Abstract
Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs.
Highlights
lipid transfer proteins (LTPs) are small, ~9 kDa proteins comprising a bundle of four α-helices packed against a C-terminal region and belong to the prolamin superfamily of allergens[10]
The displacement curves for peach and wheat LTP differed (Fig. 1B), the latter showing an increase in fluorescence as the displacing ligand was titrated in, which gradually decreased
Wheat LTP has previously been shown to bind multiple lipids and the bell-shaped curve could be explained by a lipid binding in the cavity alongside the cis-parinaric acid (CPA), which may result in more effective exclusion of solvent from the pocket, increasing the fluorescence of the CPA11,26–28
Summary
LTPs are small, ~9 kDa proteins comprising a bundle of four α-helices packed against a C-terminal region and belong to the prolamin superfamily of allergens[10]. Eight conserved cysteines are characteristic of the superfamily, notably the Cys-Cys and Cys-X-Cys motifs, (where X represents any other residue) The structures of several free and liganded LTPs have been determined, including those from barley, wheat and peach[11,12,13] and a post-translationally modified form of barley LTP1, LTP1b, in which a lipid-like adduct is attached to the protein via the side chain of Asp 714,15. Amino acid side chain mobility may play an important role in determining susceptibility to hydrolysis by intestinal proteases trypsin and chymotrypsin, with the increased susceptibility of the lipid adducted LTP1b suggesting ligand occupancy might enhance digestion by increasing polypeptide mobility[21]. We have tested this hypothesis by investigating the effect of ligand binding on the susceptibility of peach and wheat LTPs to simulated gastroduodenal digestion, using the widely found plant lipid linoleic acid
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