CHGA Human, HEK
Description
Fundamental Characteristics of Human Chromogranin A
Chromogranin A (CHGA), also known as pituitary secretory protein I (SP-I), belongs to the granin family of regulated secretory proteins predominantly expressed in neuroendocrine cells throughout the body. CHGA exhibits several distinctive characteristics common to the granin family: an acidic isoelectric point, capacity to bind calcium ions, ability to form aggregates, and the presence of multiple dibasic cleavage sites that facilitate proteolytic processing into bioactive peptides . The human CHGA gene is organized into eight exons and seven introns, with the resulting protein having a molecular weight of 48-52 kDa in its unmodified form, though post-translational modifications significantly increase its observed molecular weight . Mature human CHGA consists of 439 amino acids and contains 10 dibasic proteolytic cleavage sites capable of yielding several smaller peptides, each displaying unique biological functions that expand the protein's physiological impact beyond its structural role . These peptides include vasostatin with vasorelaxant properties, catestatin that inhibits catecholamine release, and pancreastatin with dysglycemic effects, demonstrating CHGA's multifunctional nature .
CHGA is expressed exclusively in the secretory dense core granules of most normal and neoplastic neuroendocrine cells, making it an excellent marker for identifying and characterizing neuroendocrine tissues and tumors . The protein is found in various neuroendocrine cells throughout the body, including those in the gastrointestinal tract, adrenal medulla, and pancreatic islets, as well as in monoamine-storing chromaffin cells where it was originally discovered . Mature human CHGA shares approximately 63% amino acid sequence identity with mouse and rat CHGA, indicating evolutionary conservation while maintaining species-specific variations that may reflect specialized functions across different mammals . This widespread distribution and conservation highlight CHGA's fundamental importance in neuroendocrine physiology and explain its utility as a biomarker for various neuroendocrine conditions and as a research target for understanding regulated secretory mechanisms.
Production and Characterization of HEK293-Expressed CHGA
The expression of recombinant human CHGA in HEK293 cells represents a significant advancement in protein production technology, enabling the generation of human CHGA with native-like post-translational modifications essential for its proper function. HEK293 cells, derived from human embryonic kidney tissue, provide a mammalian expression environment that preserves the natural processing pathways for human proteins, making them an ideal host for CHGA production . This expression system supports proper glycosylation patterns and formation of disulfide bonds, which are critical for CHGA's biological activity and structural integrity in ways that bacterial expression systems cannot achieve. The resultant recombinant protein typically comprises the mature sequence of human CHGA (Leu19-Gly457) with a C-terminal His-tag added for purification purposes, yielding a construct of approximately 445 amino acids with a calculated molecular mass of 49.7-50.2 kDa, though the observed molecular weight is considerably higher (60-80 kDa) due to glycosylation .
The production process involves transfection of HEK293 cells with expression vectors containing the human CHGA sequence, followed by culture in controlled conditions, protein expression, and subsequent purification steps. Purification typically employs affinity chromatography utilizing the His-tag, followed by additional purification steps to achieve high purity, generally greater than 90-95% as determined by SDS-PAGE analysis and visualized with silver staining or Coomassie Blue staining . Quality control measures include endotoxin testing, with specifications typically requiring less than 1.0 EU/μg as determined by the LAL method, ensuring the product's suitability for sensitive research applications . N-terminal sequence analysis confirms the identity of the recombinant protein, with Leu19 being the first amino acid of the mature protein, consistent with proper signal peptide processing in the HEK293 expression system .
The purified protein is commonly lyophilized from a buffered solution (typically PBS at pH 7.0) and can be stored in this form at -80°C until reconstitution, maintaining stability for up to 12 months under proper storage conditions . Reconstituted protein remains stable at 4°C for approximately one week, providing reasonable working stability for experimental applications . Functional characterization of HEK293-expressed CHGA demonstrates proper biological activity, particularly its ability to bind to Secretogranin III (SCG3), a known physiological interaction partner, with an ED50 (effective dose for 50% response) measured at 40-360 ng/mL in functional ELISA assays . This detailed characterization ensures that recombinant CHGA from HEK293 cells accurately represents the native human protein for research applications.
Role in Secretory Vesicle Biogenesis
Chromogranin A serves as a master regulator of secretory vesicle biogenesis in neuroendocrine cells, a function that has been demonstrated through both in vitro and in vivo studies . CHGA aggregates in the acidic environment of developing vesicles and induces budding of the trans-Golgi network membranes to form dense-core granules, essentially driving the creation of the regulated secretory pathway . The N-terminal region of CHGA binds tightly to lipid-rich microdomains of trans-Golgi network membranes, influencing the transport of pro-hormones into secretory granules or, in the adrenal medulla, into chromaffin granules . This granulogenic function explains why CHGA is considered both necessary and sufficient for the formation of a regulated secretory pathway in various cell types, with CHGA gene ablation in mice leading to reduced secretory granule formation and altered neuroendocrine function .
Beyond initial granule formation, CHGA plays a critical role in maintaining the secretory capacity of neuroendocrine cells by replenishing secretory granules following exocytosis. This replenishment function operates particularly through serpinin, a C-terminal peptide derived from CHGA that mediates inhibition of granule degradation processes . Additionally, CHGA functions as a calcium-binding protein within secretory granules, regulating calcium homeostasis that is crucial for proper vesicle function and controlled exocytosis . The protein's capacity to bind calcium contributes to the condensation of granule contents and maintenance of the appropriate intragranular environment required for efficient hormone and neurotransmitter storage and subsequent release upon stimulation .
Bioactive Peptides and Their Functions
As a prohormone, CHGA undergoes proteolytic processing at multiple dibasic sites to generate several biologically active peptides, each with distinct physiological functions. Table 1 summarizes the major bioactive peptides derived from human CHGA.
Table 1: Major Bioactive Peptides Derived from Human CHGA
These peptides collectively enable CHGA to influence multiple physiological systems, including cardiovascular regulation, glucose metabolism, immune function, and neurotransmitter release. Catestatin, for example, acts as an autocrine negative feedback regulator of catecholamine release and demonstrates anti-hypertensive properties, while pancreastatin influences glucose homeostasis by modulating insulin sensitivity . The multifunctional nature of these CHGA-derived peptides explains the diverse physiological impacts of altered CHGA expression in both experimental models and human pathological conditions.
Protein-Protein Interactions
CHGA engages in several important protein-protein interactions that influence its trafficking, processing, and function within the neuroendocrine system. A particularly significant interaction occurs between CHGA and Secretogranin III (SCG3), with CHGA binding strongly to SCG3 in the intragranular environment . This interaction is functionally important as it targets CHGA to secretory granules in pituitary and pancreatic endocrine cells, demonstrating CHGA's involvement in protein sorting mechanisms that maintain neuroendocrine cell specialization . Recombinant human CHGA from HEK293 cells shows specific binding to SCG3 with an ED50 of 40-360 ng/mL in functional ELISA assays, providing a quantitative measure of this important interaction that can be used to assess the functional integrity of recombinant CHGA preparations .
Other molecular interactions involve CHGA-derived peptides with their respective receptors and targets. Catestatin, for instance, interacts with nicotinic acetylcholine receptors to inhibit catecholamine release, while vasostatin binds to endothelial cells to mediate its vasorelaxant effects . These peptide-specific interactions expand the functional repertoire of CHGA beyond its structural role in secretory granules and explain its wide-ranging physiological influences across multiple organ systems. The detailed characterization of these interactions using recombinant CHGA has significantly advanced our understanding of neuroendocrine regulatory networks and identified potential therapeutic targets for conditions involving dysregulated neuroendocrine function.
Expression Patterns and Tissue Distribution
Chromogranin A exhibits distinct expression patterns across various tissues and cell types, with particularly high expression in neuroendocrine cells throughout the body. Research using CHGA reporter systems has provided detailed characterization of CHGA-expressing cells, revealing complex distribution patterns with cell type-specific expression levels . In the gastrointestinal tract, CHGA expression follows a distinctive pattern with variations across different regions and enteroendocrine cell populations. Table 2 summarizes the expression patterns of CHGA across major tissues and cell types based on recent research findings.
Table 2: CHGA Expression Patterns in Human Tissues and Cell Types
In the stomach, CHGA is highly expressed in almost all histamine-storing enterochromaffin (EC)-like cells, at lower levels in the majority of serotonin-storing EC cells and ghrelin cells, in a small fraction of somatostatin cells, but is absent from gastrin cells . This heterogeneous expression pattern continues throughout the gastrointestinal tract, with selective but weak expression in EC cells of the small intestine and exclusive expression in EC cells of the colon, while being absent from peptide-storing enteroendocrine cells in these regions . In contrast, the pancreas shows a different pattern, with CHGA expression in β-cells, α-cells, and a fraction of pancreatic polypeptide cells .
These detailed expression patterns, revealed through studies utilizing recombinant CHGA and reporter systems, provide crucial insights into the functional organization of the neuroendocrine system across different tissues. The cell type-specific expression levels suggest specialized roles for CHGA in different neuroendocrine populations and help explain the varied clinical manifestations of conditions involving CHGA dysregulation . Understanding these expression patterns is essential for interpreting CHGA measurements in diagnostic applications and for developing targeted therapeutic approaches for neuroendocrine disorders.
Diagnostic Applications in Neuroendocrine Tumors
Chromogranin A has established itself as an invaluable clinical biomarker for neuroendocrine tumors (NETs), with elevated levels detected in patients with both neuroendocrine and non-neuroendocrine tumors . The protein's exclusive expression in neuroendocrine cells and co-secretion with hormones and neurotransmitters makes it particularly suitable for detecting and monitoring conditions involving abnormal neuroendocrine activity. Increased CHGA levels have been observed in various neuroendocrine tumors, including carcinoid tumors, pheochromocytomas, paragangliomas, and other NETs, with CHGA measurements in blood or tissue samples providing diagnostic and prognostic information . The availability of well-characterized recombinant human CHGA produced in HEK293 cells has significantly contributed to the development and standardization of assays for measuring CHGA in clinical samples, ensuring reliable diagnostic and monitoring protocols .
Beyond its role as a circulating biomarker, tissue expression of CHGA serves as an important diagnostic indicator in histopathological evaluation of suspected neuroendocrine neoplasms. Coexpression of CHGA and neuron-specific enolase (NSE) is common in neuroendocrine neoplasms, providing a characteristic immunohistochemical profile for these tumors . Additionally, research has demonstrated that full-length CHGA containing its C-terminal region can impair angiogenesis and tumor growth, suggesting potential therapeutic applications beyond its diagnostic utility . This anti-angiogenic property may contribute to the clinical behavior of CHGA-expressing tumors and offers insights into potential treatment approaches targeting CHGA-mediated pathways.
Cardiovascular and Metabolic Implications
Beyond oncology, CHGA has significant implications for cardiovascular and metabolic disorders, with research establishing connections between CHGA variants and conditions such as hypertension and metabolic syndrome. Studies have shown that plasma CHGA levels parallel catecholamine release in human populations, establishing phenotypic links between CHGA and human hypertension . Genetic studies have identified specific CHGA promoter haplotypes associated with altered CHGA expression levels that correlate with clinical parameters including plasma glucose levels, diastolic blood pressure, and body mass index . One particular haplotype (haplotype 2, containing variant T alleles at -1018 and -57 bp) exhibits higher promoter activity and is associated with elevated plasma CHGA levels and metabolic markers, suggesting that CHGA genetic variations may contribute to cardiovascular and metabolic risk assessment .
The CHGA-derived peptide catestatin has emerged as particularly relevant to cardiovascular regulation, functioning as an endogenous inhibitor of catecholamine release with potent anti-hypertensive properties . Reduced plasma catestatin levels have been observed in subjects with established hypertension and their at-risk siblings, suggesting a potential mechanistic link between CHGA processing and blood pressure regulation . These findings highlight the complex relationship between CHGA expression, processing to bioactive peptides, and cardiovascular homeostasis, offering potential targets for therapeutic intervention in hypertension and related disorders. The availability of recombinant human CHGA from HEK293 cells facilitates detailed investigation of these relationships by providing consistent material for mechanistic studies and assay development.
Genetic Regulation and Promoter Analysis
Recent research has made significant progress in understanding the genetic regulation of CHGA expression, with important implications for both normal physiological variation and disease susceptibility. Detailed analysis of the human CHGA promoter has identified multiple single-nucleotide polymorphisms (SNPs) that influence transcriptional activity and CHGA expression levels . A comprehensive study revealed 20 common SNPs in the CHGA locus, including 8 variants in the 1.2-kbp proximal promoter region . From these variations, researchers have identified several promoter haplotypes that display differential activities in neuronal cells, with particular haplotypes showing enhanced promoter activity under both basal conditions and pathophysiological states such as inflammation and hypoxia .
Of particular interest, transcription factor c-Rel has been identified as playing a significant role in activating specific CHGA promoter haplotypes, particularly haplotype 2 (containing variant T alleles at -1018 and -57 bp), which exhibits the highest promoter activity . Additionally, the transcription factor LEF1 (Lymphoid Enhancer Factor-1) has been shown to differentially regulate CHGA expression based on the G-462A polymorphism (rs9658634), with preferential effects on the A allele compared to the G allele . These findings provide mechanistic insights into how genetic variations in the CHGA promoter region influence gene expression and potentially contribute to disease susceptibility. Individuals carrying promoter haplotype 2 demonstrate higher plasma CHGA levels, plasma glucose levels, diastolic blood pressure, and body mass index, suggesting this genetic variant (occurring in a large proportion of the world population) may identify individuals at higher risk for cardiovascular and metabolic disorders .
Advanced Technologies for CHGA Research
Technological advancements have significantly enhanced our ability to investigate CHGA biology and its clinical implications. The development of reporter systems for chromogranin A has enabled visualization and tracking of CHGA-expressing cells in various tissues and experimental models . These reporter systems have facilitated detailed characterization of enteroendocrine cell populations throughout the gastrointestinal tract, providing unprecedented insights into their distribution and functional properties . For instance, the ChgA-humanized Renilla reniformis (hr)GFP reporter mouse has allowed researchers to identify and characterize serotonin and histamine-secreting enteroendocrine cells across different regions of the gastrointestinal tract, revealing complex patterns of CHGA expression that correlate with specific hormone production .
Single-cell transcriptomics applied to CHGA-expressing cells has further expanded our understanding of neuroendocrine cell diversity. Analysis of CHGA-positive enteroendocrine cells from human intestinal organoids has revealed distinct subpopulations with unique gene expression signatures, contributing to a more nuanced understanding of neuroendocrine cell heterogeneity . The integration of CHGA data with broader genomic and epigenomic datasets, such as the FANTOM5 enhancer atlas, provides context for understanding CHGA regulation within the landscape of human gene expression across diverse cell types and tissues . These advanced technologies, combined with the availability of well-characterized recombinant CHGA from HEK293 cells, are accelerating our understanding of CHGA biology and its implications for health and disease.
Product Specs
Chromogranin A (CgA) is a protein found in neuroendocrine cells throughout the body. These cells are responsible for releasing hormones and other signaling molecules. CgA is particularly abundant in the digestive system, adrenal glands, and pancreas. Measuring CgA levels is valuable in diagnosing and monitoring neuroendocrine tumors (NETs), which are cancers that arise from these cells. Elevated CgA levels can indicate the presence and aggressiveness of NETs, helping healthcare professionals make informed treatment decisions.
This product consists of a specifically designed version of the human Chromogranin A (CgA) protein produced in a laboratory setting using HEK cells. This recombinant CgA protein encompasses amino acids 19 to 457 of the full-length human CgA sequence, resulting in a single polypeptide chain with a molecular weight of 49.7 kDa. A 6-amino acid Histidine tag is attached to the C-terminus of the protein to facilitate its purification and detection. Notably, this recombinant CgA protein undergoes glycosylation, a process of adding sugar molecules, making it structurally similar to the naturally occurring CgA found in the human body.
The CHGA protein has undergone filtration using a 0.4 µm filter and subsequent lyophilization from a solution containing 0.5mg/ml CHGA in phosphate-buffered saline (PBS) at a pH of 7.0.
To create a working solution of around 0.5 mg/ml, it's advised to introduce deionized water to the lyophilized pellet and allow it to fully dissolve.
For long-term storage, maintain the lyophilized protein at -20°C. After reconstituting the protein, divide it into smaller portions to minimize freeze-thaw repetitions. The reconstituted protein can be kept at 4°C for a limited period, showing stability for up to two weeks without noticeable changes.
The purity of this product is greater than 95%, as assessed by SDS-PAGE, a widely used technique for determining protein purity.
CHGA, CGA, Chromogranin-A, Vasostatin I, SP-I, Pituitary secretory protein I.
HEK293 cells.
LPVNSPMNKG DTEVMKCIVE VISDTLSKPS PMPVSQECFE TLRGDERILS ILRHQNLLKE LQDLALQGAK ERAHQQKKHS GFEDELSEVL ENQSSQAELK EAVEEPSSKD VMEKREDSKE AEKSGEATDG ARPQALPEPM QESKAEGNNQ APGEEEEEEE EATNTHPPAS LPSQKYPGPQ AEGDSEGLSQ GLVDREKGLS AEPGWQAKRE EEEEEEEEAE AGEEAVPEEE GPTVVLNPHP SLGYKEIRKG ESRSEALAVD GAGKPGAEEA QDPEGKGEQE HSQQKEEEEE MAVVPQGLFR GGKSGELEQE EERLSKEWED SKRWSKMDQL AKELTAEKRL EGQEEEEDNR DSSMKLSFRA RAYGFRGPGP QLRRGWRPSS REDSLEAGLP LQVRGYPEEK KEEEGSANRR PEDQELESLS AIEAELEKVA HQLQALRRGH HHHHH
Q&A
What is Chromogranin A (CHGA) and what is its molecular significance?
Chromogranin A is a ~50-kDa soluble, acidic glycoprotein that serves as an essential component in the formation of catecholamine secretory vesicles in neuronal, endocrine, and neuroendocrine tissues. It functions as a prohormone, undergoing proteolytic cleavage to generate multiple biologically active peptides including vasostatin (exhibiting antiadrenergic activity), parastatin (inhibiting parathyroid hormone secretion), pancreastatin (inhibiting insulin secretion), and catestatin (inhibiting catecholamine secretion) .
CHGA is encoded by the CHGA/CgA gene located on chromosome 14, and its expression correlates directly with the quantity of secretory vesicles in neuroendocrine cells . The molecular structure contains numerous sites susceptible to enzymatic proteolytic activity, resulting in the formation of various bioactive peptides that are believed to act as precursors to active proteins .
Why are HEK-293 cells commonly used for CHGA expression studies?
HEK-293 cells have become a preferred host system for heterologous expression of membrane proteins like CHGA for several important reasons:
-
High transfection efficiency compared to other mammalian cell lines
-
Faithful translation and processing of proteins, including post-translational modifications
-
Advantageous cell size, morphology, and division rate for experimental manipulation
-
Low expression of native channels, making them particularly suitable for current-voltage measurements in electrophysiological studies
-
Ability to express complex membrane proteins including those from different species
These characteristics make HEK-293 cells particularly valuable for functional studies of CHGA, especially when conducting single-cell applications such as electrophysiology or fluorescent imaging experiments .
What are the key considerations for designing expression vectors for CHGA in HEK cells?
When designing expression vectors for CHGA studies in HEK-293 cells, researchers should consider:
-
Appropriate promoter selection: Strong promoters like CMV are typically used for high expression levels in HEK cells
-
Inclusion of suitable identification tags: Fluorescent tags (GFP, YFP) or epitope tags (FLAG, His) facilitate detection and purification
-
Codon optimization: Adjusting codons for optimal expression in human cells, especially when expressing CHGA variants from other species
-
Kozak sequence inclusion: Enhances translation initiation efficiency
-
Regulatory elements: Including appropriate enhancers and terminators for stable expression
As demonstrated in transient expression studies, careful vector design significantly impacts the successful expression of complex membrane proteins in HEK-293 cells . For CHGA specific studies, vectors containing the full-length human CHGA sequence with appropriate tags have been successfully used to study genetic variants like the 3′-UTR C+87T (rs7610) .
What common methods are used to detect CHGA expression in transfected HEK cells?
Several methods are employed to detect CHGA expression in transfected HEK-293 cells:
-
Fluorescence microscopy: When CHGA is fused to fluorescent proteins like GFP, direct visualization of subcellular localization and expression levels becomes possible
-
Western blotting: Using anti-CHGA antibodies to detect protein expression levels
-
RT-PCR and qPCR: Measuring CHGA mRNA expression levels
-
Immunocytochemistry: Using specific antibodies to visualize CHGA localization
-
Functional assays: Including luciferase reporter assays when CHGA is fused to luciferase, allowing quantification of expression
For more sensitive detection, research has successfully utilized chimeric luciferase/CHGA reporters to quantify expression differences between genetic variants, such as the T allele versus C allele at position C+87T in the 3′-UTR .
How do microRNAs regulate CHGA expression, and what methodologies are used to study these interactions?
MicroRNAs (miRNAs) play a critical role in post-transcriptional regulation of CHGA expression. Research has identified that miR-107 interacts with the 3′-UTR of CHGA, particularly at the C+87T (rs7610) variant site, with differential effects depending on the allele present.
Methodological approaches to study these interactions include:
-
Bioinformatic prediction: Identifying potential miRNA binding sites in the CHGA 3′-UTR
-
Luciferase reporter assays: Constructing chimeric luciferase/CHGA 3′-UTR reporters containing either the T or C allele at position C+87T to quantify expression differences when miR-107 levels are altered
-
Cotransfection experiments: Using specific miRNA oligonucleotides (such as hsa-miR-107) with eukaryotic CHGA plasmids
-
In vitro transcription/translation: Examining the direct effects of miRNA expression on CHGA variant expression
-
Transgenic animal models: Creating humanized CHGA locus models to study the effects of miRNA inhibitors on physiological parameters
Research has demonstrated that the CHGA 3′-UTR C+87T variant disrupts an miR-107 binding motif, with the T allele showing greater sensitivity to changes in miR-107 expression levels than the C allele. This cis:trans (mRNA:miR) interaction appears to regulate the association of CHGA with blood pressure and hypertensive nephropathy .
What experimental protocols are recommended for optimizing transient transfection of CHGA in HEK-293 cells?
Optimizing transient transfection of CHGA in HEK-293 cells requires careful consideration of multiple factors:
| Transfection Parameter | Optimization Approaches |
|---|---|
| Cell density | 70-80% confluency at time of transfection |
| DNA quality | High purity (A260/A280 ratio ~1.8-2.0) |
| DNA:transfection reagent ratio | Typically 1:3 ratio, but requires optimization |
| Transfection reagent selection | Compare lipid-based (Lipofectamine), polymer-based (PEI), and physical methods |
| Incubation time | Usually 24-72 hours post-transfection |
| Serum conditions | Reduced serum during transfection, full serum for expression |
| Selection markers | For stable transfection (if needed) |
A step-by-step optimized protocol should include:
-
Seed HEK-293 cells at appropriate density 24 hours before transfection
-
Prepare DNA-transfection reagent complexes in serum-free media
-
Add complexes to cells and incubate for 4-6 hours
-
Replace with complete growth medium
-
Analyze expression after appropriate incubation period (typically 24-72 hours)
For CHGA expression specifically, researchers have successfully used both transient and stable transfection approaches in HEK-293 cells to study variant effects and functional properties .
How can researchers investigate the functional consequences of CHGA genetic variants using HEK cell models?
Investigating functional consequences of CHGA genetic variants in HEK cell models involves several sophisticated approaches:
-
Site-directed mutagenesis: Creating specific CHGA variants (like C+87T) in expression vectors
-
Luciferase reporter assays: Quantifying differential expression of variants using chimeric constructs
-
RNA stability assays: Measuring mRNA half-life differences between variants
-
miRNA interaction studies: Assessing how variants affect miRNA binding using synthetic miRNA mimics or inhibitors
-
Protein expression and localization: Using Western blotting and immunofluorescence to assess expression levels and subcellular distribution
-
Secretion assays: Measuring secreted CHGA and its processed peptides in cell culture supernatants
-
Electrophysiological measurements: Assessing functional consequences of variants on cellular physiology
For example, researchers have used chimeric luciferase/CHGA 3′-UTR reporters in HEK cells to demonstrate that the T allele at position C+87T is more responsive to changes in miR-107 expression levels compared to the C allele, providing a molecular mechanism for the association of this variant with hypertension and renal disease .
What approaches are used to study the interaction between CHGA-derived peptides and their receptors in HEK expression systems?
Studying interactions between CHGA-derived peptides (catestatin, vasostatin, pancreastatin, parastatin) and their receptors in HEK expression systems employs several methodological approaches:
-
Heterologous receptor expression: Expressing putative receptors for CHGA-derived peptides in HEK cells
-
Calcium imaging: Monitoring intracellular calcium changes in response to peptide exposure
-
FRET/BRET binding assays: Measuring direct binding between fluorescently labeled peptides and receptors
-
Signaling pathway analysis: Assessing downstream signaling pathways activated by peptide-receptor interactions
-
Receptor mutagenesis: Identifying critical receptor domains involved in peptide binding
-
Competitive binding assays: Using labeled and unlabeled peptides to determine binding kinetics and affinity
-
Electrophysiology: Measuring changes in membrane potential or ionic currents in response to peptide application
These methods help elucidate the molecular mechanisms by which CHGA-derived peptides exert their biological effects. For instance, catestatin has been shown to inhibit catecholamine secretion from chromaffin cells, while pancreastatin exhibits a strong inhibitory effect on insulin secretion .
How can researchers translate findings from HEK-CHGA studies to in vivo physiological effects?
Translating HEK-CHGA findings to in vivo physiological effects requires building bridges between cellular observations and whole-organism physiology:
-
Humanized mouse models: Using BAC transgenesis to create mice expressing human CHGA variants, as demonstrated with the humanized CHGA mouse strain carrying the T/T genotype at C+87T
-
miRNA modulators in vivo: Administering miRNA inhibitors (like hsa-miR-107 antagomir) to study effects on blood pressure regulation in transgenic animals
-
Telemetric physiological monitoring: Continuous measurement of blood pressure and heart rate in conscious mice to assess physiological effects of CHGA variants
-
Tissue-specific expression analysis: Examining CHGA mRNA and protein levels in relevant tissues (adrenal, brain stem) after experimental manipulations
-
Comparison between in vitro and in vivo findings: Correlating HEK cell expression data with physiological observations
Research has shown that in humanized CHGA mice (T/T genotype at C+87T), administration of hsa-miR-107 antagomir resulted in prolonged decreases in both systolic and diastolic blood pressure, effects not observed in wild-type mice. These findings were correlated with elevated CHGA mRNA expression in adrenal and brain stem tissues 5 hours post-treatment, demonstrating successful translation from cellular mechanisms to physiological effects .
What are the critical controls needed when studying CHGA microRNA interactions in HEK cells?
When investigating CHGA-microRNA interactions in HEK cells, several critical controls must be included:
-
Empty vector controls: Transfection with vectors lacking the CHGA 3′-UTR to control for non-specific effects
-
Mutated binding site controls: Creating constructs with mutations in the predicted miRNA binding sites
-
Non-targeting miRNA controls: Using miRNAs that don't target the CHGA sequence
-
Dose-response assessments: Testing various concentrations of miRNA mimics or inhibitors
-
Time-course experiments: Evaluating effects at different time points post-transfection
-
Endogenous miRNA expression analysis: Quantifying baseline expression of the miRNA of interest in HEK cells
-
Rescue experiments: Determining if phenotypes can be rescued by restoring normal miRNA levels
-
Cross-species controls: Comparing effects on human CHGA versus mouse Chga to validate species-specific interactions
For example, in studies of miR-107 interaction with the CHGA 3′-UTR, researchers demonstrated significantly greater effects of miR-107 expression changes on the T allele variant compared to the C allele at position C+87T, establishing the specificity of this interaction .
What are common challenges in achieving optimal CHGA expression in HEK cells and how can they be overcome?
Researchers frequently encounter several challenges when expressing CHGA in HEK-293 cells:
| Challenge | Troubleshooting Approach |
|---|---|
| Low transfection efficiency | Optimize cell density, DNA:reagent ratio; consider electroporation for difficult constructs |
| Protein misfolding | Reduce expression temperature to 30-32°C; add chemical chaperones |
| Cytotoxicity | Reduce expression time; use weaker promoters; create stable cell lines |
| Poor protein detection | Optimize tag position (N- vs C-terminal); use different antibodies or detection methods |
| Inconsistent expression | Standardize cell passage number; use single-cell cloning for stable lines |
| Non-physiological overexpression | Use inducible expression systems; titrate transfection amount |
For CHGA specifically, optimization of the transfection protocol based on the HEK-293T cell line has proven effective since these cells naturally express both CHGA and hsa-miR-107, making them particularly suitable for studying their interactions .
How can researchers differentiate between CHGA-specific effects and artifacts in HEK expression systems?
Distinguishing genuine CHGA-specific effects from artifacts in HEK expression systems requires rigorous controls:
-
Multiple detection methods: Corroborate findings using different techniques (Western blotting, qPCR, immunofluorescence)
-
Dose-dependent responses: Establish concentration-response relationships for CHGA-related effects
-
Specific inhibitors: Use targeted inhibitors of CHGA-derived peptides or their signaling pathways
-
siRNA knockdown validation: Confirm effects by specifically knocking down CHGA expression
-
Rescue experiments: Restore function by re-introducing wild-type CHGA in knockdown cells
-
Alternative cell lines: Verify findings in other cell types to rule out HEK-specific artifacts
-
Validation in primary cells: Confirm key findings in primary cells that naturally express CHGA
-
In vivo correlation: Connect cellular observations with animal model phenotypes
These approaches help ensure that observed effects are genuinely related to CHGA biology rather than artifacts of the expression system.
What analytical approaches can resolve contradictory data when studying CHGA variants in different experimental setups?
When faced with contradictory data from different experimental approaches studying CHGA variants, researchers should employ:
-
Systematic meta-analysis: Compare methodologies, cell types, and experimental conditions across studies
-
Standardization of protocols: Establish consistent experimental conditions to minimize variation
-
Statistical power analysis: Ensure adequate sample sizes to detect true effects
-
Bayesian analysis: Incorporate prior probability distributions to better interpret contradictory results
-
Multivariate analysis: Examine interactions between multiple variables that may explain discrepancies
-
Independent validation: Confirm key findings through independent laboratories
-
Alternative methodologies: Apply complementary techniques to address the same question
-
Genetic background consideration: Account for differences in genetic background when using different cell lines or animal models
For instance, contradictory findings regarding CHGA variant effects might be resolved by considering differences in miRNA expression levels across experimental systems, as demonstrated by the differential effects of miR-107 on the CHGA 3′-UTR C+87T variant depending on cellular context .
What emerging technologies show promise for advancing CHGA research in HEK expression systems?
Several cutting-edge technologies are poised to advance CHGA research using HEK expression systems:
-
CRISPR/Cas9 gene editing: Creating precise CHGA variants or knockouts in HEK cells
-
Single-cell transcriptomics: Examining heterogeneity in CHGA expression and processing
-
Optogenetics: Controlling CHGA expression or secretion with light-sensitive proteins
-
Organ-on-chip technologies: Studying CHGA in more physiologically relevant microenvironments
-
Proximity labeling methods: Identifying novel CHGA-interacting proteins
-
Super-resolution microscopy: Visualizing CHGA trafficking and secretory vesicle dynamics
-
Nanobody-based detection: Developing more specific tools for CHGA visualization
-
AI-driven predictive modeling: Forecasting functional consequences of CHGA variants
These technologies could overcome current limitations in studying the mechanisms underlying CHGA function and the roles of its genetic variants in disease pathogenesis .
How might studying CHGA in HEK cells contribute to understanding chronic renal disease progression?
CHGA research in HEK cells offers several promising avenues for understanding chronic renal disease progression:
-
Genetic variant functional analysis: Elucidating how CHGA 3′-UTR variants like C+87T affect expression and contribute to renal disease susceptibility
-
miRNA-based regulatory mechanisms: Understanding how miR-107 and related miRNAs modulate CHGA expression in renal contexts
-
Long-term expression models: Developing stable HEK cell lines expressing CHGA variants to model chronic effects
-
Co-culture systems: Combining HEK cells with renal cell types to study paracrine effects
-
Humanized mouse validation: Translating HEK cell findings to animal models for long-term renal outcomes
-
Patient-derived variant screening: Testing patient-specific CHGA variants in HEK cells to predict disease risk
Research has already established associations between CHGA genetic variation and hypertensive nephropathy in African Americans, with the 3′-region of CHGA linked to end-stage renal disease (ESRD) and the rate of chronic GFR decline. Furthermore, CHGA genetic variation has been shown to predict renal injury in IgA nephropathy with malignant hypertension in Chinese populations .
What methodological advances are needed to better translate HEK-CHGA findings to clinical applications?
To enhance translation of HEK-CHGA research to clinical applications, several methodological advances are needed:
-
Patient-derived cell models: Using induced pluripotent stem cells (iPSCs) from patients with CHGA variants
-
High-throughput variant screening: Developing platforms to rapidly test multiple CHGA variants
-
Physiologically relevant culture conditions: Incorporating flow, mechanical stretch, or hypoxia to better mimic in vivo conditions
-
Multi-omics integration: Combining transcriptomic, proteomic, and metabolomic data from CHGA studies
-
Longitudinal in vitro models: Developing systems to study long-term effects of CHGA variants
-
Improved animal models: Creating more sophisticated humanized CHGA mouse models with tissue-specific expression
-
Biomarker validation pipelines: Establishing standardized approaches to validate CHGA-related biomarkers
-
Clinical correlation databases: Building repositories linking CHGA variants to clinical outcomes
As noted in existing research, while studies have clearly shown effects of miR-107 on human CHGA gene expression influencing blood pressure, the specific effects of the cis/trans CHGA/miR-107 interaction on progressive renal disease require additional long-term rodent experiments spanning months to years to fully understand the mechanisms underlying renal disease associations .
Product Science Overview
Chromogranin A (CgA) is a member of the granin family of proteins, which are found in the secretory vesicles of neuroendocrine cells. It was first identified in the bovine adrenal medulla in 1967 and has since been recognized as a precursor to several functional peptides, including vasostatin-1, vasostatin-2, pancreastatin, catestatin, and parastatin . These peptides play crucial roles in modulating neuroendocrine functions.
Chromogranin A is an intrinsically disordered protein, meaning it does not have a fixed three-dimensional structure under physiological conditions. This characteristic allows it to be cleaved into various bioactive peptides that perform diverse functions. For instance, vasostatin-1 and vasostatin-2 inhibit vasoconstriction, while catestatin acts as an inhibitor of catecholamine release .
Human recombinant Chromogranin A is often expressed in Human Embryonic Kidney (HEK) 293 cells. These cells are widely used in biotechnology for the production of recombinant proteins due to their high transfection efficiency and ability to perform post-translational modifications similar to those in human cells . The recombinant form of Chromogranin A produced in HEK cells is used in various research and clinical applications, including the study of neuroendocrine tumors and cardiovascular diseases .
Chromogranin A serves as a biomarker for several diseases, particularly neuroendocrine tumors (NETs). Elevated levels of circulating CgA can be detected in patients with gastroenteropancreatic NETs and correlate with tumor burden . Additionally, CgA has been implicated in cardiovascular diseases, including essential hypertension, where it may play a role in regulating blood pressure .
The discovery of Chromogranin A marked a significant milestone in the field of neuroendocrinology. Initially identified as a mysterious protein released from the adrenal medulla, it was later found to be a key player in the regulation of neuroendocrine functions. Over the years, research has expanded our understanding of its structure, function, and clinical significance .
