Structural Characterization of Act c 10.0101 and Pun g 1.0101-Allergens from the Non-Specific Lipid Transfer Protein Family

Abstract

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins' sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.

Keywords: cross-reactivity; food allergy; natural source; nsLTP; structure.

Figure 1

(a) Sequence alignment of non-specific lipid transfer proteins (nsLTPs) originating from pomegranate and kiwifruits. Disulfide bridges observed in Act c 10.0101 are marked in orange. Secondary structure elements are indicated below the sequence alignment. Helices are shown in blue and loops in green. Yellow color indicates residues that are different between Act c 10.0101 and Act d 10.0101 or Act d 10.0201. (b) Cartoon representation of Act c 10.0101 shown in two different orientations. Disulfide bridges are marked in orange. (c) Surface representation of Act c 10.0101 showing distribution of charges.

Figure 2

Results of mass spectrometric analysis of (a) Act c 10.0101 and (b) Pun g 1.0101 samples used for crystallization experiments. Calculated molecular weights for Act c 10.0101 and Pun g 1.0101 that have intact disulfide bridges are 9453 and 9342 Da, respectively. When there is no formation of disulfide bridges, the calculated molecular weights are 9461 and 9350 Da for Act c 10.1010 and Pun g 1.0101, respectively.

Figure 3

(a) Superposition of Act c 10.0101 and Pun g 1.0101 models shown in cartoon representation. Act c 10.0101 is shown in yellow, while Pun g 1.0101 models corresponding to two different conformations of the molecules are presented in different shades of pink. Regions of the proteins showing the biggest changes in conformation correspond to the Ω loops and were marked with the black square. (b) Close-up view of the Ω loops. Residues from Act c 10.0101 are marked using black labels, while Pun g 1.0101 residues are labeled in pink. (c) Models of Act c 10.0101 and Pun g 1.0101 in cartoon (top) and surface (bottom) representations. The models are shown in an orientation that shows the role of the Ω loops in closing and opening access to the ligand binding cavity.

Figure 4

Phylogenetic tree constructed with sequences of nsLTPs that are reported as allergens. Dots next to proteins’ names indicate orders from which particular allergens originate. Stars mark proteins that have their structures determined and reported to the Protein Data Bank [51].

Figure 5

Lipid binding modes observed in nsLTPs. (a) Covalent binding of oxylipin by barley nsLTP (PDB code: 3GSH). Model of barley nsLTP is shown in blue. Structure of Act c 10.0101 is shown in gold. Asp7 (barley), Asp8 (Act c 10.0101), and oxylipin are shown in stick representation. Position of the conserved Asp is marked with a circle. (b) Complex of maize nsLTP with α-linolenic acid (PDB code: 1FK6). (c) Complex of rice nsLTP with two molecules of palmitoleic acid (PDB code: 1UVB). (d) Complex of wheat nsLTP with two molecules of lyso-myristoyl-phosphatidylcholine (PDB code: 1BWO). (e) Superposition of Cor a 8 (PDB code: 4XUW; grey), Pru p 3 (PDB code: 2ALG; yellow), and models corresponding to two different conformations of Pun g 1.0101 (pink). Conserved Tyr81 is shown in stick representation. (f) Superposition of Cor a 8 and Pru p 3. Lipids bound by Pru p 3 are shown in purple. The figure shows that the conformation of the conserved residues that is present in the Cor a 8 structure is not compatible with ligand binding.

Figure 6

Plots showing Allergens’-Relative Identity, Similarity and Cross-reactivity (A-RISC) indexes of allergens from the nsLTP family. (a) Comparison of the family members to Act c 10.0101 and (b) Pun g 1.0101.

Figure 7

Surface residue conservation between Act c 10.0101 and Pru p 3.0102 (a), as well as between Pun g 1.0101 and Pru p 3.0102 (b). Identical residues are marked in red, while similar residues are marked in blue.