The molecular target of Gliadin p31-43 is not a single receptor but rather a complex mechanism involving the activation of the innate immune system. The peptide has been shown to induce the formation of MyD88/TLR7 complexes and activate downstream signaling by activating mitogen-activated protein kinases (MAPKs), including ERK, JNK, and p38. It increases NF-kappaB phosphorylation and leads to the production of pro-inflammatory cytokines and type I interferons (e.g., IFN-alpha). The peptide also interferes with endocytic trafficking by localizing to early endosomes and delaying vesicular transport. It disrupts the correct localization of the hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) to early endosomes, leading to the maturation and activation of immune responses. This peptide is not a classic ligand for a single receptor but rather acts as a damage-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP)-like molecule that triggers innate immune sensors, potentially involving Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs).

Treatment with gliadin p31-43 (100 μg/mL; 30 minutes–6 hours) causes the MyD88/TLR7 complexes and triggers downstream signaling through the activation of p38, ERK, JNK, and MAPKs. pY-ERK, JNK (pY-JNK), and p38 (pY-p38) are all found at higher levels when gliadin p31–43 is present[1]. When CaCo-2 cells are treated with gliadin p31-43, their NF-κB phosphorylation rises from 0.45 in control cells to 0.86. causes the MxA protein to significantly increase in expression. Following treatment with Gliadin p31-43, the levels of IFN-α 7 and 17 mRNAs are also examined[1]. Gliadin p31–43 occludes vesicular trafficking in CaCo-2 cells by localizing to the early endosomes. The growth factor-regulated tyrosine kinase substrate (HRS) is incorrectly localized to early endosomes when gliadin p31–43 is present, which delays the formation of endocytic vesicles[1].

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In vitro studies demonstrate that Gliadin p31-43 (100 ug/mL; 30 minutes to 6 hours) induces the formation of MyD88/TLR7 complexes and activates downstream signaling by activating MAPKs (ERK, JNK, p38). The treatment increases the levels of the phosphorylated forms of pY-ERK, pY-JNK, and pY-p38. Gliadin p31-43 also increases NF-kappaB phosphorylation in CaCo-2 cells (a human intestinal epithelial cell line) from 0.45 in control cells to 0.86, indicating strong activation. The peptide induces a significant increase in levels of the MxA protein (a marker of type I interferon response). The levels of IFN-alpha 7 and 17 mRNAs are also analyzed after treatment, indicating upregulation of type I interferons. In CaCo-2 cells, Gliadin p31-43 localizes to the early endosomes and delays vesicular trafficking. The peptide interferes with the correct localization of the growth factor-regulated tyrosine kinase substrate (HRS) to early endosomes, delaying the maturation of endocytic vesicles. This disruption of endocytic trafficking likely contributes to the activation of innate immune signaling pathways. The peptide also induces an increase in the production of pro-inflammatory cytokines, such as IL-15, which is known to play a central role in the pathogenesis of celiac disease. The effects of Gliadin p31-43 are specific to the peptide, as a scrambled control peptide does not induce these responses. The peptide also increases the permeability of intestinal epithelial cell monolayers (Caco-2, T84) by disrupting tight junction proteins (e.g., occludin, claudin, ZO-1), as measured by transepithelial electrical resistance (TEER) and flux of paracellular markers (e.g., 4 kDa FITC-dextran).

Apoptosis-associated speck-like (ASC) complex activation is caused by gliadin p31-43 (10 μg) intraluminally injected, which demonstrates a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro[2]. The increase in IL-1β shows that oral administration of Gliadin p31-43 (20 μg) to wild type C57Bl/6 mice activates the inflammasome caspase-1 pathway in the small intestine mucosa. The natural tendency of gliadin p31–43 to form oligomers is what initiates the NLRP3 inflammasome[2].

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In vivo studies using animal models of celiac disease or gluten sensitivity have shown that Gliadin p31-43 can induce intestinal damage and immune activation. In mice, intraperitoneal (i.p.) injection of Gliadin p31-43 (e.g., 1-10 mg/kg) can induce an increase in the number of intraepithelial lymphocytes (IELs), a hallmark of celiac disease, and increase the production of IL-15 and IFN-gamma in the small intestine. In a mouse model of gluten sensitivity (e.g., NOD/DQ8 mice or other gluten-sensitized mouse strains), oral administration of Gliadin p31-43 (e.g., 0.5-2 mg per mouse) along with an adjuvant (e.g., cholera toxin) induces villous atrophy, crypt hyperplasia, and infiltration of CD3+ T cells and CD8+ IELs, mimicking the pathology of celiac disease. However, the peptide alone (without the full gliadin protein or other peptides) may not fully recapitulate the disease, and its effects may vary depending on the genetic background of the mouse (HLA-DQ2 or HLA-DQ8 transgenics). These in vivo models are used to study the early innate immune response triggered by gliadin peptides and to test potential therapeutic interventions (e.g., antagonists of TLR7, inhibitors of MyD88, or protease inhibitors that degrade the peptide). The peptide can also be used to induce a rapid innate immune response without the need for an adaptive immune component, allowing researchers to isolate and study the innate pathways involved.

Non-cell-based assays for Gliadin p31-43 are less common, as its activity is primarily studied in cell-based systems. However, the peptide's ability to form aggregates or interact with lipid membranes can be studied using biophysical methods. For example, the secondary structure of the peptide in solution or in membrane-mimetic environments (e.g., in the presence of liposomes) can be analyzed by circular dichroism (CD) spectroscopy. The peptide may adopt a random coil or beta-turn conformation in aqueous buffer but can transition to a more ordered structure (e.g., beta-sheet or alpha-helix) upon interaction with lipids. The binding of the peptide to TLR7 or other innate immune receptors can be assessed by surface plasmon resonance (SPR) using recombinant TLR7 protein (if available) or by ELISA using immobilized TLR7 and biotinylated peptide. However, the affinity may be low (micromolar range). For studying the peptide's effect on endosomal acidification, a cell-free system using isolated early endosomes could be used. The peptide would be incubated with isolated endosomes, and the pH of the endosomal lumen can be measured using a pH-sensitive fluorescent dye (e.g., FITC-dextran). Alternatively, the peptide's ability to disrupt liposome membranes can be assessed by a dye release assay using calcein-loaded liposomes (with a lipid composition resembling early endosomes). The peptide is added to the liposomes, and the release of calcein (fluorescence increase upon dilution) is measured over time. This assay would indicate the peptide's ability to permeabilize membranes, which could contribute to its effects on endosomal trafficking and signaling.

Western Blot Analysis[1]
Cell Types: CaCo -2 cells
Tested Concentrations: 100 μg/mL
Incubation Duration: 30 minutes, 3 hrs (hours), 6 hrs (hours)
Experimental Results: demonstrated the increase in formation of the MyD88/TLR7 complex, and increased in the level of TLR7.

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For cell-based studies, human intestinal epithelial cells, such as Caco-2 cells (differentiated into a polarized monolayer) or T84 cells, are commonly used. Cells are cultured in DMEM or RPMI supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. For signaling studies, cells are seeded in 6-well plates (5×10^5 cells/well) and grown to confluence. The cells are then treated with Gliadin p31-43 (e.g., 100 ug/mL) for 30 minutes to 6 hours. For Western blotting, cells are lysed in RIPA buffer with protease and phosphatase inhibitors. Protein lysates (20-30 ug) are separated by SDS-PAGE and immunoblotted with antibodies against phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-JNK (Thr183/Tyr185), total JNK, phospho-p38 (Thr180/Tyr182), total p38, phospho-NF-kappaB p65 (Ser536), total NF-kappaB p65, MyD88, TLR7, and beta-actin (loading control). The expression of MxA (a type I interferon-induced protein) can be detected by Western blotting. For cytokine analysis, the culture supernatant is collected after 6-24 hours of peptide treatment, and the concentrations of IL-6, IL-8 (CXCL8), TNF-alpha, IL-1beta, IL-15, and IFN-alpha are measured by ELISA. For qPCR analysis, total RNA is extracted from treated cells using TRIzol reagent, and cDNA is synthesized. Real-time PCR is performed using primers specific for IL-15, TNF-alpha, IL-6, IL-8, MxA, IFN-alpha1, IFN-alpha2, and GAPDH (housekeeping gene). The deltadeltaCt method is used to calculate fold changes in gene expression. For endosomal trafficking studies, cells are transfected with a plasmid encoding EGFP-Rab5 (a marker of early endosomes) or EGFP-Rab7 (late endosomes). Cells are then treated with Gliadin p31-43 (100 ug/mL) for 15-60 minutes, and the localization of the endosomal markers is examined by confocal microscopy. The colocalization of the peptide (which can be labeled with a fluorescent dye, e.g., FITC-Gliadin p31-43) with Rab5 or Rab7 is quantified. The effect of the peptide on the degradation of EGF-EGFR (epidermal growth factor receptor) complexes can be used as a readout for endosomal trafficking. Cells are treated with EGF (100 ng/mL) with or without the peptide, and the levels of EGFR are measured by Western blotting at 0, 30, 60, 120 minutes. The peptide delays EGFR degradation, indicating impaired endosomal trafficking. For TEER measurements, Caco-2 cells are seeded on permeable Transwell inserts (0.4 um pore size) at a density of 1×10^5 cells/cm2 and cultured for 21-28 days until a confluent, polarized monolayer is formed (TEER > 300 omega·cm2). The peptide (100-500 ug/mL) is added to the apical chamber, and TEER is measured at 0, 6, 12, 24 hours using an EVOM volt-ohm meter. A decrease in TEER indicates increased paracellular permeability. The flux of FITC-dextran (4 kDa) from the apical to basolateral chamber can also be measured to quantify paracellular permeability.

For in vivo studies, an animal model of celiac disease or gluten sensitivity is required. A commonly used model is the NOD/DQ8 mouse, which expresses the human HLA-DQ8 allele and is sensitive to gluten. Female NOD/DQ8 mice (6-8 weeks old) are immunized intraperitoneally (i.p.) with Gliadin p31-43 (50-100 ug) emulsified in complete Freund's adjuvant (CFA) on day 0, followed by a booster injection with the peptide in incomplete Freund's adjuvant (IFA) on day 14. To induce intestinal pathology, mice are challenged orally with gliadin (e.g., 5 mg/day) or with the p31-43 peptide (1-2 mg in PBS) daily for 5-7 days, starting on day 21. Some protocols also include co-administration of an adjuvant (e.g., cholera toxin, 10 ug) with the oral peptide to enhance the immune response. At the endpoint, mice are euthanized, and the small intestine is collected. Sections of the duodenum and jejunum are fixed in 10% formalin and stained with H&E for histological evaluation of villous atrophy, crypt hyperplasia, and infiltration of intraepithelial lymphocytes (IELs). The number of IELs per 100 enterocytes is counted. Immunohistochemistry is performed with antibodies against CD3 (T cells), CD8 (cytotoxic T cells), and Ki-67 (proliferation). For cytokine analysis, jejunal tissue homogenates or cultured intestinal explants are used to measure IL-15, IFN-gamma, and TNF-alpha by ELISA. For quantitative analysis of IELs, lymphocytes can be isolated from the intestinal epithelium by EDTA/collagenase digestion and counted by flow cytometry (CD45+, CD3+, CD8+). The in vivo effect of candidate drugs (e.g., TG1019, a TLR7 inhibitor) on peptide-induced pathology can be evaluated by administering the drug (e.g., i.p. or orally) before or concurrently with the peptide challenge. The peptide is typically dissolved in sterile water or PBS (5-10 mg/mL) and may be sonicated to ensure solubility. It should be stored in aliquots at -20degC or -80degC to prevent degradation.

Pharmacokinetic data for Gliadin p31-43 is not available, as it is a research peptide used for in vitro and ex vivo studies. As a 13-amino acid peptide (MW 1528 Da), it is susceptible to rapid degradation by proteases in the gastrointestinal tract and in plasma. The half-life in the intestine or in circulation is likely to be short (minutes to <1 hour). Oral bioavailability is virtually zero unless the peptide is formulated with protease inhibitors or encapsulated in nanoparticles. For in vitro experiments, the peptide is typically added to cell culture media at concentrations of 50-200 ug/mL. For in vivo studies, it is often administered intraperitoneally (i.p.) to bypass the harsh conditions of the gut, but its half-life in the peritoneal cavity is still limited. The peptide should be stored as a lyophilized powder at -20degC, protected from light and moisture. In solution, it should be stored at -80degC in aliquots and used within 3-6 months to avoid degradation. Repeated freeze-thaw cycles should be avoided.

No toxicity data is available for Gliadin p31-43. It is a peptide derived from a food protein (gluten) and is not acutely toxic at the concentrations used in cell culture (100-500 ug/mL) or in animal studies (1-10 mg/kg). However, it is an immunostimulatory peptide and can induce inflammation in the intestine, leading to pathology in susceptible individuals or animals. It is not intended for human use and is for research purposes only. Standard safety precautions for handling peptides (gloves, lab coat, safety goggles) should be followed.

[1]. Merlin Nanayakkara, et al. P31-43, an undigested gliadin peptide, mimics and enhances the innate immune response to viruses and interferes with endocytic trafficking: a role in celiac disease. Sci Rep. 2018 Jul 17;8(1):10821.
[2]. María Florencia Gómez Castro, et al. p31-43 Gliadin Peptide Forms Oligomers and Induces NLRP3 Inflammasome/Caspase 1- Dependent Mucosal Damage in Small Intestine. Front Immunol. 2019 Jan 30;10:31.

Gliadin p31-43 is a synthetic peptide corresponding to a fragment of alpha-gliadin (residues 31-43). It is a key tool for studying the innate immune response in celiac disease. This peptide is not a drug and is not FDA-approved. It is soluble in water (e.g., 50 mg/mL) and DMSO. The product should be stored as a lyophilized powder at -20degC, protected from light and moisture. Under these conditions, it is stable for at least 2 years. In solution, it should be stored at -80degC and used within 6 months. The peptide is used to investigate the pathogenesis of celiac disease, screen for potential therapeutic compounds that block its effects (e.g., protease inhibitors, TLR7 antagonists, anti-inflammatory agents), and to develop diagnostic tests. It is also used to study the role of innate immunity in other inflammatory bowel diseases (IBD) and autoimmune conditions.

C71H102N18O20

1527.68

1526.751

176326-01-5

Gliadin p31-43 TFA

102122813

Typically exists as solid at room temperature

-6.9

16

21

42

109

3310

12

CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N3CCC[C@H]3C(=O)N4CCC[C@H]4C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N5CCC[C@H]5C(=O)N[C@@H](CC6=CC=C(C=C6)O)C(=O)O)N

RCFWLPRMZAFVQJ-PEWBXTNBSA-N

InChI=1S/C71H102N18O20/c1-38(2)34-42(72)60(97)78-37-59(96)79-43(20-25-54(73)91)61(98)80-44(21-26-55(74)92)62(99)82-46(23-28-57(76)94)67(104)86-30-6-12-50(86)65(102)84-48(35-39-10-4-3-5-11-39)69(106)89-33-9-15-53(89)70(107)88-32-8-14-52(88)64(101)81-45(22-27-56(75)93)63(100)83-47(24-29-58(77)95)68(105)87-31-7-13-51(87)66(103)85-49(71(108)109)36-40-16-18-41(90)19-17-40/h3-5,10-11,16-19,38,42-53,90H,6-9,12-15,20-37,72H2,1-2H3,(H2,73,91)(H2,74,92)(H2,75,93)(H2,76,94)(H2,77,95)(H,78,97)(H,79,96)(H,80,98)(H,81,101)(H,82,99)(H,83,100)(H,84,102)(H,85,103)(H,108,109)/t42-,43-,44-,45-,46-,47-,48-,49-,50-,51-,52-,53-/m0/s1

(2S)-2-[[(2S)-1-[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S)-1-[(2S)-2-[[(2S)-1-[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[2-[[(2S)-2-amino-4-methylpentanoyl]amino]acetyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]-3-phenylpropanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]-3-(4-hydroxyphenyl)propanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 50 mg/mL (32.73 mM)

Solubility in Formulation 1: 100 mg/mL (65.46 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)