ENA 78 Human (8-78 a.a.)
Description
Structure and Bioactivity of ENA-78 Human (8-78 a.a.)
ENA-78 (Epithelial Neutrophil-Activating Peptide-78), also known as CXCL5, is a mature, bioactive chemokine derived from a 114-amino acid (aa) precursor. Following signal peptide cleavage, the functional form spans residues 8–78 (71 aa), with a molecular weight of 8.3–12 kDa . Key structural features include:
Bioactivity:
-
Chemotaxis: Recruits neutrophils via CXCR2 receptors, inducing calcium flux and exocytosis .
-
Angiogenesis: Promotes endothelial cell migration and vascular remodeling .
-
Potency Variation: Truncated forms (ENA-74/70) exhibit enhanced neutrophil activation .
Expression and Regulation of ENA-78
ENA-78 is upregulated in inflammatory environments and expressed by:
Clinical Associations:
Neutrophil Activation
ENA-78 mimics IL-8 and GRO-α, acting through CXCR2 to:
-
Induce Chemotaxis: Concentration-dependent recruitment of neutrophils (ED50: 2–40 ng/mL) .
-
Enhance Inflammation: Synergizes with other chemokines (e.g., IL-8) to amplify immune responses .
Angiogenesis
ENA-78 stimulates endothelial cell proliferation and vessel formation, contributing to tumor vascularization .
Cancer Progression
Overexpression in tumors correlates with metastasis and poor prognosis, particularly in lung, breast, and colorectal cancers .
Rheumatoid Arthritis Models
In adjuvant-induced arthritis (AIA), anti-ENA-78 antibodies administered pre-symptomatically reduced joint inflammation, while post-onset treatment showed no effect .
Diagnostic and Therapeutic Potential
| Application | Approach | Outcome |
|---|---|---|
| Biomarker | Quantikine ELISA for serum/synovial fluid | Detects ENA-78 levels (16–1000 pg/mL) |
| Therapeutic Target | Neutralizing antibodies | Reduces neutrophil recruitment in AIA |
Research Tools and Applications
R&D Systems’ Quantikine and DuoSet ELISA kits offer:
Product Specs
Recombinant Human Epithelial Neutrophil-Activating Protein 78, produced in E. coli, is a single, non-glycosylated polypeptide chain consisting of 71 amino acids (8-78 a.a.). This protein has a molecular weight of 7.8 kDa. The CXCL5 is purified using proprietary chromatographic techniques.
(a) Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) analysis.
(b) SDS-PAGE analysis.
Q&A
What is the structural composition of human ENA-78?
Full-length human ENA-78/CXCL5 is 114 amino acids in length with a predicted molecular weight of 12 kDa. Following removal of the signal peptide, the bioactive form becomes 78 amino acids in length. This chemokine can be further N-terminally cleaved by proteolytic enzymes such as Cathepsin G and Chymotrypsin to generate shorter variants including ENA-74 (74 aa) and ENA-70 (70 aa). These N-terminally truncated forms demonstrate increased biological potency compared to the full 78 aa form . The specific 8-78 a.a. variant represents one of the naturally occurring truncated forms with enhanced biological activity.
Which cell types express ENA-78 and under what conditions?
ENA-78 expression occurs in multiple cell types, with significant upregulation at sites of inflammation. Expression has been documented in:
-
Multiple hematopoietic cell lineages
-
Fibroblasts
-
Endothelial cells
-
Vascular smooth muscle cells
The production of ENA-78 is stimulated by pro-inflammatory cytokines, particularly interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα). This induction pattern indicates its role in inflammatory cascades, with stimulation leading to significant increases in expression across diverse cell types .
How does one accurately measure ENA-78 concentrations in biological samples?
Several validated methodologies exist for quantifying ENA-78 in biological specimens:
-
ELISA-based detection systems: The Quantikine Human ENA-78 Immunoassay represents a standardized 4.5-hour solid-phase ELISA designed specifically for human ENA-78 measurement in cell culture supernatants, serum, and plasma samples . The table below demonstrates the precision of this assay:
| Sample Type | Intra-Assay Precision (CV%) | Inter-Assay Precision (CV%) |
|---|---|---|
| Cell Culture Supernates | 4.1-5.8% | 8.9-9.8% |
| Serum/Plasma | 3.8-8.3% | 6.7-9.3% |
-
Simple Plex Cartridge Systems: These automated microfluidic immunoassays provide enhanced precision with:
| Intra-Assay Precision | Inter-Assay Precision |
|---|---|
| Sample | 1 |
| Mean (pg/mL) | 32.6 |
| Standard Deviation | 2.14 |
| CV% | 6.5 |
-
High-throughput protein arrays: For multiplex analysis, antibody-coated glass slide arrays can simultaneously assess ENA-78 alongside other cytokines, as demonstrated in studies of diabetic wound healing .
What methodological considerations are important when studying ENA-78 in disease models?
When investigating ENA-78 in disease contexts, researchers should consider:
-
Appropriate biological matrices: ENA-78 can be measured in various specimens including serum, plasma, synovial fluid, and wound exudates. Sample processing protocols must be standardized to ensure consistent results.
-
Statistical approaches: Studies analyzing ENA-78 typically employ:
-
Student's t-test or Fisher-Pitman permutation test for normally distributed variables
-
Non-parametric Mann-Whitney U test for non-normally distributed data
-
Correlation analysis using Pearson or Spearman methods depending on data distribution
-
Binary logistic regression for assessing independent predictors
-
ROC curve analysis for determining clinically relevant cutoff points
-
-
Sample dilution protocols: Depending on the biological specimen and expected concentration range, dilution factors ranging from 1:2 to 1:100 may be necessary for accurate quantification .
How does ENA-78 compare functionally to other CXC chemokines?
ENA-78 belongs to the CXC subfamily of chemokines and shares several functional properties with related molecules:
-
Receptor specificity: ENA-78 signals primarily through the CXCR2 chemokine receptor .
-
Biological activity assessment: The functional potency of ENA-78 can be experimentally determined through neutrophil chemotaxis assays using concentration ranges of 2.0-40.0 ng/ml .
-
Species-specific considerations: Despite earlier assumptions, genome-wide analysis and current consensus indicate that human ENA-78 does not have a true murine ortholog . This has important implications for translational research and necessitates careful consideration when designing animal model experiments.
What is the role of ENA-78 in rheumatoid arthritis pathogenesis?
ENA-78 demonstrates significant involvement in rheumatoid arthritis (RA) pathophysiology:
-
Expression pattern: Increased expression of ENA-78 has been documented in inflamed synovial tissue and fluid in human RA compared to osteoarthritis .
-
Animal model findings: In rat adjuvant-induced arthritis (AIA), an ENA-78-like protein shows:
-
Therapeutic implications: Administration of anti-human ENA-78 antibodies prior to disease onset modified AIA severity, while post-onset administration showed no significant effect. This temporal specificity suggests a critical role for ENA-78 in the initiation phase of inflammatory arthritis rather than in established disease .
How does ENA-78 contribute to cancer progression?
ENA-78 has been implicated in neoplastic processes through multiple mechanisms:
-
Prognostic significance: Elevated CXCL5 expression in tumor tissues correlates with poor prognosis in pancreatic cancer, as demonstrated by Kaplan-Meier survival analysis .
-
Pathophysiological mechanisms: ENA-78 contributes to cancer progression through:
-
Research methodologies: Investigating ENA-78 in cancer contexts typically involves techniques such as:
What is the significance of ENA-78 in wound healing processes?
Recent research has identified ENA-78 as a novel predictor of wound healing outcomes:
-
Clinical relevance: In diabetic foot ulcers, ENA-78 levels in wound exudate and plasma correlate with healing outcomes.
-
Analytical approaches: Studies investigating this association have employed:
-
Correlations with clinical parameters: The relationship between ENA-78 levels and various clinical factors has been analyzed:
| Variable | ENA-78 Plasma | ENA-78 Exudate |
|---|---|---|
| Age | Rho=-0.042, P=0.666 | Rho=-0.161, P=0.095 |
| HbA1C | Rho=-0.206, P=0.031 | Rho=-0.266, P=0.005 |
| Diabetes duration | Rho=0.133, P=0.168 | Rho=0.011, P=0.907 |
| Hypertension | Rho=-0.160, P=0.097 | Rho=-0.058, P=0.551 |
This data indicates significant inverse correlations between ENA-78 levels and glycemic control (HbA1C), suggesting potential mechanistic links between diabetic status and chemokine-mediated healing processes.
What are the optimal recombinant ENA-78 preparations for in vitro studies?
When designing experiments utilizing recombinant ENA-78:
-
Source considerations: E. coli-expressed recombinant human ENA-78 represents a commonly used research preparation .
-
Endotoxin testing: High-quality preparations should demonstrate endotoxin levels <0.1 ng/μg (1 EU/μg) when assessed using the LAL gel clot method .
-
Functional validation: Biological activity of recombinant preparations should be confirmed through neutrophil chemotaxis assays, typically using concentration ranges of 2.0-40.0 ng/ml .
How should researchers design experiments to study ENA-78's role in inflammatory conditions?
Experimental design for investigating ENA-78 in inflammatory contexts should consider:
-
Appropriate controls: Matched controls for age, sex, disease duration, and relevant clinical parameters are essential for meaningful comparisons .
-
Temporal dynamics: As demonstrated in arthritis models, ENA-78 levels change throughout disease progression, necessitating time-course analyses rather than single-point measurements .
-
Intervention timing: Therapeutic interventions targeting ENA-78 show differing efficacy depending on the disease stage, highlighting the importance of strategic timing in experimental interventions .
Product Science Overview
CXCL5 is produced by various cell types, including epithelial cells, in response to inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) . The protein consists of 78 amino acids and has a molecular weight of approximately 8.3 kDa . The recombinant form of CXCL5, specifically the 8-78 amino acid sequence, is often used in research to study its biological functions and therapeutic potential.
CXCL5 is primarily involved in the recruitment and activation of neutrophils, a type of white blood cell essential for the immune response. Upon binding to its receptor, CXCR2, on the surface of neutrophils, CXCL5 induces chemotaxis, guiding these cells to the site of infection or injury . This process is vital for the body’s defense against pathogens and the resolution of inflammation.
Research has shown that CXCL5 plays a significant role in various inflammatory diseases and conditions. For instance, elevated levels of CXCL5 have been observed in patients with acute coronary syndromes (ACS), suggesting its involvement in endothelial inflammation . Additionally, studies have indicated that genetic variations in the CXCL5 gene may influence the prognosis of ACS and the effectiveness of statin therapy .
Given its role in inflammation and immune response, CXCL5 is a potential target for therapeutic interventions. Modulating the activity of CXCL5 or its receptor, CXCR2, could provide new treatment options for inflammatory diseases, including cardiovascular diseases and chronic inflammatory conditions .
