Recombinant Human Cysteine-rich with EGF-like domain protein 2 (CRELD2)

What is the structural composition of CRELD2 and how is it characterized experimentally?

Human CRELD2 is an approximately 60 kDa glycoprotein containing two EGF-like domains and two FU domains. The mature human protein spans from Ala21-Leu353 and shares approximately 78% amino acid sequence identity with mouse and rat CRELD2 . Alternative splicing generates multiple isoforms with substitutions, deletions, and/or insertions in the C-terminal half of the molecule .

For experimental characterization, researchers typically use recombinant CRELD2 with tags for detection and purification. Commercial sources provide the protein with C-terminal 6-His tags, expressed in mammalian systems to ensure proper folding and post-translational modifications . When designing experiments with recombinant CRELD2, it's crucial to consider that carrier-free versions (without BSA) are recommended for applications where BSA might interfere with experimental outcomes .

Methodologically, CRELD2's localization can be visualized through immunofluorescence microscopy using specific antibodies against CRELD2, showing its distribution primarily in the ER and Golgi apparatus under normal conditions, with extracellular detection under ER stress .

How is CRELD2 expression regulated at the transcriptional level?

CRELD2 expression is primarily regulated through ER stress-responsive elements in its promoter region. Genomic sequence analysis of the mouse Creld2 gene promoter has identified a putative ER stress-responsive element (ERSE; CGTGG-N9-ATTGG) that is highly conserved across species . This conservation indicates CRELD2's evolutionary importance in ER stress responses.

Methodologically, CRELD2 transcriptional regulation can be studied using:

• Luciferase reporter assays with wild-type and mutated ERSE sequences

• Chromatin immunoprecipitation to confirm transcription factor binding

• RT-qPCR following treatment with ER stress inducers

Under ER stress conditions induced by agents such as thapsigargin (Tg, a Ca²⁺-ATPase inhibitor), tunicamycin (Tm, an inhibitor of protein glycosylation), or brefeldin A (BFA, an inhibitor of ER-Golgi transport), Creld2 mRNA levels increase significantly . This induction occurs primarily through ATF6 binding directly to the ERSE in the Creld2 gene promoter. Experiments show that mutations in this ERSE considerably decrease both basal promoter activity and responsiveness to ER stress stimuli .

What mechanisms control CRELD2 retention in the ER versus its secretion?

CRELD2 contains a four-amino acid sequence (R/H)EDL at its C-terminus that functions as an ER retention motif similar to the well-known KDEL motif in other ER-resident proteins . The regulation of CRELD2 secretion versus retention involves a competitive mechanism with other ER-resident proteins.

• Multiple ER stress-inducible proteins including CRELD2 and GRP78 are upregulated

• The relative expression of KDELRs does not increase proportionally

• GRP78, with its higher-affinity KDEL motif, competes with CRELD2 for binding to KDELRs

• This competition allows CRELD2 to escape ER retention and be secreted

Experimental evidence supports this mechanism: modifying CRELD2's C-terminal region (deleting the four C-terminal amino acids or adding tag-peptides) dramatically enhances its secretion. Additionally, overexpression of wild-type GRP78 (but not mutant GRP78 lacking the KDEL sequence) significantly increases CRELD2 secretion .

This mechanism can be studied using:

• Site-directed mutagenesis of the REDL motif

• Co-immunoprecipitation with KDELRs

• Pulse-chase experiments to track secretion kinetics

• Competitive binding assays between CRELD2 and other ER-resident proteins

What is CRELD2's role in skeletal development and metabolism?

CRELD2 plays crucial roles in skeletal development through multiple mechanisms. Studies using tissue-specific knockout models have demonstrated that:

• Cartilage-specific deletion of Creld2 results in disrupted endochondral ossification

• Bone-specific deletion leads to osteopenia with low bone density and altered trabecular architecture

CRELD2 functions as a novel chaperone for the low-density lipoprotein receptor-related protein 1 (LRP1), promoting its transport to the cell surface. LRP1, in turn, directly regulates WNT4 expression in chondrocytes through TGF-β1 signaling . This mechanism highlights CRELD2's role in promoting skeletal cell differentiation and maturation.

Additionally, CRELD2 modulates calcium signaling in osteoclasts. Overexpression of Creld2 in osteoclasts impairs calcium release from the ER, which is essential for activating calcineurin, a key phosphatase in osteoclastogenesis . This leads to reduced dephosphorylation of nuclear factor of activated T cells 1 (NFATc1), preventing its nuclear localization and activation as a pro-osteoclastogenic transcription factor .

Methodologically, these findings demonstrate the importance of using both tissue-specific knockout models and overexpression systems to fully understand CRELD2's tissue-specific functions, as they may differ significantly between cell types.

How does CRELD2 regulate intracellular signaling pathways?

CRELD2 activates multiple signaling pathways with distinct temporal patterns. Phosphoproteomics and immunoblotting experiments have revealed that recombinant CRELD2 rapidly promotes phosphorylation of AMP-activated protein kinase catalytic subunit alpha (AMPKα) at threonine 172 . More slowly but with sustained effect, it induces AKT phosphorylation at threonine 308 and serine 473, events required for maximal AMPK and AKT activation .

Interestingly, comparative phosphoproteomic analysis demonstrates that CRELD2 and VEGFA (vascular endothelial growth factor A) have distinct albeit overlapping phosphoproteome signatures. The number of phosphosites significantly altered in CRELD2-stimulated cells is smaller than in VEGFA-stimulated cells, suggesting CRELD2 has more targeted effects .

For investigating CRELD2's signaling effects, researchers can employ:

• Phosphoproteomics with liquid chromatography and tandem mass spectrometry (LC-MS/MS)

• Principal component analysis and hierarchical clustering to distinguish signaling patterns

• Kinase inhibitor studies to validate predicted signaling pathways

• Western blotting to verify specific phosphorylation events

These methodological approaches can help delineate CRELD2's unique signaling profile compared to other growth factors and identify potential therapeutic targets.

What insights have CRELD2 knockout models provided about its physiological functions?

CRELD2 knockout models have revealed unexpected roles in metabolism and stress responses. A complete Creld2 knockout (Creld2-/-) mouse model showed:

• Reduced body weight under both chow diet and high-fat diet (HFD) conditions

• Development of insulin resistance despite lower body weight

• Reduced hepatic lipid content

• Significantly reduced adipose tissue weights

Contrary to expectations, 12 weeks of HFD did not induce ER stress in the liver of these mice, and neither transcriptome nor protein expression analysis revealed a lipid-driven ER stress response in their livers .

These findings suggest that:

• CRELD2 plays roles beyond ER stress, potentially in regulating whole-body energy balance

• It serves as a protective factor during acute ER stress

• Its functions may be context-dependent and tissue-specific

For researchers, these results emphasize the importance of studying CRELD2 under both basal and stressed conditions, and examining multiple metabolic parameters beyond the primary phenotype.

What experimental approaches are optimal for studying CRELD2 secretion and its extracellular functions?

Studying CRELD2 secretion and its extracellular functions requires a multi-faceted approach:

• Secretion induction protocols:

  • Treatment with ER stress inducers (tunicamycin, thapsigargin, brefeldin A)

  • Serum starvation

  • Disease models with known ER stress components

• Secretion detection methods:

  • Western blotting of conditioned media

  • ELISA for quantitative assessment

  • Immunohistochemistry to detect extracellular matrix localization

• Functional analysis of secreted CRELD2:

  • Recombinant protein treatment followed by phosphoproteomic analysis

  • Receptor identification through crosslinking and co-immunoprecipitation

  • Neutralizing antibody studies to block secreted CRELD2 function

Experiments have shown that CRELD2 secretion is dramatically induced upon ER stress both in vitro and in vivo. In cell culture, chemical agents like tunicamycin, thapsigargin, and brefeldin A or serum deprivation increase CRELD2 secretion . In disease models such as matrilin-3 (Matn3) and collagen type X alpha 1 chain (Col10a1) mutant growth plates, CRELD2 is detectable at significant levels in the extracellular matrix but not in wild-type controls .

For biomarker applications, urinary CRELD2 excretion coincides with podocyte ER stress during the development of proteinuria and can be detected at early disease stages. Early postoperative urinary CRELD2 elevation is significantly associated with severe acute kidney injury (AKI) and other adverse outcomes following pediatric cardiac surgery .

These findings suggest researchers should consider both tissue-specific and systemic readouts when studying CRELD2 as a potential biomarker or therapeutic target.

How does CRELD2 modulate calcium signaling and what are the implications for cell function?

CRELD2 plays a crucial role in modulating calcium signaling, with significant implications for diverse cellular functions. Research in osteoclasts has revealed that CRELD2 overexpression impairs calcium release from the ER, which has downstream effects on calcium-dependent signaling pathways .

Specifically, CRELD2 inhibits calcium-dependent activation of calcineurin, a phosphatase essential for osteoclast differentiation. This inhibition blocks the dephosphorylation of nuclear factor of activated T cells 1 (NFATc1), preventing its nuclear localization and activation as a transcription factor . The mechanism represents a novel regulatory pathway in which an ER-resident calcium-binding chaperone modulates calcium flux during cellular differentiation.

To study CRELD2's effects on calcium signaling, researchers can employ:

• Real-time calcium imaging with fluorescent indicators

• Patch-clamp electrophysiology to measure calcium currents

• Calcium-dependent enzyme activity assays (e.g., calcineurin phosphatase activity)

• Nuclear translocation assays for calcium-dependent transcription factors

• Calcium chelation experiments to confirm calcium dependency of observed effects

The implications of CRELD2's calcium regulatory functions extend beyond bone to potentially include:

• Neuronal excitability and neurotransmission

• Cardiac contractility and rhythm

• Immune cell activation and inflammatory responses

• Cellular stress responses where calcium signaling is pivotal

These findings underscore the importance of considering CRELD2 not just as an ER stress protein but as an active modulator of fundamental calcium-dependent cellular processes.

What is the potential of CRELD2 as a biomarker for ER stress-related diseases?

CRELD2 shows significant promise as a biomarker for ER stress-related diseases, particularly in kidney disorders. Several characteristics make it a compelling biomarker candidate:

• Specificity: CRELD2 is dramatically induced and secreted under ER stress conditions

• Detectability: It can be measured in urine, making it a non-invasive biomarker

• Early appearance: CRELD2 elevation precedes clinical symptoms or histological changes

• Mechanistic relevance: Its presence directly reflects underlying ER stress pathology

In kidney disease models, urinary CRELD2 excretion coincides with podocyte ER stress during proteinuria development and can be detected at early disease stages. In tubular cell ER stress, urinary CRELD2 increases prior to kidney function decline or histological changes in ischemia/reperfusion-induced acute kidney injury (AKI) .

Clinical studies have shown that early postoperative urinary CRELD2 elevation is significantly associated with severe AKI and other adverse outcomes following pediatric cardiac surgery. Urinary CRELD2 effectively distinguishes between controls and patients with early kidney disease .

For researchers investigating CRELD2 as a biomarker, recommended approaches include:

• Longitudinal sampling to establish temporal relationships between CRELD2 elevation and disease progression

• Multivariate analysis comparing CRELD2 with established biomarkers

• Determination of sensitivity, specificity, and predictive values in diverse patient populations

• Correlation of CRELD2 levels with treatment responses and long-term outcomes

While these findings suggest CRELD2 has strong biomarker potential, larger multi-institutional studies with diverse patient cohorts are needed before clinical implementation.

What are the optimal protocols for working with recombinant CRELD2 in experimental systems?

When working with recombinant CRELD2 in experimental systems, researchers should consider several key methodological aspects:

• Selection of appropriate recombinant forms:

  • Carrier-free (CF) versions (without BSA) should be used for applications where BSA might interfere with results

  • BSA-containing formulations provide enhanced stability for general cell culture applications or ELISA standards

  • Tag placement can affect functionality; C-terminal 6-His tags are commonly used

• Storage and handling:

  • Lyophilized protein should be reconstituted at 250 μg/mL in PBS

  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles

  • The product is typically shipped at ambient temperature but should be stored at recommended temperatures upon receipt

• Effective concentration ranges:

  • For cellular assays, the typical effective range is 1-5 μg/mL

  • Dose-response experiments are recommended to determine optimal concentrations for specific cell types

• Functional verification:

  • Confirm biological activity through phosphorylation of known targets like AMPKα (at 5 minutes) and AKT (at 15 minutes)

  • Verify ER stress modulation using markers like GRP78 expression

When designing experiments with recombinant CRELD2, it's important to consider its dual localization. Extracellular application of recombinant CRELD2 mimics the secreted form's effects, while transfection or viral delivery of CRELD2 expression constructs allows study of its intracellular functions. For comprehensive understanding, both approaches may be necessary within the same experimental system.

How can researchers effectively study the interaction between CRELD2 and other ER chaperones?

Studying the interactions between CRELD2 and other ER chaperones requires specialized methodological approaches due to the complex environment of the ER and the dynamic nature of chaperone interactions. Effective research strategies include:

• Co-immunoprecipitation (Co-IP) assays:

  • Use native conditions to preserve physiological interactions

  • Include appropriate controls for non-specific binding

  • Consider crosslinking approaches for transient interactions

  • Perform reciprocal Co-IPs (pulling down each protein and probing for the other)

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins can identify proteins in close proximity to CRELD2

  • APEX2-based proximity labeling for temporal control of labeling reactions

  • These methods are particularly valuable for identifying weak or transient interactions

• Fluorescence-based interaction assays:

  • Förster resonance energy transfer (FRET) for live-cell monitoring of protein-protein interactions

  • Fluorescence recovery after photobleaching (FRAP) to assess chaperone complex dynamics

  • Split-fluorescent protein complementation to visualize interactions in living cells

• Proteomic approaches:

  • Mass spectrometry following Co-IP to identify interaction partners

  • Quantitative proteomics comparing wild-type and CRELD2-deficient cells

  • SILAC or TMT labeling for comparative analyses across conditions

Research has shown that CRELD2 interacts with several chaperones and enzymes required for overcoming cellular stress, including GRP78, thioredoxin domain-containing 5, and glutathione S-transferase Mu2 . These interactions appear to be functionally significant, as CRELD2-deficient mice show exacerbated ER stress responses when challenged with tunicamycin .

When studying these interactions, researchers should consider both basal and stress-induced conditions, as the interactome of CRELD2 likely changes significantly during ER stress responses.

What considerations are important when developing tissue-specific CRELD2 knockout models?

Developing tissue-specific CRELD2 knockout models requires careful planning to ensure meaningful results. Key considerations include:

• Selection of appropriate Cre-driver lines:

  • Choose tissue-specific promoters with well-characterized expression patterns

  • Consider temporal control (inducible systems) to distinguish developmental versus adult functions

  • Validate specificity and efficiency of recombination in target tissues

• Genetic background considerations:

  • Maintain consistent genetic background through backcrossing

  • Use littermate controls (Cre-negative floxed animals) to control for Cre expression effects

  • Consider strain-specific differences in ER stress responses

• Phenotypic analysis strategy:

  • Employ comprehensive phenotyping beyond the primary tissue of interest

  • Include metabolic parameters (glucose tolerance, insulin sensitivity, body composition)

  • Assess both basal conditions and responses to various stressors

  • Examine multiple timepoints to capture progressive or age-dependent phenotypes

• Molecular validation approaches:

  • Confirm CRELD2 deletion at protein and mRNA levels

  • Assess compensation by related proteins

  • Evaluate ER stress markers in targeted tissues

Previous studies have shown distinct phenotypes with tissue-specific CRELD2 deletions:

• Cartilage-specific deletion disrupts endochondral ossification

• Bone-specific deletion leads to osteopenia

• Complete knockout affects metabolism with reduced body weight and adiposity despite insulin resistance

These diverse findings highlight the importance of tissue-specific approaches to fully understand CRELD2's context-dependent functions. Additionally, researchers should consider complementary gain-of-function models (tissue-specific overexpression) to provide a complete picture of CRELD2's roles in different tissues.

What are the most promising therapeutic applications targeting CRELD2 or its pathways?

Based on current understanding of CRELD2's functions, several promising therapeutic applications emerge:

• Cardiovascular disease therapy:

  • CRELD2 functions as an angiogenic growth factor promoting ischemic heart repair

  • Therapeutic administration of recombinant CRELD2 might enhance cardiac recovery after ischemic events

  • CRELD2-based therapies could potentially target key signaling pathways (AMPK, AKT) without the broader effects of VEGF

• Bone and cartilage disorders:

  • Modulating CRELD2 activity could potentially treat osteoporosis by influencing osteoclast differentiation

  • For skeletal developmental disorders, targeting the CRELD2-LRP1-WNT4 axis might provide novel therapeutic approaches

• ER stress-related conditions:

  • CRELD2-based therapies might enhance cellular resilience to ER stress

  • Particularly relevant for conditions like diabetes, neurodegenerative diseases, and inflammatory disorders where ER stress plays a pathogenic role

• Diagnostic applications:

  • Development of CRELD2-based biomarker assays for early detection of kidney diseases

  • Potential application in monitoring ER stress levels in various pathological conditions

For researchers pursuing these therapeutic directions, methodological considerations include:

• Development of recombinant CRELD2 variants with enhanced stability or targeted activity

• Creation of tissue-specific delivery systems to minimize off-target effects

• Design of small molecule modulators of CRELD2 expression or function

• Establishment of reliable biomarker assays for patient stratification and treatment monitoring

The dual nature of CRELD2 as both an intracellular chaperone and secreted signaling molecule opens unique therapeutic possibilities, potentially allowing separate targeting of these distinct functions for different therapeutic applications.

How might CRELD2 function differ across diverse tissue types and pathological conditions?

CRELD2 exhibits notable tissue-specific and context-dependent functions that merit systematic investigation. Current evidence suggests significant variations in CRELD2's roles across tissues:

• Skeletal system:

  • In cartilage: Functions as an LRP1 chaperone regulating noncanonical WNT signaling

  • In bone: Affects trabecular architecture; deletion leads to osteopenia

  • In osteoclasts: Modulates calcium release and calcineurin signaling

• Liver:

  • Influences lipid metabolism; knockout mice show reduced hepatic lipid content

  • Provides protection during acute ER stress responses

  • May influence insulin sensitivity and glucose homeostasis

• Cardiovascular system:

  • Functions as an angiogenic growth factor

  • Activates distinct phosphorylation cascades from traditional growth factors like VEGF

• Kidney:

  • Highly expressed during normal human fetal and postnatal kidney development

  • Secreted during podocyte and tubular cell ER stress

  • Serves as a potential biomarker for kidney injury

These diverse functions suggest CRELD2 may have evolved tissue-specific roles beyond its general ER stress response function. For comprehensive characterization of these differences, researchers should consider:

• Comparative transcriptomic and proteomic analyses across tissues under basal and stressed conditions

• Identification of tissue-specific interaction partners

• Examination of cell type-specific signaling outcomes following CRELD2 stimulation

• Investigation of developmental versus adult functions in each tissue context

Understanding these tissue-specific differences will be crucial for developing targeted therapeutic approaches with minimal off-target effects.

What novel technologies might advance our understanding of CRELD2 biology?

Emerging technologies offer exciting opportunities to advance CRELD2 research:

• Single-cell multi-omics:

  • Single-cell RNA sequencing to identify cell populations responsive to CRELD2

  • Single-cell proteomics to map cell type-specific signaling networks

  • Spatial transcriptomics to visualize CRELD2 expression patterns in intact tissues

  • Integration of these approaches to create comprehensive cellular atlases of CRELD2 function

• Advanced protein imaging techniques:

  • Super-resolution microscopy to visualize CRELD2 localization within ER subdomains

  • Live-cell imaging with tagged CRELD2 to track its trafficking during ER stress

  • Correlative light and electron microscopy to examine ultrastructural changes associated with CRELD2 function

• CRISPR-based technologies:

  • CRISPRi/CRISPRa for precise temporal control of CRELD2 expression

  • Base editing to introduce specific mutations modeling human variants

  • CRISPR screens to identify genes that modulate CRELD2 function

  • In vivo CRISPR editing for tissue-specific and temporal knockout studies

• Protein engineering approaches:

  • Development of biosensors to monitor CRELD2 conformational changes or activity

  • Creation of optogenetically controllable CRELD2 variants

  • Design of engineered CRELD2 proteins with enhanced or selective functions

• Computational biology tools:

  • Molecular dynamics simulations to understand CRELD2 structural changes during ER stress

  • Network analysis to position CRELD2 within broader cellular stress response pathways

  • Machine learning approaches to predict CRELD2 interactions and functions

These technological advances could help resolve key outstanding questions about CRELD2, including the molecular basis of its dual functions as both an ER chaperone and secreted signaling molecule, its tissue-specific roles, and its potential as a therapeutic target or biomarker in various diseases.