His-tagged CTGF?

CTGF has a known tendency to aggregate due to its multiple binding domains and cysteine-rich structure. Researchers can implement several strategies to minimize aggregation of His-tagged CTGF:

• Optimization of expression conditions:

  • Lower induction temperature (16-18°C) during recombinant expression

  • Reduce expression time to prevent inclusion body formation

  • Consider using fusion partners (e.g., thioredoxin, SUMO) that enhance solubility

  • Employ specialized expression strains designed for disulfide-rich proteins

• Buffer formulation optimization:

  • Include mild non-ionic detergents (0.01-0.05% Tween-20)

  • Add stabilizing agents such as glycerol (10-20%) or arginine (50-100 mM)

  • Optimize pH based on CTGF's isoelectric point

  • Consider adding low concentrations of reducing agents (0.1-1 mM DTT) to prevent disulfide-mediated aggregation

• Purification strategy modifications:

  • Implement multi-step chromatography (IMAC followed by SEC)

  • Use on-column refolding for proteins recovered from inclusion bodies

  • Consider affinity purification under denaturing conditions followed by controlled refolding

  • Filter solutions through 0.22 μm filters immediately before storage

• Storage and handling considerations:

  • Store at moderate protein concentration (0.5-1 mg/ml) to reduce aggregation potential

  • Aliquot to avoid freeze-thaw cycles

  • Include carrier proteins (e.g., BSA at 0.1%) for dilute solutions

  • Monitor aggregation status using dynamic light scattering before experiments

Implementing these strategies ensures the availability of monomeric, properly folded His-tagged CTGF for functional studies, enhancing reproducibility and reliability of experimental results.

What are the most effective methods for analyzing the phosphorylation state of CTGF and its impact on function?

CTGF can undergo post-translational modifications including phosphorylation, which may significantly alter its function. Researchers can employ these methods to analyze CTGF phosphorylation:

• Phosphorylation site mapping:

  • Mass spectrometry-based phosphoproteomics:

    • Enrich phosphopeptides using titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC)

    • Perform LC-MS/MS analysis with electron transfer dissociation (ETD) fragmentation

    • Use parallel reaction monitoring (PRM) for targeted quantification of specific phosphosites

  • Site-directed mutagenesis:

    • Create phosphomimetic (Ser/Thr to Asp/Glu) and phosphodeficient (Ser/Thr to Ala) mutants

    • Compare functional properties with wild-type His-tagged CTGF

• Phosphorylation state-specific detection:

  • Phospho-specific antibodies:

    • Develop antibodies against predicted phosphorylation sites

    • Validate antibody specificity using phosphatase-treated samples and phosphosite mutants

  • Phos-tag SDS-PAGE:

    • Separate phosphorylated from non-phosphorylated CTGF isoforms

    • Quantify relative abundance of phosphorylated species

    • Combine with western blotting for specific detection

• Kinase and phosphatase identification:

  • In vitro kinase assays:

    • Screen candidate kinases using purified His-tagged CTGF as substrate

    • Monitor incorporation of radioactive phosphate or use phospho-specific antibodies

  • Kinase inhibitor studies:

    • Treat cells with specific kinase inhibitors and monitor CTGF phosphorylation

    • Correlate changes in phosphorylation with functional outcomes

• Functional analysis of phosphorylation:

  • Compare binding properties of phosphorylated versus non-phosphorylated CTGF

  • Assess impact on cellular responses (e.g., cell survival, gene expression)

  • Determine effect on CTGF stability and turnover

  • Analyze phosphorylation changes in disease states (e.g., MDD, ALL)

These methodologies provide complementary information about CTGF phosphorylation and its functional significance, offering insights into how this post-translational modification may contribute to CTGF's role in various pathological conditions.

How can researchers utilize CTGF as a potential biomarker or therapeutic target in psychiatric disorders?

Based on evidence linking CTGF to major depressive disorder, researchers can develop approaches for its utilization as a biomarker or therapeutic target:

• Biomarker development strategies:

  • Correlate CTGF levels in accessible biofluids (CSF, blood) with neuroimaging measures of amygdala activity

  • Establish reference ranges for CTGF in healthy controls versus MDD patients

  • Develop simplified assays (ELISA, electrochemiluminescence) for clinical application

  • Conduct longitudinal studies to determine whether CTGF levels predict treatment response

  • Integrate with other biomarkers for improved sensitivity and specificity

• Anti-CTGF therapeutic approaches:

  • Neutralizing antibodies:

    • FG-3019 (pamrevlumab) has shown antidepressant effects in animal models

    • Optimize delivery methods for CNS penetration

    • Design dosing regimens based on temporal dynamics of CTGF expression

  • Small molecule inhibitors:

    • Design compounds targeting CTGF binding interfaces

    • Screen for molecules that disrupt CTGF-receptor interactions

    • Evaluate blood-brain barrier penetration and CNS bioavailability

• Gene therapy approaches:

  • RNA interference (RNAi) to knockdown CTGF expression

  • CRISPR-based repression of CTGF in specific brain regions

  • Viral vector delivery systems for targeted modulation in relevant neural circuits

• Companion diagnostics development:

  • Identify patient subgroups most likely to benefit from CTGF-targeted therapies

  • Develop CTGF expression assays to guide treatment selection

  • Monitor CTGF levels during treatment to assess target engagement

The translational potential is supported by findings that CTGF administration increases depression-like behavior in rats, while anti-CTGF antibody treatment decreases depression-like behavior . This bidirectional modulation suggests CTGF represents a viable therapeutic target with clear biological rationale.

What emerging technologies show promise for studying CTGF function in complex neural circuits?

Several cutting-edge technologies are particularly valuable for investigating CTGF function in neural circuits:

• Optogenetic and chemogenetic approaches:

  • Combine CTGF expression control with circuit manipulation

  • Use Cre-dependent CTGF expression in specific neural populations

  • Implement activity-dependent CTGF expression systems to mimic stress-induced changes

  • This permits temporal and spatial precision in studying CTGF's effects on circuit function

• Advanced imaging techniques:

  • Multiphoton calcium imaging to monitor circuit activity while manipulating CTGF

  • Expansion microscopy for nanoscale visualization of CTGF localization at synapses

  • Light sheet microscopy for whole-brain CTGF expression mapping

  • Correlative light and electron microscopy to link CTGF localization with ultrastructural features

• Spatial transcriptomics and proteomics:

  • Visium spatial gene expression analysis to map CTGF expression in brain regions

  • Imaging mass cytometry for multi-parameter protein analysis in tissue sections

  • Digital spatial profiling to correlate CTGF with other markers in microdissected regions

  • These approaches reveal regional heterogeneity that is missed in bulk tissue analyses

• Organoid and assembloid models:

  • Human brain organoids to study CTGF in development and disease

  • Brain region-specific organoids (amygdala, hippocampus) to model area-specific effects

  • Assembloids combining different brain regions to study circuit-level effects

  • These models bridge the gap between animal studies and human biology

These technologies enable unprecedented insight into how CTGF functions within the complex cellular and circuit environment of the brain, potentially revealing novel mechanisms underlying its role in psychiatric disorders.