Proguanylin Human
Description
Biochemical Structure and Processing
Proguanylin originates from a 116-amino acid precursor in mice (residues 22–116) and 115 residues in humans (22–115) . Key processing events include:
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Proteolytic cleavage: Generates shorter peptides, including the canonical 15-amino acid guanylin (positions 102–116 in mice) .
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Tissue-specific processing: Luminal intestinal extracts contain processed forms (e.g., 101–116, 103–116), while plasma retains full-length proguanylin .
| Proguanylin Forms | Detection Site | Bioactivity |
|---|---|---|
| Full-length (22–115/116) | Plasma, tissues | Endocrine signaling |
| Processed (e.g., 101–116) | Intestinal lumen | Local fluid secretion |
Intestinal Fluid Homeostasis
Proguanylin-derived guanylin activates GC-C, increasing cyclic GMP (cGMP) to:
Metabolic and Systemic Effects
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Obesity and Cancer: Reduced guanylin expression correlates with obesity-driven colorectal cancer . Forced guanylin re-expression suppresses tumor growth in high-fat diet mice .
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Renal Function: Elevated plasma proguanylin occurs in chronic renal failure, suggesting utility as a renal biomarker .
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Appetite Regulation: GC-C activation stimulates glucagon-like peptide-1 (GLP-1) secretion, indirectly influencing satiety .
Production Sites
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Intestinal Epithelium: Goblet cells, Paneth-like cells, and mature enterocytes (except duodenal enterocytes) in transgenic mouse models .
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Polarized Secretion: In Ussing chamber studies, human and murine tissues release 4–5x more proguanylin into the apical (luminal) compartment than basolateral (blood) side .
| Compartment | Proguanylin Concentration (Murine Jejunum) |
|---|---|
| Apical | 1,200–1,800 ng/mL |
| Basolateral | 200–400 ng/mL |
Disease Associations
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Inflammatory Bowel Disease (IBD): Serum proguanylin levels are reduced in active ulcerative colitis (UC) and Crohn’s disease (CD) compared to healthy controls .
| Parameter | UC Patients (Pre-Treatment) | Healthy Controls |
|---|---|---|
| Proguanylin (ng/mL) | 5.27 (3.80–6.65) | 11.35 ± 2.59 |
| S100A12 (ng/mL) | 39.36 (25.15–70.99) | 19.74 ± 8.07 |
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Surgical Impact: Total gastrectomy and intestinal transplantation elevate plasma proguanylin (26.5 ± 6.9 ng/mL post-gastrectomy vs. 15.2 ± 2.7 ng/mL in controls) .
Quantification Assays
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ELISA: The BioVendor Human Proguanylin ELISA demonstrates high precision, with spiking recoveries of 85.9–106.1% and linearity up to 98.4% recovery in serially diluted samples .
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LC-MS/MS: Identifies processed peptides in intestinal tissues but detects only full-length proguanylin in plasma .
Research Gaps and Therapeutic Potential
Product Specs
Recombinant Human Proguanylin, produced in E. coli, is a single, non-glycosylated polypeptide chain containing 104 amino acids. This includes a 10 amino acid N-terminal His tag and amino acids 22-115 of the Proguanylin sequence. The calculated molecular mass is 11.5 kDa.
Proguanylin is filtered through a 0.4 µm filter and lyophilized in deionized water at a concentration of 0.5 mg/ml.
To create a working stock solution, add deionized water to the lyophilized pellet to achieve a concentration of approximately 0.5 mg/ml and ensure complete dissolution. Please note that Proguanylin is not sterile. Before use in cell culture, it must be filtered through an appropriate sterile filter.
Lyophilized Proguanylin should be stored at -20°C. After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles. The reconstituted protein remains stable at 4°C for a limited period, showing no significant change after one week.
Purity is determined to be greater than 90.0% as assessed by SDS-PAGE.
MKHHHHHHAS VTVQDGNFSF SLESVKKLKD LQEPQEPRVG KLRNFAPIPG EPVVPILCSN PNFPEELKPL CKEPNAQEIL QRLEEIAEDP GTCEICAYAA CTGC.
Q&A
What is the molecular structure of human proguanylin?
Human proguanylin is a 116-amino acid prohormone that acts as a bioactive form of human guanylin. The recombinant form used in research settings is typically a 11.5 kDa protein containing 104 amino acid residues . The peptide structure consists of a signal sequence (residues 1-21) followed by the proguanylin sequence itself (residues 22-116 in mouse, 22-115 in human) . Understanding this molecular structure is essential for designing experiments that investigate proguanylin's role in physiological processes.
What is the relationship between proguanylin and guanylin?
Proguanylin is the 116-amino acid prohormone form, while guanylin is the processed 15-amino acid bioactive peptide derived from the C-terminus of the proguanylin molecule . Guanylin acts as an endogenous ligand for the intestinal cell receptor guanylyl cyclase C (GC-C) . When guanylin binds to GC-C, it activates a cGMP-mediated second messenger system that culminates in chloride and bicarbonate efflux through the cystic fibrosis transmembrane regulator (CFTR) . Researchers should note that proguanylin is the predominant secreted form, while processed guanylin appears to act locally at its site of action.
Which specific cell types express proguanylin in the intestine?
Transgenic mouse models with fluorescent reporter protein expression driven by the proguanylin promoter have revealed that proguanylin is expressed throughout the small intestine and colon in specific cell types. These include goblet cells and Paneth(-like) cells throughout the intestinal tract. Additionally, mature enterocytes express proguanylin in all intestinal regions except the duodenum . This differential expression pattern suggests specialized roles for proguanylin-producing cells in different regions of the intestine. Researchers investigating cell-specific expression should employ co-localization studies with appropriate cell-type markers.
Is proguanylin secretion regulated by negative feedback inhibition?
Studies using the C2/bbe1 cell line model for intestinal villous epithelial cells have investigated potential feedback regulation. Interestingly, proguanylin synthesis and secretion were not decreased following activation of guanylyl cyclase C-mediated chloride secretion . This finding implies that negative-feedback inhibition is not a primary regulatory mechanism for proguanylin secretion. Researchers should consider alternative regulatory mechanisms when investigating the control of proguanylin production and release.
What roles does proguanylin play outside the intestine?
Beyond its intestinal functions, proguanylin has been found to play an endocrine role by regulating the function of other tissues such as the kidney and liver . It serves as a significant marker in renal insufficiency, with plasma levels increasing in patients with chronic renal failure undergoing hemodialysis . Additionally, serum levels of proguanylin rise in patients with Cohn syndrome, suggesting potential use as a marker in cardiac failure diagnosis and therapy . Some research also suggests links between proguanylin and metabolism or food intake, with conflicting findings regarding its effects on appetite regulation .
How is proguanylin processed in different physiological compartments?
LC-MS/MS analysis has identified processed forms of proguanylin in tissue and luminal extracts, but only full-length proguanylin has been detected in plasma . This compartmentalization of processing suggests that proguanylin is activated locally at or near its site of action rather than systemically. The enzymatic pathway for processing proguanylin to generate bioactive guanylin remains incompletely characterized. Researchers investigating proguanylin processing should consider using protease inhibitors and cellular fractionation approaches to identify the specific enzymes and subcellular locations involved.
How do proguanylin levels change in renal disease?
Proguanylin serves as a significant biomarker in renal insufficiency. Plasma levels of proguanylin increase significantly in patients with chronic renal failure who are undergoing hemodialysis . This elevation may reflect altered clearance of proguanylin by the kidneys or could represent a compensatory response to disturbed fluid and electrolyte homeostasis. For clinical research, proguanylin measurements should be evaluated alongside other markers of renal function to establish correlations and potential causative relationships.
How do surgical interventions affect circulating proguanylin levels?
Human studies have shown that plasma proguanylin levels increase following total gastrectomy or intestinal transplantation . This finding indicates that the intestine is a major source of circulating proguanylin, and surgical modifications to the gastrointestinal tract can significantly impact systemic proguanylin levels. Moreover, unlike many metabolic hormones, proguanylin levels appear to be largely unresponsive to nutrient ingestion . This characteristic distinguishes proguanylin from many classical gut hormones that respond acutely to feeding.
What methods are available for measuring proguanylin in biological samples?
The primary method for measuring proguanylin is enzyme-linked immunosorbent assay (ELISA). The BioVendor Human Proguanylin ELISA uses a sandwich format where samples are incubated in microplate wells pre-coated with polyclonal anti-human proguanylin antibody . After washing, HRP-conjugated polyclonal anti-human proguanylin antibody is added, followed by substrate reaction and spectrophotometric measurement at 450 nm . For more detailed molecular characterization, liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be employed to distinguish between full-length proguanylin and its processed forms .
How does sample matrix affect proguanylin measurement?
The biological matrix used for proguanylin analysis significantly impacts measurement results. A comparative study of 10 individuals showed variable correlation between serum and different plasma types (EDTA, citrate, and heparin) . The data revealed substantial differences as presented in the following table:
| Volunteer | Serum (ng/ml) | EDTA Plasma (ng/ml) | Citrate Plasma (ng/ml) | Heparin Plasma (ng/ml) |
|---|---|---|---|---|
| 1 | 8.7 | 5.9 | 4.9 | 7.9 |
| 2 | 7.3 | 17.8 | 15.8 | 27.8 |
| 3 | 15.4 | 19.3 | 16.6 | 19.7 |
| 4 | 6.5 | 8.0 | 4.6 | 6.2 |
| 5 | 13.3 | 10.5 | 10.4 | 11.2 |
| 6 | 10.9 | 11.5 | 8.7 | 12.1 |
| 7 | 11.2 | 10.3 | 9.5 | 9.6 |
| 8 | 8.8 | 10.3 | 8.9 | 17.0 |
| 9 | 4.3 | 11.5 | 8.0 | 11.0 |
| 10 | 21.0 | 13.8 | 20.4 | 22.2 |
| Mean | 10.7 | 11.9 | 10.8 | 14.5 |
| % of Serum | 100% | 111% | 100% | 135% |
These variations highlight the importance of consistent sample collection protocols within studies and caution when comparing results across studies using different matrices .
What performance characteristics should be validated for proguanylin assays?
When developing or evaluating proguanylin assays, several performance characteristics should be validated:
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Spiking Recovery: Serum samples spiked with different amounts of human proguanylin should show recovery rates between 85-106% .
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Linearity: Serial dilution of samples should demonstrate consistent performance, with recovery values typically between 92-103% .
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Precision: Intra-assay and inter-assay variability should be assessed to ensure reproducibility.
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Specificity: Cross-reactivity with related peptides should be determined.
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Reference Range: Preliminary population data from 234 unselected donors (142 women + 92 men, ages 5-85) can serve as a reference population .
Thorough validation of these parameters ensures reliable and comparable results across different research studies.
How does proguanylin secretion differ from classical hormone secretion?
Unlike many classical hormones that respond acutely to specific stimuli, proguanylin secretion appears to be largely constitutive and requires endoplasmic reticulum to Golgi transport . Research indicates that proguanylin secretion is not acutely regulated by salt or other stimuli that might be expected to influence intestinal fluid balance . This constitutive secretion pattern represents a distinctive regulatory paradigm compared to many other gut hormones. For investigating secretory mechanisms, researchers should consider pulse-chase experiments and secretory pathway inhibitors to characterize trafficking pathways.
What is the significance of finding only full-length proguanylin in plasma?
The observation that only full-length proguanylin is detected in plasma, while processed forms are found in tissue and luminal extracts, has important physiological implications . This finding supports the notion that the primary site of action for proguanylin is the gut itself, with processing occurring locally at the site of action rather than systemically . This compartmentalization of processing may serve as a regulatory mechanism, limiting the systemic effects of the active peptide. For researchers investigating the endocrine actions of proguanylin, it is essential to consider whether circulating proguanylin requires local processing in target tissues to exert its effects.
Product Science Overview
The structure of proguanylin consists of a three-helix bundle, a small three-stranded β-sheet, and an unstructured linker region . The hormone guanylin is located at the COOH terminus of the prohormone and is involved in interactions with the NH2-terminal residues, which shield parts of the hormone surface . These interactions explain the negligible bioactivity of the prohormone and highlight the importance of the NH2-terminal residues in the disulfide-coupled folding of proguanylin .
Proguanylin itself exhibits negligible GC-C-activating potency . The bioactive form of the hormone is released through proteolytic processing, which involves the cleavage of proguanylin by specific proteases . This processing liberates the active hormone guanylin, which can then bind to and activate GC-C, leading to an increase in intracellular cyclic GMP (cGMP) levels . The activation of GC-C results in the secretion of fluid and electrolytes into the intestinal lumen, thereby regulating intestinal fluid balance .
Proguanylin and its active form, guanylin, have been implicated in various physiological processes, including the regulation of salt and water homeostasis through an intestinal-renal axis . The hormone’s ability to regulate GC-C activity makes it a potential target for therapeutic interventions in conditions such as chronic constipation and irritable bowel syndrome .
Recombinant proguanylin is produced using recombinant DNA technology, which involves the insertion of the proguanylin gene into a suitable expression system, such as bacteria or yeast, to produce the protein in large quantities. This recombinant form is used in research to study the structure, function, and biological activity of proguanylin and its role in various physiological processes .
