Identification of DPP-IV inhibitory peptides derived from buffalo colostrum: Mining through bioinformatics, in silico and in vitro approaches.

Bioactive peptides derived from foods provide physiological health benefits beyond nutrition. This study focused on profiling small peptide inhibitors against two key serine proteases, dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). DPP-IV is a well-known protein involved in diverse pathways regulating inflammation, renal, cardiovascular physiology, and glucose homeostasis. POP is yet another key target protein for neurodegenerative disorders. The study evaluated peptide libraries of buffalo colostrum whey and fat globule membrane proteins derived from pepsin and pepsin-pancreatin digestion through in silico web tools and structure-based analysis by molecular docking and binding free-energy estimation, followed by in vitro assay for DPP-IV inhibition for the lead peptides. The bioinformatic study indicated 49 peptides presented motifs with DPP-IV inhibition while 5 peptides with sequences for POP inhibition. In the molecular docking interactions study, 22 peptides interacted with active site residues of DPP-IV and 3 peptides with that of POP. The synthesized peptides, SFVSEVPEL and LTFQHNF inhibited DPP-IV in vitro with an IC50 of 193.5 µM and 1.782 mM, respectively. The study revealed the key residues for inhibition of DPP-IV and POP thus affirming the DPP-IV inhibitory potential of milk-derived peptides.

One-step high efficiency separation of prolyl endopeptidase from Aspergillus niger and its application.

Prolyl endopeptidase from Aspergillus niger (An-PEP) is an enzyme that recognizes C-terminal peptide bonds of amino acid chains and cleaves them by hydrolysis. An aqueous two-phase system (ATPS) was used to separate An-PEP from fermentation broth. Through single factor experiments, the ATPS containing 16 % (w/w) PEG2000 and 15 % (w/w) (NH4)2SO4 at pH 6.0 obtained the recovery of 79.74 ± 0.16 % and the purification coefficient of 7.64 ± 0.08. It was then used to produce soy protein isolate peptide (SPIP) by hydrolysis of soy protein isolate (SPI), and SPIP-Ferrous chelate (SPIP-Fe) was prepared with SPIP and Fe2+. The chelation conditions were optimized by RSM, as the chelation time was 30 min, chelation temperature was 25 °C, SPIP mass to VC mass was two to one and pH was 6.0. The obtained chelation rate was 82.56 ± 2.30 %. The change in the structures and functional features of SPIP before and after chelation were investigated. The FTIR and UV-Vis results indicated that the chelation of Fe2+ and SPIP depended mainly on the formation of amide bonds. The fluorescence, SEM and amino acid composition analysis results indicated that Fe2+ could induce and stabilize the surface conformation and change the amino acid distribution on the surfaces of SPIP. The chelation of SPIP and Fe2+ resulted in the enhancement of radical scavenging activities and ACE inhibitory activities. This work provided a new perspective for the further development of peptide-Fe chelates for iron supplement.

Study of the Conformational Dynamics of Prolyl Oligopeptidase by Mass Spectrometry: Lessons Learned.

Ion mobility mass spectrometry (IM-MS) can be used to analyze native proteins according to their size and shape. By sampling individual molecules, it allows us to study mixtures of conformations, as long as they have different collision cross sections and maintain their native conformation after dehydration and vaporization in the mass spectrometer. Even though conformational heterogeneity of prolyl oligopeptidase has been demonstrated in solution, it is not detectable in IM-MS. Factors that affect the conformation in solution, binding of an active site ligand, the stabilizing Ser554Ala mutation, and acidification do not qualitatively affect the collision-induced unfolding pattern. However, measuring the protection of accessible cysteines upon ligand binding provides a principle for the development of MS-based ligand screening methods.

Similar metabolic pathways are affected in both Congenital Myasthenic Syndrome-22 and Prader-Willi Syndrome.

Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl-/- and PWS-IC-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders.

5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.

Unraveling the allosteric mechanisms of prolyl endopeptidases for celiac disease therapy: Insights from molecular dynamics simulations.

Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic strategy for celiac disease. However, the mechanisms governing the interaction of these enzymes with their substrates remain unclear. In this study, conventional molecular dynamics simulations with a microsecond timescale and targeted molecular dynamics simulations were performed to investigate the native states of mxPEP and scPEP enzymes, as well as their allosteric binding with a representative substrate, namely, Z-Ala-Pro-p-nitroanilide (pNA). The simulations reveal that the native scPEP is in an open state, while the native mxPEP is in a closed state. When pNA approaches a closed mxPEP, it binds to an allosteric pocket located at the first and second ß-sheet of the ß-propeller domain, inducing the opening of this enzyme. Neither enzyme is active in the open or partly-open states. Enzymatic activity is enabled only when the catalytic pocket in the closed state fully accommodates the substrates. The internal capacity of the catalytic pocket of PEP in the closed state determines the maximum size of the gluten peptides that the enzymes can catalyze. The present work provides essential molecular dynamics information for the redesign or engineering of PEP enzymes.

Novel benzimidazole derivatives as effective inhibitors of prolyl oligopeptidase: synthesis, in vitro and in silico analysis.

Background: This research aims to discover novel derivatives having potential therapeutic applications in treating conditions related to prolyl oligopeptidase (POP) dysfunction. Method: Novel benzimidazole derivatives have been synthesized, characterized and screened for their in vitro POP inhibition. Results: All these derivatives showed excellent-to-good inhibitory activities in the range of IC50 values of 3.61 ± 0.15 to 43.72 ± 1.18 µM, when compared with standard Z-prolyl-prolinal. The docking analysis revealed the strong interactions between our compounds and the target enzyme, providing critical insights into their binding affinities and potential implications for drug development. Conclusion: The significance of these compounds in targeting POP enzyme offers promising prospects for future research in the field of neuropharmacology.

Efficient degradation of soybean protein B3 subunit in soy sauce by ultrasound-assisted prolyl endopeptidase and its primary mechanism.

Prolyl endopeptidase can partially degrade soybean protein B3 subunit and alleviate soy sauce secondary precipitate. In this study, the influences of ultrasound-assisted prolyl endopeptidase on the degradation of soybean protein B3 subunit of soy sauce and primary mechanism were investigated using SDS-PAGE, MALDI-TOF-MS, circular dichromatic spectrometer, fluorescence spectra, etc. Results showed that ultrasound-assisted prolyl endopeptidase enhanced 72% degradation rate of B3 subunit and reduced soy sauce secondary precipitate remarkably, meanwhile significantly increased content of organic taste compounds of soy sauce compared with control (p < 0.05). Sonication markedly reduced percentage of α-helix and increased percentage of random coil, made hydrophobic amino acids inside prolyl endopeptidase exposed to its surface and enhanced its flexibility, which facilitated the binding of prolyl endopeptidase active center with B3 subunit and finally enhanced the latter's degradation rate and appearance quality of soy sauce. This work laid a foundation for solving soy sauce secondary precipitate.

D-Aspartate Depletion Perturbs Steroidogenesis and Spermatogenesis in Mice.

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17ß-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction.

High-level production of Aspergillus niger prolyl endopeptidase from agricultural residue and its application in beer brewing.

BACKGROUND: Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. RESULTS: Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. CONCLUSION: Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.

Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aß) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

Inhibition of Prolyl Oligopeptidase Restores Prohibitin 2 Levels in Psychosis Models: Relationship to Cognitive Deficits in Schizophrenia.

Cognitive impairment represents one of the core features of schizophrenia. Prolyl Oligopeptidase (POP) inhibition is an emerging strategy for compensating cognitive deficits in hypoglutamatergic states such as schizophrenia, although little is known about how POP inhibitors exert their pharmacological activity. The mitochondrial and nuclear protein Prohibitin 2 (PHB2) could be dysregulated in schizophrenia. However, altered PHB2 levels in schizophrenia linked to N-methyl-D-aspartate receptor (NMDAR) activity and cognitive deficits are still unknown. To shed light on this, we measured the PHB2 levels by immunoblot in a postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects, in the frontal pole of mice treated with the NMDAR antagonists phencyclidine and dizocilpine, and in rat cortical astrocytes and neurons treated with dizocilpine. Mice and cells were treated in combination with the POP inhibitor IPR19. The PHB2 levels were also analyzed by immunocytochemistry in rat neurons. The PHB2 levels increased in DLPFC in cases of chronic schizophrenia and were associated with cognitive impairments. NMDAR antagonists increased PHB2 levels in the frontal pole of mice and in rat astrocytes and neurons. High levels of PHB2 were found in the nucleus and cytoplasm of neurons upon NMDAR inhibition. IPR19 restored PHB2 levels in the acute NMDAR inhibition. These results show that IPR19 restores the upregulation of PHB2 in an acute NMDAR hypoactivity stage suggesting that the modulation of PHB2 could compensate NMDAR-dependent cognitive impairments in schizophrenia.

The Role of Tß4-POP-Ac-SDKP Axis in Organ Fibrosis.

Fibrosis is a pathological process in which parenchymal cells are necrotic and excess extracellular matrix (ECM) is accumulated due to dysregulation of tissue injury repair. Thymosin ß4 (Tß4) is a 43 amino acid multifunctional polypeptide that is involved in wound healing. Prolyl oligopeptidase (POP) is the main enzyme that hydrolyzes Tß4 to produce its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which is found to play a role in the regulation of fibrosis. Accumulating evidence suggests that the Tß4-POP-Ac-SDKP axis widely exists in various tissues and organs including the liver, kidney, heart, and lung, and participates in the process of fibrogenesis. Herein, we aim to elucidate the role of Tß4-POP-Ac-SDKP axis in hepatic fibrosis, renal fibrosis, cardiac fibrosis, and pulmonary fibrosis, as well as the underlying mechanisms. Based on this, we attempted to provide novel therapeutic strategies for the regulation of tissue damage repair and anti-fibrosis therapy. The Tß4-POP-Ac-SDKP axis exerts protective effects against organ fibrosis. It is promising that appropriate dosing regimens that rely on this axis could serve as a new therapeutic strategy for alleviating organ fibrosis in the early and late stages.

Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger.

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

Recent Progress on Biological Activity of Amaryllidaceae and Further Isoquinoline Alkaloids in Connection with Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive age-related neurodegenerative disease recognized as the most common form of dementia among elderly people. Due to the fact that the exact pathogenesis of AD still remains to be fully elucidated, the treatment is only symptomatic and available drugs are not able to modify AD progression. Considering the increase in life expectancy worldwide, AD rates are predicted to increase enormously, and thus the search for new AD drugs is urgently needed. Due to their complex nitrogen-containing structures, alkaloids are considered to be promising candidates for use in the treatment of AD. Since the introduction of galanthamine as an antidementia drug in 2001, Amaryllidaceae alkaloids (AAs) and further isoquinoline alkaloids (IAs) have been one of the most studied groups of alkaloids. In the last few years, several compounds of new structure types have been isolated and evaluated for their biological activity connected with AD. The present review aims to comprehensively summarize recent progress on AAs and IAs since 2010 up to June 2021 as potential drugs for the treatment of AD.

Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line.

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

Structure-activity relationship and biochemical evaluation of novel fibroblast activation protein and prolyl endopeptidase inhibitors with α-ketoamide warheads.

Peptidomimetic inhibitors of fibroblast activation protein (FAP) are regarded as promising tools for tumor targeting in vivo. Even though several peptidomimetic compounds with nanomolar potency have been described, broad chemical space for further modification remained unexplored. Therefore, we set to analyze the structure-activity relationship (SAR) of pseudopeptide compound series with α-ketoamide warheads in order to explore the contributions of the P1' and P2' moieties to the inhibitory potency. A series of novel inhibitors bearing varied P1' and/or P2' moieties was synthesized by combining a Passerini reaction-Amine Deprotection-Acyl Migration (PADAM) approach with peptide coupling and subsequent oxidation. The resulting compounds inhibited FAP and the related prolyl endopeptidase (PREP) with potencies in the nanomolar to sub-nanomolar range. The most potent FAP inhibitor IOCB22-AP446 (6d, IC50 = 89 pM) had about 36-fold higher inhibition potency than the most potent inhibitor published to date. The compounds were selective over FAP's closest homolog DPP-IV, were stable in human and mouse plasma and in mouse microsomes, and displayed minimal cytotoxicity in tissue cultures.

Altered Expression of DAAM1 and PREP Induced by Cadmium Toxicity Is Counteracted by Melatonin in the Rat Testis.

Cadmium (Cd) is one of the most toxic pollutants for health due to its accumulation in several tissues, including testis. This report confirms that Cd increased oxidative stress and apoptosis of germ and somatic cells and provoked testicular injury, as documented by biomolecular and histological alterations, i.e., CAT and SOD activity, the protein level of steroidogenic enzymes (StAR and 3ß-HSD), and morphometric parameters. Additionally, it further documents the melatonin (MLT) coadministration produces affects in mitigating Cd-induced toxicity on adult rat testis, as demonstrated by the reduction of oxidative stress and apoptosis, with reversal of the observed histological changes; moreover, a role of MLT in partially restoring steroidogenic enzymes expression was evidenced. Importantly, the cytoarchitecture of testicular cells was perturbed by Cd exposure, as highlighted by impairment of the expression and localization of two cytoskeleton-associated proteins DAAM1 and PREP, which are involved in the germ cells' differentiation into spermatozoa, altering the normal spermatogenesis. Here, for the first time, we found that the co-treatment with MLT attenuated the Cd-induced toxicity on the testicular DAAM1 and PREP expression. The combined findings provide additional clues about a protective effect of MLT against Cd-induced testicular toxicity by acting on DAAM1 and PREP expression, encouraging further studies to prove its effectiveness in human health.

Intrinsic basis of thermostability of prolyl oligopeptidase from Pyrococcus furiosus.

Salt-bridges play a key role in the thermostability of proteins adapted in stress environments whose intrinsic basis remains to be understood. We find that the higher hydrophilicity of PfP than that of HuP is due to the charged but not the polar residues. The primary role of these residues is to enhance the salt-bridges and their ME. Unlike HuP, PfP has made many changes in its intrinsic property to strengthen the salt-bridge. First, the desolvation energy is reduced by directing the salt-bridge towards the surface. Second, it has made bridge-energy more favorable by recruiting energetically advantageous partners with high helix-propensity among the six possible salt-bridge pairs. Third, ME-residues that perform intricate interactions have increased their energy contribution by making major changes in their binary properties. The use of salt-bridge partners as ME-residues, and ME-residues' overlapping usage, predominant in helices, and energetically favorable substitution are some of the favorable features of PfP compared to HuP. These changes in PfP reduce the unfavorable, increase the favorable ME-energy. Thus, the per salt-bridge stability of PfP is greater than that of HuP. Further, unfavorable target ME-residues can be identified whose mutation can increase the stability of salt-bridge. The study applies to other similar systems.

Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.