Plant-Derived Proteins and Peptides as Potential Immunomodulators.

The immune response of humans may be modulated by certain biopeptides. The present study aimed to determine the immunomodulatory potential of plant-derived food proteins and hydrolysates obtained from these proteins via monocatalytic in silico hydrolysis (using ficin, stem bromelainm or pepsin (pH > 2)). The scope of this study included determinations of the profiles of select bioactivities of proteins before and after hydrolysis and computations of the frequency of occurrence of selected bioactive fragments in proteins (parameter A), frequency/relative frequency of the release of biopeptides (parameters AE, W) and the theoretical degree of hydrolysis (DHt), by means of the resources and programs available in the BIOPEP-UWM database. The immunomodulating (ImmD)/immunostimulating (ImmS) peptides deposited in the database were characterized as well (ProtParam tool). Among the analyzed proteins of cereals and legumes, the best precursors of ImmD immunopeptides (YG, YGG, GLF, TPRK) turned out to be rice and garden pea proteins, whereas the best precursors of ImmS peptides appeared to be buckwheat (GVM, GFL, EAE) and broad bean (LLY, EAE) proteins. The highest number of YG sequences was released by stem bromelain upon the simulated hydrolysis of rice proteins (AE = 0.0010-0.0820, W = 0.1994-1.0000, DHt = 45-82%). However, antibacterial peptides (IAK) were released by ficin only from rice, oat, and garden pea proteins (DHt = 41-46%). Biopeptides (YG, IAK) identified in protein hydrolysates are potential immunomodulators, nutraceuticals, and components of functional food that may modulate the activity of the human immune system. Stem bromelain and ficin are also active components that are primed to release peptide immunomodulators from plant-derived food proteins.

Characteristics of Potential Protein Nutraceuticals of Plant Origin with Antioxidant Activity.

This study used selected plant proteins and the tools available in the BIOPEP-UWM database to profile proteins and release antioxidant nutraceuticals from their primary structures. The frequency of the occurrence of fragments with antioxidant activity in a protein sequence (the A parameter) was determined. A simulated monocatalytic proteolysis was carried out using ficin or stem bromelain or pepsin (pH > 2), and the theoretical degree of hydrolysis (DHt) and the frequency (including relative frequency) of the release of fragments with a particular antioxidant activity by a selected enzyme (the AE and W parameters, respectively). Both barley hordoindolines and the protein group of "actins and other rice proteins" were characterised by the best antioxidant potential. On the other hand, among the main analysed cereal protein groups or species, the best nutraceutical sources included kafirins, rice glutelins and α-gliadins. Potentially the most nutraceutical molecules were released by pepsin (HL, VY, PHQ and PWQ biopeptides) from gliadins, but the most analysed proteins were hydrolysed (66% on average) and the DHt for ficin and bromelain amounted to 27% and 31%, respectively. However, based on the calculated AE mean values, it can be concluded that nutraceuticals were more frequently released from rice protein structures (IY and VY biopeptides), and less frequently released from barley and other cereal protein species, which may be of significance in the context of designing nutraceutical food.

Isolation of Oat (Avena sativa L.) Total Proteins and Their Prolamin Fractions for 2D Electrophoresis.

Appropriate sample preparation is essential to obtaining good results of two-dimensional gel electrophoresis (2-DE). For various reasons (particularly phenolic compounds, proteolytic enzymes, and cell-wall mucilages) the extraction of proteins from plant material, among them oat proteins, is difficult. During isolation all soluble substances that may interfere with the analysis (especially isoelectric focusing) are removed, and proteins of interest are separated from the remains. However, the applied procedure of isolation cannot be too extensive, because additional stages cause loss of the proteins.In this chapter, we describe a simple procedure for the isolation of oat total proteins and their prolamin fractions prior to 2-DE, without necessity of considerable purification. It can be used for oat protein fractionation, measurement of oat protein concentration, and their 2-DE analysis, with particular reference to prolamin fractions. The presented routine includes modified methods of plant seed proteins extraction and sequential Osborne extraction, based on oat protein solubility differences.

2-DE Separation and Identification of Oat (Avena sativa L.) Proteins and Their Prolamin Fractions.

At present two-dimensional polyacrylamide gel electrophoresis (2-DE) is the most widely used proteomic tool, which enables simultaneous separation of even thousands of proteins with a high degree of resolution. The quality of 2-DE separation depends on the type of biological material used as a protein source. The presence of interfering compounds (e.g., phenols, as it is the fact in plant material including oat seeds) impedes 2-DE run. With the use of this technique it is possible to analyze the complex protein mixtures, characteristic protein fractions, as well as individual proteins.The purpose of this chapter is to describe the 2-DE technique (the separate stages of the first and the second dimension) for determining the oat protein composition (oat seed proteome), separation and preliminary identification of oat prolamin fractions. Electrophoretically separated proteins are identified on the basis of pI markers (identifying the location of both ends of an IPG strip) and on 2D SDS-PAGE standards. The gel images of oat proteins are analyzed with the help of ImageMaster 2D Platinum 6.0 program (Amersham Bioscience, part of GE Healthcare, Uppsala, Sweden). It allows finding unique spot identifiers for the occurrence of oat prolamin fractions in oat total proteins. The characteristic spots of similar shape and intensity (anchoring spots) and characteristic groups of spots can be searched for the purpose of identification.

Selected Bioinformatic Tools and MS (MALDI-TOF, PMF) Techniques Used in the Strategy for the Identification of Oat Proteins After 2-DE.

Computer analysis of protein maps obtained from the separation of proteins with two-dimensional polyacrylamide gel electrophoresis (2-DE), in combination with mass spectrometry (MS) analysis and selected bioinformatic tools is used in the strategy for the identification of oat proteins. In proteomic research the most often used MS technique is the combination of ion sources: matrix-assisted laser desorption/ionization (MALDI) and the analyzer of the time of flight (TOF), i.e., MALDI-TOF MS.This chapter describes the possibilities of the use of selected bioinformatic tools (UniProtKB database, ProtParam, Compute pI/MW programs) for initial identification of separated oat proteins (especially prolamin fractions) with the 2-DE technique. Also the procedure of preparation of samples obtained from cut out protein spots for analysis with the MALDI-TOF MS and peptide mass fingerprinting (PMF) technique is presented.Among oat prolamins separated with the 2-DE technique (see Chapter 17 ), 13 protein spots are considered to be the most characteristic (range of MW 27.0-34.6 kDa, pI 5.7-7.6) for this fraction of proteins. Among them there are four protein spots (MW 27.0-28.0 kDa) and two spots (MW 31.4-32.1 kDa) which can correspond to avenins (Accession numbers (AC) in UniProtKB: L0L5I0, I4EP88, I4EP64, L0L4I8 and F2Q9W5, L0L6J0, respectively).

Proteomic analysis of albumin and globulin fractions of pea (Pisum sativum L.) seeds.

BACKGROUND: Proteomic analysis is emerging as a highly useful tool in food research, including studies of food allergies. Two-dimensional gel electrophoresis involving isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis is the most effective method of separating hundreds or even thousands of proteins. In this study, albumin and globulin tractions of pea seeds cv. Ramrod were subjected to proteomic analysis. Selected potentially alergenic proteins were identified based on their molecular weights and isoelectric points. MATERIAL AND METHODS: Pea seeds (Pisum sativum L.) cv. Ramrod harvested over a period of two years (Plant Breeding Station in Piaski-Szelejewo) were used in the experiment. The isolated albumins, globulins and legumin and vicilin fractions of globulins were separated by two-dimensional gel electrophoresis. Proteomic images were analysed in the ImageMaster 2D Platinum program with the use of algorithms from the Melanie application. The relative content, isoelectric points and molecular weights were computed for all identified proteins. Electrophoregrams were analysed by matching spot positions from three independent replications. RESULTS: The proteomes of albumins, globulins and legumin and vicilin fractions of globulins produced up to several hundred spots (proteins). Spots most characteristic of a given fraction were identified by computer analysis and spot matching. The albumin proteome accumulated spots of relatively high intensity over a broad range of pi values of ~4.2-8.1 in 3 molecular weight (MW) ranges: I - high molecular-weight albumins with MW of ~50-110 kDa, II - average molecular-weight albumins with MW of ~20-35 kDa, and III - low molecular-weight albumins with MW of ~13-17 kDa. 2D gel electrophoregrams revealed the presence of 81 characteristic spots, including 24 characteristic of legumin and 14 - of vicilin. CONCLUSIONS: Two-dimensional gel electrophoresis proved to be a useful tool for identifying pea proteins. Patterns of spots with similar isoelectric points and different molecular weights or spots with different isoelectric points and similar molecular weights play an important role in proteome analysis. The regions characteristic of albumin, globulin and legumin and vicilin fractions of globulin with typical MW and pi values were identified as the results of performed 2D electrophoretic separations of pea proteins. 2D gel electrophoresis of albumins and the vicilin fraction of globulins revealed the presence of 4 and 2 spots, respectively, representing potentially allergenic proteins. They probably corresponded to vicilin fragments synthesized during post-translational modification of the analysed protein.