In vitro digestibility and IgE reactivity of enzymatically cross-linked heterologous protein polymers
Introduction
Transglutaminase (TGase) catalyzes acyl transfer reactions between the γ-carboxamide group of glutamine residues and the ε-amino group of lysine residues, which results in the formation of inter-molecular and intra-molecular cross-linking in proteins (Agyare and Damodaran, 2010, DeJong and Koppelman, 2002, Han and Damodaran, 1996). In the case of inter-molecular cross-linking, which is the predominant route, the reaction produces branched-chain protein polymers (Han & Damodaran, 1996).
The TGase-mediated protein cross-linking reaction has been extensively studied in the context of improving the functional properties of proteins (Lauber et al., 2003, Liu and Damodaran, 1999, Kuraishi et al., 2001, Muguruma et al., 2003, Ali et al., 2010, Chambi and Grosso, 2006, Damodaran and Agyare, 2013). Another potential application of this enzymatic cross-linking reaction, which has not been explored much, is in reduction or elimination of the allergenicity of food proteins (Buchert et al., 2007, Malandain, 2005, Pedersen et al., 2004). The allergenicity of cow’s milk proteins, egg, wheat, and soy proteins in children is attributed mainly to the IgE-mediated hypersensitivity to some of the protein components in these foods (Exl & Fritsche, 2001). Elicitation of allergenic response of a protein could be both due to a sequence-specific epitope (i.e., linear epitope) or a spatial structure-dependent non-sequential epitope (i.e., conformational epitope). It might be possible to mask these immune reactive epitopes using TGase-mediated cross-linking of proteins into high molecular weight polymers. In addition to masking the exposure of both conformational and linear epitopes in the oral cavity during food consumption, these cross-linked proteins may remain hypoallergenic during their transit through the gastro-intestinal digestive system since the amino acid profiles of peptides released from cross-linked proteins would be different from those released from non-cross-linked proteins.
Stanic et al. (2010) reported that polymerization of β-casein using four different enzymes, including TGase, caused a modest decrease in their IgE binding potential. Similar results were also observed with purified β-lactoglobulin (Olivier, Villas-Boas, Netto, & Zollner, 2012). On the other hand, while treatment of wheat flour with TGase caused a 70% decrease in immunoreactivity of glutenin in non-competitive ELISA (Leszczyńska, Łącka, & Bryszewska, 2006), the IgE binding potential of ω-gliadin increased upon TGase treatment (Palosuo et al., 2003). Results of these studies suggested that homologous polymerization of single proteins using TGase did decrease the extent of IgE binding, but a large fraction of conformational and linear epitopes in those homologous polymers were still accessible to IgE binding.
The hypothesis of the present study is that TGase-catalyzed heterologous polymerization between two structurally very different proteins may substantially decrease the accessibility of conformational and linear epitopes to IgE binding. To test this hypothesis, the IgE binding ability of heterologously cross-linked polymers (derived from TGase-catalyzed polymerization of whey protein isolate (WPI) + soy protein isolate (SPI), sodium caseinate (CN) + SPI, and CN + WPI mixtures) was investigated. The digestibility of these heterologous polymers under simulated gastric-duodenal conditions, and the IgE reactivity of the digests were also studied.
Section snippets
Materials
Soy protein isolate (SPI) was extracted from defatted soy flour (Archer Daniel Midland Co., Decatur, IL, USA) as described elsewhere (Thanh & Shibasaki, 1976) with slight modifications. Briefly, 50 g soy flour was dissolved in 1 L of 30 mM Tris-HCl buffer at pH 8.0 and extracted for 1 h at room temperature. The slurry was then centrifuged at 7000g for 30 min and the supernatant was adjusted to pH 4.6 with 2 N HCl to precipitate proteins. The precipitated protein was separated by centrifugation at 7000
SDS-PAGE of SPI - WPI homo- and heterologous conjugates
The SDS-PAGE profile of polymers produced by TGase in WPI and SPI single protein systems and in SPI + WPI mixed protein systems are shown in Fig. 1A. In single protein systems, TGase was able to polymerize WPI and SPI to high molecular weight polymers. However, there were significant differences in the extent of polymerization of these proteins: Whereas almost all the subunits of SPI (Fig. 1A, lane 1), with the exception of a small amount of the basic subunit of 11S (m.w. ∼18 kDa), were polymerized
Discussion
Transglutaminase (TGase) catalyzes formation of a covalent isopeptide bond between primary amines (including ε-amino group of lysine residues) and the γ-carboxamide group of glutamine residues in protein, leading to formation of cross-linked protein polymers. These polymers are not linear in nature as found in native proteins, but are branched-chain polymers. Depending on the extent of polymerization as well as the tertiary structure of the monomer, the branched-chain polymers of a protein can
Acknowledgment
The National Institute of Food and Agriculture, United States Department of Agriculture, Hatch project 227287, supported this work.
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