Peptides Derived from Soy and Lupin Protein as Dipeptidyl-Peptidase

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Peptides Derived from Soy and Lupin Protein as Dipeptidyl-Peptidase IV Inhibitors: In Vitro Biochemical Screening and In Silico Molecular Modeling Study Carmen Lammi, Chiara Zanoni, Anna Arnoldi, and Giulio Vistoli J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04041 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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Journal of Agricultural and Food Chemistry

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Peptides Derived from Soy and Lupin Protein as Dipeptidyl-Peptidase

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IV Inhibitors: In Vitro Biochemical Screening and In Silico Molecular

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Modeling Study

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Carmen Lammi, Chiara Zanoni, Anna Arnoldi,* Giulio Vistoli

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Department of Pharmaceutical Sciences, University of Milan, Milan, Italy

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*Corresponding author: Anna Arnoldi, Department of Pharmaceutical Sciences, University of

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Milan, via Mangiagalli 25, 20133 Milan, Italy. Tel +390250319372, fax +390250319343; e-mail

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[email protected]

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Abstract

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Dipeptidyl peptidase IV (DPP-IV) is a new molecular target correlated with the development of

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type 2 diabetes. Literature describes the identification of some inhibitory peptides from the

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hydrolysis of different food proteins. This paper reports a study on six peptides from soybean and

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lupin proteins, i.e. Soy 1 (IAVPTGVA), Soy 2 (YVVNPDNDEN), Soy 3 (YVVNPDNNEN), Lup

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1 (LTFPGSAED), Lup 2 (LILPKHSDAD), and Lup 3 (GQEQSHQDEGVIVR), which were

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screened for their capacity to inhibit the activity of DPP-IV, using an in vitro bioassay against

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human recombinant DPP-IV. Two peptides Soy 1 and Lup 1 resulted to be efficient inhibitors with

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IC50 values equal to 106 and 228 µM, respectively. A molecular docking analysis predicted the key

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molecular interactions, stabilizing the active peptides within DPP-IV enzyme. Soy and lupin

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proteins may be sources of DPP-IV inhibitory peptides potentially useful for the prevention of type

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2 diabetes.

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KEYWORDS: bioactive peptide, dipeptidyl peptidase IV inhibitor, lupin, soy, type 2 diabetes ACS Paragon Plus Environment


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INTRODUCTION

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Plant proteins are useful in the prevention of cardiovascular disease and diabetes.1 In particular,

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some studies provided evidence that soy protein and/or peptides exert a hypoglycemic activity

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either in suitable animal models2, 3 or in type-2 diabetic patients.4, 5 Moreover, some peptides from

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soy protein improve glucose uptake in HepG2 cells6 and peptide mixtures obtained by pepsin-

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pancreatin hydrolysis of soy protein improve glucose uptake in muscle L6 cells.7 Other

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investigations support the hypoglycemic activity of lupin protein.8,

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demonstrated that a specific lupin protein fraction decreases blood glucose concentration in rats10

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and has a relevant post-prandial hypoglycemic effect in humans.10 All these pieces of evidence

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suggest that the consumption of soy and lupin proteins may be beneficial for the prevention of type

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2 diabetes.

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Dipeptidyl peptidase IV (DPP-IV) is a new molecular target correlated with the development of

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type 2 diabetes.11 It is a serine exopeptidase that cleaves Xaa-proline or Xaa-alanine dipeptides

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from the N-terminus of polypeptides. Among all DPP-IV substrates, the most widely investigated

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are glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), two

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incretins playing essential roles in maintaining glucose homeostasis.12,13 Together, they stimulate

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the insulin biosynthesis at pancreatic level and are responsible for up to 70% of insulin secretion

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following a meal.14, 15 Since after secretion GLP-1 and GIP are rapidly degraded by DPP-IV,16 the

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inhibition of this enzyme improves the glucose tolerance in diabetic patients by enhancing the

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insulinotropic effects of GLP-117 and lowers blood glucose via stimulation of insulin and inhibition

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of glucagon. For this reason DPP-IV inhibitors have emerged as a new class of oral antidiabetic

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agents,18 with excellent therapeutic potentials in the management of type 2 diabetes.11,

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synthetic DPP-IV inhibitors sitagliptin and vildagliptin are currently the most widely investigated

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new drugs for the treatment of type 2 diabetes.20

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Interestingly, some food proteins appear to be sources of DPP-IV inhibitory peptides, which may be

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released from their sequences by enzymatic hydrolysis. For example, DPP-IV inhibitory peptides ACS Paragon Plus Environment

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In particular, it has been

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The

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have been isolated and characterized from the proteins of cow milk,21 bovine proteins,22,

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milk,24 silver carp,25 tuna,26 salmon,27 rice,28 black bean,29 and amaranth.30

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Owing to our interest for the role of plant proteins and peptides in the prevention of

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hypercholesterolemia and hyperglycemia, in the last few years, we have collected some bioactive

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peptides from soy (Soy 1-3) and lupin proteins (Lup 1-3) and investigated their biological activities

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(Table 1). Soy 1 (IAVPTGVA) corresponds to position 142-149 of glycinin (UniProtK P04776.2)

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and modulates cholesterol and glucose biosyntheses in HepG2 cells.6, 31, 32 Soy 2 (YVVNPDNDEN)

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corresponds to position 232-241 of the α subunit of β-conglycinin (UNIProtKB P13916), whereas

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Soy 3 (YVVNPDNNEN) corresponds to positions 310-319 of the α' subunit of β-conglycinin

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(UNIProtKB P11827). Both are able to inhibit the activity of 3-hydroxymethylglutarylCoA

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reductase (HMGCoAR) and to modulate cholesterol biosynthesis in HepG2 cells.33 Interestingly, an

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investigation in Caco2 cells has shown that they are potentially absorbed at intestine level.34 Lup 1

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(LTFPGSAED), Lup 2 (LILPKHSDAD), and Lup 3 (GQEQSHQDEGVIVR) correspond to

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positions 484-492, 235-244, and 362-375, respectively, of β-conglutin (UniProtKB Q53HY0.2), a

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7S storage protein. Using monolayers of Caco2 cells grown in bicameral system, we have recently

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demonstrated that these lupin peptides are transferred from the apical to the basolateral chamber and

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that the basolateral solution inhibits the activity of HMGCoAR and modulate cholesterol

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metabolism in HepG2 cells.35, 36

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BIOPEP (www.uwm.edu.pI/biochemia)37 is an open access database enabling to hypothesize the

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potential biological activities of peptides based on the presence of specific amino acid sequences. A

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screening of the structures of these peptides with BIOPEP suggested that their structures were

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compatible with a potential DPP-IV inhibitory activity. In case of Soy 1 the sequences were VA,

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VP, IA, AV, GV, PT, TG; for Soy 2 they were VV, NP, DN, ND, VN,YV; for Soy 3 they were VV,

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NP, DN, NE, NN, VN, YV; for Lup 1 they were FP, AE, LT, PG, TF; for Lup 2 they were LP, AD,

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HS, IL, KH, LI, PK; and for Lup 3 they were GQ, VR, EG, GV, QD, QE, QS, SH, VI. It was thus

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decided to evaluate experimentally their potential inhibitory activity using a commercial in vitro ACS Paragon Plus Environment

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goat


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bioassay against human recombinant DPP-IV. Subsequently, the interaction of two active peptides

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and an inactive one with the enzyme was investigated employing an in silico molecular model and

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scoring approach. The present work describes the results of these investigations.

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MATERIAL & METHODS

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Materials. Tris-HCl, ethylenediaminetetraaceticacid (EDTA), and NaCl were from Sigma-Aldrich

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(St. Louis, MO, USA). The peptides Soy 1-3 and Lup 1-3 (Table 1) were synthesized by the

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company PRIMM (Milan, Italy) with >95% purity, assessed by HPLC.

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DPP-IV activity assay. The DPP-IV enzyme and the substrate solution (5 mM H-Gly-Pro-AMC)

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were provided by Cayman Chemicals (Michigan, USA). The experiments were carried out in

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triplicate in a half volume 96 well solid plate (white). Each reaction (50 µL) was prepared adding

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the reagents in a microcentrifuge tube in the following order: 1 X assay buffer [20 mM Tris-HCl,

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pH 8.0, containing 100 mM NaCl, and 1 mM EDTA] (30 µL), 100 µM of each soy and lupin

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peptide [Soy 1-3 or Lup 1-3] or vehicle (10 µL) and finally the DPP-IV enzyme (10 µL).

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Subsequently, the samples were mixed and 50 µL of each reaction were transferred in each plate

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well. Each reaction was started by adding 50 µL of substrate solution to each well and incubated at

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37 °C for 30 minutes. Fluorescence signals were measured using the Synergy H1 fluorescent plate

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reader from Biotek (excitation and emission wavelengths 360 and 465 nm, respectively). In order to

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build the dose-inhibition curves of the active peptides Soy 1 and Lup 1, concentrations in the range

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10-1000 µM were tested using the same procedure described above.

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Statistically Analysis. Statistical analyses were carried out by One-way ANOVA using Graphpad

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Prism 6 (Graphpad, La Jolla, CA, USA) followed by Dunnett’s test. Values were expressed as

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means ± SEM; P-values < 0.05 were considered to be significant. ACS Paragon Plus Environment

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Computational methods. By applying a computational strategy already adopted in a previous

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study,33 two active peptides and an inactive one (i.e., Soy 1, Lup 1, and Lup 2) were built in

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canonical α-helix by using the Peptide Builder function of the VEGA suite of programs38 and their

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conformational profiles were explored by a MonteCarlo procedure, which produced 10,000

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conformers by randomly rotating the backbone torsions only. The obtained geometries were then

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clustered according to their similarity to discard redundant ones; here, two conformations were

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considered as non-redundant when they differed by more than 60 degrees in at least one backbone

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torsion angle. For each cluster, the lowest energy structure was collected and memorized.

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Among the resolved human DPP-IV structures, the study involved the complex between the enzyme

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and the long-acting inhibitor omarigliptin (PDB Id: 4PNZ) chosen due to its very high resolution 39.

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After deleting water molecules, ions and crystallization additives, the selected dimer bound to

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omarigliptin was completed by adding the hydrogen atoms and then optimized by keeping fixed the

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backbone atoms to preserve the resolved folding. The inhibitor was finally deleted and the obtained

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protein structure underwent the following docking simulations.

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Docking simulations were carried out by using PLANTS and involved the 20 lowest energy

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conformations as derived by the previous MonteCarlo analysis in order to minimize the biasing

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effects of the starting conformation on the obtained results40. In detail, the search was focused on a

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12.0 Å radius sphere around the bound omarigliptin thus including the entire binding cavity.

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PLANTS was used with default settings and without geometric constraints, speed 1 was used and 5

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poses were generated for each conformer and scored by using the PLP function. The obtained poses

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were evaluated by considering both the docking scores and the conformational energies of the

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docked conformers. The best generated poses was then minimized keeping fixed all atoms inside a

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12.0 Å radius sphere around the bound peptide.

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The computed best DPP-IV / Soy 1 complex was neutralized, inserted into a 100 Å side cubic box

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containing about 19000 water molecules and, after a preliminary minimization, underwent 10 ns

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MD run by using NAMD with the same characteristics already described in a previous study.33

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RESULTS

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Biochemical evaluation of the inhibition of DPP-IV activity. Figure 1 shows the results of the

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experiments aimed to evaluate the inhibitory activity of soybean and lupin peptides against

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recombinant DPP-IV using H-Gly-Pro-AMC as substrate. The enzymatic reaction was monitored

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measuring the fluorescence signals (465 nm) due to the release of free AMC group after the

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cleavage of the peptide H-Gly-Pro catalyzed by DPP-IV. Each peptide was screened at the final

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concentration of 100 µM, in parallel with sitagliptin as positive control (0.1 µM). Two peptides

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showed inhibitory activity: Soy 1 reduced the DPP-IV activity by 46% and Lup 1 by 35%. On the

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contrary, Soy 2, Soy 3, Lup 2, and Lup 3 were inactive, whereas the positive control sitagliptin

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inhibited the DPP-IV activity by 88% (Figure 1). Dose-response curves were then built for the two

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active peptides (Figure 2): Soy 1 displayed the highest inhibitory activity with an estimated IC50

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value of 106 µM, whereas Lup 1 was less efficient, since its IC50 was 228 µM.

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Molecular modeling investigation. With the objective of revealing the key interactions elicited by

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the simulated peptides and better rationalizing their different behavior, a molecular docking study

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was undertaken. Even though performed a posteriori and without a predictive character, these

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results are useful to delve into the recognition processes between DPP-IV and peptide inhibitors and

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as such they may be useful when screening novel peptide libraries. Figure 3 shows the putative

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complex between Soy 1 and DPP-IV revealing the key ionic interactions, which involve both

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peptide charged termini and seem to play a largely predominant role. In detail, the amino terminus

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is engaged in a double salt bridge involving Glu205 and Glu206, while the carboxyl terminus

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stabilizes an ion-pair with Arg358. The key role of these ionic contacts is confirmed by the ACS Paragon Plus Environment

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comparison of the computed pose of Soy 1 with the crystallographic pose of omarigliptin shown in

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Figure 4, which highlights the perfect matching of the Soy 1 N-terminus with the amino group of

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omarigliptin as well as of the Soy 1 C-terminus with the sulphonyl group of omarigliptin. Apart

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from Thr5, which reinforces the contacts elicited by the ammonium head by approaching Glu205,

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the remaining part of the peptide appears to be marginally involved in the complex stabilization. In

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fact, the central residues might even play a negative role, since peptide apolar residues are seen to

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contact protein polar residues as in the case of Pro4, which unfittingly approaches Glu206 and

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Ser209. Moreover, Soy 1 does not contain any aromatic side-chains able to elicit π−π stacking

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interactions with the numerous aromatic residues lining the enzyme cavity.

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This pattern of key interactions can easily rationalize the different inhibitory activities observed for

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the other simulated peptides. In detail, the marked difference in the inhibition activity between Lup

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2 and Lup 1 seems to be mostly attributable to the interfering effect of the central Lys5 residue in

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Lup 2, which stabilizes the ionic contacts normally involving the amino terminus, which is

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therefore constrained to detrimentally approach Arg125. In detail, the carboxyl terminus of both

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Lup 2 and Lup 1 peptides interacts with Arg358 and Arg356 and is engaged in an extended ionic

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network also involving the side-chain of the C-terminal residue (Asp10 in Lup 2 and Asp9 in Lup

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1) and Arg429. As mentioned above, the greatest differences concern the contacts stabilized by the

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amino terminus, since it elicits the already described ion-pairs with Glu205 and Glu206 in the

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active peptide Lup 1, whereas in the inactive Lup 2 peptide it is replaced by Lys5 and is confined

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to a more lateral pose, where it approaches Arg125, while contacting Glu205. In the putative

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complexes of both Lup 2 and Lup 1, the central residues seem to play a relevant role. In detail, the

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negatively charged residue in the C-terminal segment of both peptides (Asp8 of Lup 2 and Glu8 of

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Lup 1) are involved in the ionic network stabilized around the carboxyl terminus as explained

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above. Moreover, Lup 1 includes an aromatic residue (Phe3) engaged in a rich set of π−π stacking

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involving Tyr547, Trp629, and His740. The key role played by the ionic interactions is further

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confirmed by the encouraging agreement found between the measured IC50 values and the ACS Paragon Plus Environment


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computed APBS scores which specifically accounts for electrostatic interactions (-75.23 kJ/mol, -

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49.14 kJ/mol and -13.38 kJ/mol for Soy 1, Lup 1, and Lup 2, respectively).

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In order to substantiate the docking results, a MD simulation involving the computed DPP-IV /

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Soy1 complex was carried out. As shown in Table 2, such a simulation confirmed the marked

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stability of the complex, which appears to be further reinforced by the stabilization of additional

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contacts during the simulation time. Table 2 reports the initial, final, and average distance values

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for some key interactions and confirms the clear stability of the already mentioned ion-pairs, which

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are stably retained throughout the MD run. In detail, Table 2 reveals the marked stability of the

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ionic contacts involving Asp205, Asp206, and Arg358, while the salt bridge with Arg356 is

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progressively lost to minimize the repulsion with the close Arg358 residue, which indeed

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approaches Glu403 at the end of simulation and forms an extended ionic network also involving

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Glu361, Arg382 and Asp588.

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Notably, Table 2 shows that the central residues, which are not engaged in clear interactions in the

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starting structure, gain at least two key H-bonds involving the hydroxyl function of Ser5 with

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Ser209 and the backbone atoms of Gly6 with Tyr588. Besides a clear rearrangement of the DPP-IV

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residues involved in the key contacts, the MD simulations reveals that the stabilization of these

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additional residues is also due to conformational shifts of the Soy 1 structure, which assumes more

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extended geometries due to turn-coil transitions. The progressive stabilization of the simulated

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complex found a convincing confirmation in the dynamic profile of the ChemPLP score function as

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computed during the MD run by the ReScore+ tool.41 Figure 5 clearly shows that after an initial

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increase, the score function progressively lowers to confirm a substantial strengthening of the

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stabilizing contacts with a gain of about 20 kcal/mol at the end of the simulation.

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Taken together, the docking results allow some general considerations. Ionic interactions stabilized

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by charged termini play a clearly crucial role even though their contribution is easily saturating and

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the stabilizing effect of the additional ionized side chains appears to be almost negligible, if not

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even negative (as seen for Lys5 in Lup 2). This effect can be explained by considering the ACS Paragon Plus Environment

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closeness between the cluster of protein negatively charged residues (i.e. Glu205 and Glu206) and

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that of positively charged residues (i.e. Arg358 and Arg356) and more generally the richness of

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ionized residues lining the enzyme cavity. In this way, the additional ionized side chains tend to

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interfere with the crucial contacts elicited by charged termini rather than playing a concrete

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stabilizing role.

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Clearly, such an interfering effect is an indirect consequence of the molecular size of the simulated

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peptides, which are excessively bulky, when considering that the enzyme cavity and the key

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stabilizing contacts are elicited by the two terminal groups, thus suggesting that the entire peptide

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should be accommodated within the catalytic cavity that is in fact arranged to accommodate

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dipeptide substrates. Together with the sequence, also the length, combined with the necessary

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involvement of both charged termini, can thus explain the inactivity of the other (non-simulated)

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longer peptides (i.e. Soy 2, Soy 3, and Lup 3) and can surely contribute to the inactivity of Lup 2.

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Finally, the non-ionized central residues of the simulated peptides appears to play very negligible

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roles and probably only Phe3 of Lup 1, which is engaged in π−π stacking interactions, should have

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a concrete stabilizing function thus differentiating Lup 1 from the other considered ligands.

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DISCUSSION

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Although the health benefits of soy and lupin protein consumption are well known, particularly in

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the area of cholesterol reduction, hypertension, and hyperglycemia prevention, this is the first study

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providing preliminary evidence that at least some peptides from soy and lupin protein may be able

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to inhibit the DPP-IV activity. This is an original contribution, since the hypoglycemic activity of

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soy protein has been previously explained with an improvement of glucose uptake in muscle cells7

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and hepatocytes,6 and that of lupin protein by an insulin-like mechanism.10

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Some years ago, a patent42 has reported the structures of 21 peptides capable of inhibiting DPP-IV

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activity. They have a hydrophobic character, a length varying from 2 to 8 amino acid residues, and



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contain a Pro residue within their sequences, which is located at the first, second, third, or fourth N-

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terminal position. Besides, the Pro residue is flanked by Leu, Val, Phe, Ala, and Gly. Indeed, our

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data are consistent with this patent. In fact, as the fourth N-terminal residue, the active peptides Soy

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1 and Lup 1 comprise a Pro, which is flanked by a Val residue in Soy 1 and by a Phe residue in

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Lup 1. Moreover, these peptides are mostly composed of hydrophobic amino acid residues, such as

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Ala, Gly, Ile, Leu, and Pro. The inactive peptides, i.e. Soy 2, Soy 3, Lup 2 and Lup 3, are probably

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too long, since they contain 10-14 amino acid residues. In addition, some of them do not respect the

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structural features indicated above: Soy 2 and Soy 3 comprise a Pro residue unfavorably located as

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fifth N-terminal residue and not flanked by any hydrophobic amino acid residue, whereas Lup 3

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does not contain any Pro residue.

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Finally, it is useful to compare the DPP-IV inhibitory activities of our peptides with other food

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peptides, such as those derived from silver carp protein,25 Atlantic salmon skin gelatin,27 bovine

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meat,22 and goat milk protein.24 Two peptides (PPL and PPG) with moderate activity have been

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identified in bovine meat protein hydrolyzates with IC50 390.1 µM and 2252.7 µM, respectively,

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using human recombinant DPP-IV.22 Four peptides (AGPPGPSG, APGPAGP, LPIIDI, and

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ALAPSTM) have been identified from the hydrolysis of silver carp protein;25 out of them LPIIDI

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and APGPAGP showed the highest DPP-IV inhibitory activity, with IC50 values equal to 105.4 and

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229.1 µM, respectively, using porcine DPP-IV,25 which are similar to those of Soy 1 and Lup 1. On

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the contrary, the peptides GPAG and GPGA from Atlantic salmon skin gelatin27 and

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INNQFLPYPY from goat milk24 appeared to be more active, showing the following IC50 inhibitory

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values: GPAG IC50 = 49.6 µM, GPGA IC50 = 41.9 µM, and INNQFLPYPY IC50 = 40.1 µM (on

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porcine DPP-IV).

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When discussing the relevance of the activity of any food component, a general issue is the

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bioavailability. In this case, the situation is favorable for Lup 1, since a very recent paper has

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already demonstrated that this peptide is able to across a monolayer of differentiated human


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enterocytes (CaCo-2 cells),35 an in vitro model of gastrointestinal absorption. Work is in progress in

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our lab to assess the bioavailability of Soy 1.

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Author Contributions

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Experiment ideation and design: CL and GV. Experiments & data analysis: biotechnology CL &

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CZ; molecular modeling GV. Figure preparation: GV and CZ. Grant retrieval: AA. Manuscript

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writing: CL, GV & AA.

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Funding

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Researches funded in part by the European Union Seventh Framework Program (FP7/2007-2013),

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under grant agreement n. 285819. We are indebted to Alpro Foundation for funding of a postdoc

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fellowship to CZ, and to Carlo Sirtori Foundation (Milan, Italy) for having provided part of

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equipment used in this experimentation.

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Notes

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The authors declare no competing financial interest.

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ABBREVIATIONS USED

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DPP-IV, dipeptidyl peptidase IV, GLP-1, glucagon-like peptide 1; GIP, glucose-dependent

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insulinotropic polypetide; AMC,.aminomethylcoumarin; EDTA, ethylenediaminetetraaceticacid.

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CAPTIONS OF FIGURES

393

Figure 1. DPP-IV inhibitory activities of Soy 1-3 and Lup 1-3. Each peptide was tested at the

394

final concentration of 100 µM, in parallel with the positive control, sitagliptin (0.1 µM). Bars

395

represent the averages ± SEM of 3 independent experiments in triplicate. ns: not significant and ***

396

P < 0.0001 versus the enzyme activity.

397

Figure 2. Dose-response curves of the inhibitory action of Soy 1 and Lup 1 peptides on DPP-

398

IV. The estimated IC50 values are equal to 106 µM and 228 µM, respectively. The data points

399

represent averages ± SEM of three independent experiments in triplicate.

400

Figure 3. Key ionic interactions stabilizing the putative complex between Soy 1 (shown by a

401

blue tube) and DPP-IV. The displayed protein residues are also involved in key interactions with

402

omarigliptin as seen in the utilized resolved DPP-IV structure.

403

Figure 4. Comparison of the computed pose of Soy 1 (colored in lime) with the

404

crystallographic pose of omarigliptin (colored in orange). Notice the perfect matching of the N-

405

terminus with the amino group of omarigliptin (red circle) as well as of the C-terminus with the

406

sulphonyl group of omarigliptin (blue circle).

407

Figure 5. Dynamic profile of the ChemPLP score as computed during the MD simulation.

408

409


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Table 1. Soy and lupin peptides.

410

Plant

Parent protein

Enzyme of

Sequence

Entry

digestion Soybean

Lupin seed

glycinin

pepsin

IAVPTGVA

Soy 1

β-conglycinin

pepsin/pancreatin

YVVNPDNDEN

Soy 2

β-conglycinin

pepsin/pancreatin

YVVNPDNNEN

Soy 3

β-Conglutin

pepsin

LTFPGSAED

Lup 1

β -Conglutin

pepsin

LILPKHSDAD

Lup 2

β -Conglutin

trypsin

GQEQSHQDEGVIVR

Lup 3

411 412 413 414 415

Table 2. Distance profile for some key interactions as derived from the MD simulation (all values are expressed in Å).

416

Distance Tyr585 – Gly6(NH) Ser209 – Ser5(OH) Arg358-Ala8(COO-) Arg356-Ala8(COO-) Glu205-Ile1(NH3+) Glu206-Ile1(NH3+)

Initial value 10.74 9.15 4.03 4.67 2.81 3.43

Final value 1.84 2.49 3.83 8.43 2.71 2.82

417


Average 5.04 ± 2.13 5.62 ± 1.81 3.83 ± 0.14 8.14 ± 1.47 2.91 ± 0.14 3.06 ± 0.35


20

418 419

Figure 1

420


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21

421 422 423

424 425

Figure 2

426



22

427

428 429 430

Figure 3

431 432


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23

433 434

Figure 4



24

435

436 437

Figure 5

438 439


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TOC

Soy

Lupin

441


Peptides Derived from Soy and Lupin Protein as Dipeptidyl-Peptidase

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