Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Chemical modification of α-chymotrypsin enabling its release from alginate hydrogel by electrochemically generated local pH change.

This study investigates the controlled release of α-chymotrypsin from an alginate hydrogel matrix. When protein molecules entrapped in the hydrogel matrix have a size smaller than the hydrogel pores, their hold/release from the polymer matrix are controlled by the electrostatic interaction between the guest molecules and host polymer. α-Chymotrypsin, as a model protein, was chemically modified with negatively charged species to change its pI and to convert its attractive interaction with a negatively charged alginate hydrogel matrix to a repulsion interaction allowing its release by pH-triggered signal. Then, bulk pH changes and electrochemically controlled local pH changes resulting from oxygen reduction were used for the controlled release of the enzyme from the alginate hydrogel. Three batches of modified α-chymotrypsin with different linker/enzyme ratios were synthesized, and their release profiles were investigated. The activity of both unmodified and modified α-chymotrypsin was evaluated using a UV-visible spectrophotometer following the standard procedure for the enzymatic assay of α-chymotrypsin (EC 3.4.21.1) and compared across all batches. Direct infusion electrospray ionization mass spectrometry (DI ESI-MS) was used to analyze the protein modifications and their impact on the isoelectric point values.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Enhancing Chymotrypsin Activity and Stability of Capillary Immobilized Enzyme Microreactors Using Zeolitic Imidazolate Frameworks as Encapsulation Materials.

Open-tubular immobilized enzyme microreactors (OT-IMERs) are some of the most widely used enzyme reaction devices due to the advantages of simple preparation and fast sample processing. However, the traditional approaches for OT-IMERs preparation had some defects such as limited enzyme loading amount, susceptibility to complex sample interference, and less stability. Here, we report a strategy for the preparation of highly active and stable OT-IMERs, in which the single-stranded DNA-enzyme composites were immobilized in capillaries and then encapsulated in situ in the capillaries via zeolitic imidazolate frameworks (ZIF-L). The phosphate groups of the DNA adjusted the surface potential of the enzyme to negative values, which could attract cations, such as Zn2+, to promote the formation of ZIF-L for enzyme encapsulation. Using chymotrypsin (ChT) as a model enzyme, the prepared ChT@ZIF-L-IMER has higher activity and better affinity than the free enzyme and ChT-IMER. Moreover, the thermal stability, pH stability, and organic solvent stability of ChT@ZIF-L-IMER were much higher than those of free enzyme and ChT-IMER. Furthermore, the activity of ChT@ZIF-L-IMER was much higher than that of ChT-IMER after ten consecutive reactions. To demonstrate the versatility of this preparation method, we replaced ChT with glucose oxidase (GOx). The stability of GOx@ZIF-L-IMER was also experimentally demonstrated to be superior to that of GOx and GOx-IMER. Finally, ChT@ZIF-L-IMER was used for proteolytic digestion analysis. The results showed that ChT@ZIF-L-IMER had a short digestion time and high digestive efficiency compared with the free enzyme. The present study broadened the synthesis method of OT-IMERs, effectively integrating the advantages of metal-organic frameworks and IMER, and the prepared OT-IMERs significantly improved enzyme stability. All of the results indicated that the IMER prepared by this method had a broad application prospect in capillary electrophoresis-based high-performance enzyme analysis.

A Multicomponent Mannich Reaction Catalyzed by Hydrolases Immobilized on Titanate Nanotubes.

This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

Thrombin has dual trypsin-like and chymotrypsin-like specificity.

BACKGROUND: The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVES: To establish if thrombin can cleave at Trp residues. METHODS: The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage. RESULTS: Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSION: The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.

Native top-down mass spectrometry for monitoring the rapid chymotrypsin catalyzed hydrolysis reaction.

Enzymes play crucial roles in life sciences, pharmaceuticals and industries as biological catalysts that speed up biochemical reactions in living organisms. New catalytic reactions are continuously developed by enzymatic engineering to meet industrial needs, which thereby drives the development of analytical approaches for real-time reaction monitoring to reveal catalytic processes. Here, taking the hydrolase- chymotrypsin as a model system, we proposed a convenient method for monitoring catalytic processes through native top-down mass spectrometry (native TDMS). The chymotrypsin sample heterogeneity was first explored. By altering sample introduction modes and pHs, covalent and noncovalent enzymatic complexes, substrates and products can be monitored during the catalysis and further confirmed by tandem MS. Our results demonstrated that native TDMS based catalysis monitoring has distinctive strength on real-time inspection and continuous observation, making it a promising tool for characterizing more biocatalysts.

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives.

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-PheP(OPh)2]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-ß-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-PheP(OPh)2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman-Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin.

Structure and activity of native and thiolated α-chymotrypsin adsorbed onto gold nanoparticles.

A detailed understanding of protein-nanoparticle interactions is critical to realize the full potential of bioconjugate-enabled technologies. Parameters that lead to conformational changes in protein structure upon adsorption must be identified and controlled to mitigate loss of biological function. We hypothesized that the installation of thiol functional groups on a protein will facilitate robust adsorption to gold nanoparticles (AuNPs) and prevent protein unfolding to achieve thermodynamic stability. Here we investigated the adsorption behavior of α-chymotrypsin (ChT) and a thiolated analog of α-chymotrypsin (T-ChT) with AuNPs. ChT, which does not present any free thiols, was modified with 2-iminothiolane (Traut's reagent) to synthesize T-ChT consisting of two free thiols. Protein adsorption to AuNPs was monitored with dynamic light scattering and UV-vis spectrophotometry, and fluorescence spectra were acquired to assess changes in protein structure induced by interaction with the AuNP. The biological function of ChT, T-ChT, and respective bioconjugates were compared using a colorimetric enzymatic assay. The thiolated analog exhibited a greater affinity for the AuNP than the unmodified ChT, as determined from adsorption isotherms. The ChT protein formed a soft protein corona in which the enzyme denatures with prolonged exposure to AuNPs and, subsequently, lost enzymatic function. Conversely, the T-ChT formed a robust hard corona on the AuNP and retained structure and function. These data support the hypothesis, provide further insight into protein-AuNP interactions, and identify a simple chemical approach to synthesize robust and functional conjugates.

Designed Trp-Cage Proteins with Antimicrobial Activity and Enhanced Stability.

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.

A non-peptide probe for detecting chymotrypsin activity based on protection-deprotection strategy in living systems.

Chymotrypsin (CHT) plays a vital role in the metabolism of organisms and affects cell proliferation and apoptosis. Abnormal levels of CHT will lead to a variety of diseases, such as inflammatory arthritis, diabetes, pharyngitis, indigestion, and pancreatic cancer. Therefore, it is significant to design an effective method for the detection of CHT in living systems. Here, we synthesized a specific deep-red non-peptide probe DT by effectively combining isophorone and p-hydroxybenzaldehyde for the detection of CHT using 3-phenylpropionate chloride as the recognition group based on a protection-deprotection strategy. The DT probe exhibited an emission range of 525-700 nm and showed excellent photostability, high sensitivity (LOD = 0.071 U mL-1), and selectivity for CHT detection. The cellular experiments demonstrated that DT could sensitively recognize CHT activity in three cell lines and the content of CHT was much higher in P815 cells than in MCF-7 and 3T3 cells. Also, DT was successfully used to visualize the endogenous CHT in zebrafish. Notably, the DT probe provided an intuitive way to visualize endogenous CHT in mouse pancreas for the first time, demonstrating the potential for application in the future clinical diagnosis of pancreatic diseases. Therefore, the small-molecule probe DT is expected to be a useful molecular tool for CHT-related disease diagnosis and drug discovery.

Insights on the interaction mechanism of exemestane to three digestive enzymes by multi-spectroscopy and molecular docking.

Exemestane is an irreversible steroidal aromatase inhibitor, typically used to treat breast cancer. As an anti-tumor drug, exemestane has more obvious side effects on the gastrointestinal tract. The purpose of this work is to investigate the combination of exemestane with three important digestive enzymes including pepsin (Pep), trypsin (Try) and α-Chymotrypsin (α-ChT) so as to analyze the mechanism of the gastrointestinal adverse effects causing by exemestane binding. Enzyme activity experiment showed that the enzyme activity of Pep was decreased in the presence of exemestane. Fluorescence spectra revealed that exemestane formed stable complexes with digestive enzymes, and the quenching mechanism of drug-digestive enzymes interaction were all static quenching. The binding constants of Pep, Try and α-ChT at 298 K were 2.34 × 105, 1.45 × 105, and 2.05 × 105 M-1, respectively. Synchronous fluorescence and 3D fluorescence spectroscopy showed that the conformation of exemestane was slightly changed after combining with digestive enzymes, and non-radiative energy transfer occurred. Circular dichroism results indicated that exemestane could change the secondary structure of digestive enzymes via increase the α-helix content and decrease in the ß-sheet content. Thermodynamic parameters (ΔH0, ΔS0, and ΔG0) revealed that exemestane interacted with α-ChT through electrostatic force, and the binding force with Pep and Try was van der Waals interactions and hydrogen, which was basically consistent with the molecular docking results.

AlphaFold - A Personal Perspective on the Impact of Machine Learning.

I outline how over my career as a protein scientist Machine Learning has impacted my area of science and one of my pastimes, chess, where there are some interesting parallels. In 1968, modelling of three-dimensional structures was initiated based on a known structure as a template, the problem of the pathway of protein folding was posed and bets were taken in the emerging field of Machine Learning on whether computers could outplay humans at chess. Half a century later, Machine Learning has progressed from using computational power combined with human knowledge in solving problems to playing chess without human knowledge being used, where it has produced novel strategies. Protein structures are being solved by Machine Learning based on human-derived knowledge but without templates. There is much promise that programs like AlphaFold based on Machine Learning will be powerful tools for designing entirely novel protein folds and new activities. But, will they produce novel ideas on protein folding pathways and provide new insights into the principles that govern folds?

Fluorescent labeling in size-controlled liposomes reveals membrane curvature-induced structural changes in the KcsA potassium channel.

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (< 30 nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.

Surface charge distribution: a key parameter for understanding protein behavior in chromatographic processes.

Multi-component adsorption of proteins still requires a better understanding of local phenomena to improve the development of predictive models. In this work, all-atom Molecular Dynamics (MD) simulations were used to investigate the influence of protein charge distribution on the adsorption capacity. The simultaneous adsorption of α-chymotrypsin and lysozyme on a cation exchanger, SP Sepharose FF, was studied through MD simulations and compared to macroscopic isotherm experiments. It appears that the charge distribution is a relevant information to better understand specific phenomena, such as a multilayer adsorption caused by the particular electrostatic profile of α-chymotrypsin. Therefore, MD simulations seem to be an interesting way to visualize and highlight these behaviors.

Marked difference in efficiency of the digestive enzymes pepsin, trypsin, chymotrypsin, and pancreatic elastase to cleave tightly folded proteins.

In order for the intestinal mucosa to absorb dietary proteins they have to be digested into single amino acids or very short peptides of a length of not more than four amino acids. In order to study the efficiency of the digestive endopeptidases to digest folded proteins we have analyzed several target proteins under different conditions, native proteins, heat denatured and acid treated. The three pancreatic serine proteases, trypsin, chymotrypsin, and pancreatic elastase, were found to be remarkable inefficient in cleaving native folded proteins whereas pepsin, which acts at a very low pH (pH 1.2) was much more efficient, possibly due to the denaturing conditions and thereby better accessibility to internal cleavage sites at the low pH. Heat treatment improved the cleavage considerably by all three pancreatic enzymes, but acid treatment followed by return to neutral pH did not have any major effect. Cleavage at the low pH when the protein is in a denatured state, is apparently very efficient. This indicates that pepsin is the prime enzyme cleaving the properly folded native proteins and that the pancreatic enzymes primarily are involved in generating single amino acids or very short peptides for efficient uptake by the intestinal mucosa.

Antibiotic-Loaded MMT/PLL-Based Coating on the Surface of Endosseous Implants to Suppress Bacterial Infections.

BACKGROUND: Bone infections remain one of the most common and serious complications of orthopedic surgery, posing a tremendous economic burden to society and patients. This is because bacteria colonize and multiply on the surface of the implant. The (MMT/PLL)8 multilayer films have been shown to effectively release antibiotics depending on the changes in the microenvironment. Here, vancomycin was loaded into the (MMT/PLL)8 multilayer films, which were prepared to be used as a local delivery system for the treatment of bone infections. METHODS: We used the layer-by-layer self-assembly method to prepare VA-loaded coatings (MMT/PLL-VA)8 consisting of montmorillonite (MMT), poly-L-lysine (PLL), and VA. The thickness and surface morphology of coatings were characterized using spectroscopic ellipsometry and scanning electron microscopy (SEM). In order to evaluate the drug release behavior from coatings in different media, we measured the size of the zone of inhibition. Additionally, in vitro antibacterial activity was assessed using the shake-flask culture method and SEM images, while that of in vivo was evaluated by establishing an animal model of bone infection. RESULTS: Our findings revealed that small-molecule antibiotics were successfully loaded into the (MMT/PLL-VA)8 multilayer film structure during the hierarchical self-assembly process and subsequently the multilayer film structure depicted linear growth behavior. The PLL in the multilayer films was progressively degraded which triggered the VA release when contacted with CMS or bacterial infections. The release of VA from multilayer film structure depends on the concentration changes of CMS. Notably, the multilayer films presented great in vitro cell compatibility. Moreover, the prepared antibacterial multilayer films showed excellent antibacterial property by killing more than 99.99% of S. aureus in 24 h. More importantly, we found that multilayer film exhibits good sterilization effect and biocompatibility under the stimulation of bacterial liquid both in vitro and in vivo antibacterial ability tests. CONCLUSION: Altogether, this study shows that (MMT/PLL-VA)8 multilayer films containing CMS and bacteria-responsive drug release properties posess high bactericidal activity and good biocompatibility. This finding provides a novel strategy for the treatment of bone infections.

Characterization of a Bowman-Birk type trypsin inhibitor purified from seeds of Solanum surattense.

A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains ß sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.

Wheat-ghretropins: novel ghrelin-releasing peptides derived from wheat protein.

Ghrelin is an endogenous orexigenic hormone mainly produced by stomach cells and is reported to influence appetite, gastrointestinal motility and growth hormone secretion. We observed that enzymatic digest of wheat gluten stimulated ghrelin secretion from mouse ghrelinoma 3-1, a ghrelin-releasing cell line. Further on, we characterized the ghrelin-releasing peptides present in the digest by comprehensive peptide analysis using liquid chromatography-mass spectrometry and structure-activity relationship. Among the candidate peptides, we found that SQQQQPVLPQQPSF, LSVTSPQQVSY and YPTSL stimulated ghrelin release. We then named them wheat-ghretropin A, B and C, respectively. In addition, we observed that wheat-ghretropin A increased plasma ghrelin concentration and food intake in mice after oral administration. Thus, we demonstrated that wheat-ghretropin stimulates ghrelin release both in vitro and in vivo. To the best of our knowledge, this is the first report of a wheat-derived exogenous bioactive peptide that stimulates ghrelin secretion.