Vicilin

Created by: Dominic DeLotto

Pine nut vicilin (PDB: 4LEJ) in Pinus koraiensis belongs to the cupin superfamily and is a potential food allergen (1). It is recognized by immunoglobulin E (IgE) as a food allergy (2). As peanuts and tree nuts account for the majority of fatal and near-fatal food allergy cases in the United States, consumption of Korean pine vicilin is a major global health concern. Unfortunately, pine nuts, similar to peanuts and other tree nuts, cause allergic reactions in certain patients, although the World Health Organization and International Union of Immunological Societies Allergen Nomenclature Subcommittee have not officially recognized pine proteins as allergens (1).

In addition to their medical importance, Korean pine nuts are economically important as a source of human food. They are a delicacy in many of the world’s cultures and are frequently used in the preparation of bakery products, vegetable dishes, and other foods. They are high in nutritional value, containing a considerable amount of vitamins (A, B1, and B2), potassium, magnesium, and other minerals as well as dietary fiber (1). The Korean pine nut is marketed globally as a gourmet product and is the most important species in today’s international pine nut trade (3). Thus, its allergenic effects influence its economic importance. To understand the allergenicity of the Korean pine vicilin, additional information of its structural properties is needed.

Korean pine nut vicilin is a 7S vicilin type seed storage globular protein, belonging to the cupin protein superfamily (4). The cupins are a large and functionally diverse superfamily of proteins that share a β-barrel structural core domain. Most cupins share the common motif of a metal-binding site in the core of the protein (5). Copper in vicilin’s core increases stability, which is hypothesized important in its allergenicity, however unknown (1). Vicilin is a homotrimeric protein with a molecular weight of 46,189.53 Da and an isoelectric point (pI) of 5.74 (6). The ExPASy server was used to determine these values.

Vicilin consists of large β-sheets that twist and coil to form a closed structure held together by hydrogen bonds (5). ). The overall structure consists of one subunit (A) with its N-terminal domain and a C-terminal domain each assuming a cupin fold (1). These domains are antiparallel β-strands with alternating polar and nonpolar amino acids. This orients the nonpolar residues into the interior of the barrel to form the hydrophilic core and the nonpolar resiudes toward the outside of the barrel on the solvent-exposed surface (5). Three of these vicilin β-sheet molecules form a doughnut-shaped homotrimer through head-to-tail association (2).

The double-stranded beta-roll that comprises vicilin’s cupin fold is very stable, which is characteristic of allergens. The N-terminal and C-terminal domains enhance vicilin’s stability because these domains are symmetrically folded about a pseudodyad axis (2). Having this stable structure allows vicilin to resist thermal denaturation and proteolysis. The stability permits sufficient immunologically active fragments to pass down the gastrointestinal tract, which leads to thermostable nature of the allergenic activity of vicilin. Such properties, coupled with the abundance of storage vicilin in the diet contribute to its ability to act as a potent allergen. However, the exact mechanism is still unclear (3).

In its native trimeric state, Korean pine vicilin has three copper ligands. The copper center consists of Cys-338, Tyr-67, His-340, and His-379. The sulfur of Cys-338, the ND1 (imidazole nitrogen) of His-340, and the NE2 of His-379 form a trigonal planar structure with the copper ion in the center, but slightly away from the plane (2). The proximal histidines minimize steric conflict in this ND1/NE2 conformation. The ND1 ligation of His-340 occupies more space (with the backbone closed) in contacting with copper. The NE2 ligation of His-376 has the histidine backbone pointing away from the copper center and has little surface area of the copper involved in the contact. The hydroxyl group of Tyr-67 provides the fourth, axial ligand with a longer coordinating distance (1). Copper with these four contact residues is called type II. These differences in contact with vicilin provide significant conformational flexibility and its ability to resist denaturation with slight changes in pH. In theory, this added stability allows vicilin to remain in its native conformation to undergo optimal activity in its allergenistic behaviors.

The copper center in Korean pine vicilin has features of both type I and type II copper sites. In both types, two histidine residues and a cysteine residue coordinate the copper center in a trigonal planar conformation. Type I is arranged with an additional axial methionine (Met-336) coordinated, distorting the geometry towards tetrahedral. Type II copper center coordinates with the same two histidine and cysteine resuidues as type I. However, type II coordinates Tyr-67, instead of Met-336, to form a square pyramidal geometry. The distance between the hydroxyl group of Tyr-67 and the copper center (2.36 A) is longer than the distance between the sulfur of Met-336 and the copper ion (2.12 A). Thus, vicilin belongs in a more divergent type II category because in the type II square pyramidal conformation, vicilin has greater compressibility and readily assists in oxidations and oxygenations (1).

Free copper ions in the cell are toxic even at low concentrations, so the ability of certain proteins to bind copper provides a mechanism to detoxify the cell. It is not well understood whether the copper center of vicilin has an important direct biological function, however in theory, contributing to its allerginicity is the hypothesis (2). As a seed storage protein, besides preserving nitrogen, carbon, and sulfur for the development of young plants, no other biological function for vicilin is actually known. All that is understood is that the copper center of Korean pine vicilin binds to the residues from both the N- and C-terminal domains, which contributes to the protein's stability. Glycerol and phosphate are two other ligand founds in the core of the protein; however, it is unknown whether these additional ligands have any biological significance. Future investigations are needed to unveil whether Korean pine vicilin’s catalytic activity requires a copper, glycerol, and phosphate, namely at the core (4).

Two of the Korean pine peptides appear to be immunodominant IgE-binding epitopes. IgE antibodies in humans can specifically recognize these epitope sites and have a long-lived interaction with the allergen. This allows IgE to trigger the MHC Class I pathway, which elicits an immune response. Epitope 15, which is located in the C-terminal end of the protein (amino acids 399-407), is in a region that shares significant sequence similarity with other known allergens (i.e. Ara h 1, PDB: 3SMH) from other legumes. The D2-D3 binding region of vicilin (amino acids 82-133) contains four epitopes. The most prevalent amino acids found in this region are aspartic acid, glutamic acid, lysine, and arginine, which comprise 40% of all amino acids found in epitopes (2).

In comparison to peanut allergen Ara h 1, a 7S globulin, the results of Dali (Z = 42.2) indicate significant tertiary structure similarities because the Z-score is above 2 (7). Ara h 1 is a seed storage globulin 7S globulin protein. Like Korean pine vicilin, Ara h 1 forms homotrimers, stabilized through hydrophobic interactions. This protein has two domains folded about a perpendicular pseudodyad axis that are stabilized by multiple hydrogen bonds. While Korean pine vicilin is hypothesized as an allergen, Ara h 1 is a known allergen. Unlike vicilin, Ara h 1 lacks a copper ligand, which questions the level of significance copper has on vicilin's allergenistic effects (1).

Results from a PSI-BLAST gave an E-value of 3E-136, indicating very similar sequence homology because it is below an E-value of 0.5 (8). In comparison to peanut allergen Ara h 1, three peptides identified in Ara h 1 (ASSEAGEIR, QFLAGK, and FGVPSGHTFY) were separately aligned with Korean pine vicilin with nearly 100% identity. Vicilin’s second peptide sequence of EFLAGK contains one amino acid residue not identical. Peanut allergen Ara h 1 contains a total of 23 linear IgE epitopes with the smallest IgE-binding sequence containing six amino acids. Aligning Korean pine vicilin with Ara h 1’s epitope sequences results in no exact matches (1). Unfortunately, very little information is available regarding conformational IgE epitopes of known allergens. To date, the understanding of Korean pine vicilin’s allergenicity remains minimal (2).