Identification and Characterization of a New Pecan [Carya illinoinensis

Apr 29, 2016 - ... Research Service, U.S. Department of Agriculture, 800 Buchanan ... School of Food Science and Technology, Jiangnan University, 2141...
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Identification and Characterization of a New Pecan [Carya illinoinensis (Wangenh.) K. Koch] Allergen, Car i 2 Yuzhu Zhang,*,† BoRam Lee,†,§ Wen-Xian Du,† Shu-Chen Lyu,‡ Kari C. Nadeau,‡ Larry J Grauke,∥ Yan Zhang,⊥ Shuo Wang,⊥ Yuting Fan,†,# Jiang Yi,∇ and Tara H. McHugh† †

Western Regional Research Center, Pacific West Area, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, California 94710, United States ‡ Division of Pediatric Immunology, Allergy, and Rheumatology, Department of Pediatrics, Stanford University School of Medicine, 269 Campus Drive, Stanford, California 94305, United States ∥ Crop Germplasm Research, Southern Plains Agricultural Research Center, USDA-ARS-SPA, 2881 F&B Road, College Station, Texas 77845, United States ⊥ Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin, 300222, China # School of Food Science and Technology, Jiangnan University, 214122, Wuxi, China ∇ College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China ABSTRACT: The 7S vicilin and 11S legumin seed storage globulins belong to the cupin protein superfamily and are major food allergens in many foods from the “big eight” food allergen groups. Here, for the first time, pecan vicilin was found to be a food allergen. Western blot experiments revealed that 30% of 27 sera used in this study and 24% of the sera from 25 patients with double-blind, placebo controlled clinical pecan allergy contained IgE antibodies specific to pecan vicilin. This allergen consists of a low-complexity region at its N-terminal and a structured domain at the C-terminal that contains two cupin motifs and forms homotrimers. The crystal structure of recombinant pecan vicilin was determined. The refined structure gave R/Rfree values of 0.218/0.262 for all data to 2.65 Å. There were two trimeric biological units in the crystallographic asymmetric unit. Pecan vicilin is also a copper protein. These data may facilitate the understanding of the nutritional value and the allergenicity relevance of the copper binding property of seed storage proteins in tree nuts. KEYWORDS: allergy, X-ray crystallography, Western blot, Carya illinoinensis (Wangenh.) K. Koch

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INTRODUCTION Plant seed storage proteins are important for the growth of the seedlings. They are also an indispensable protein source for human nutrition. Unfortunately, a number of seed storage proteins, including the 7S and 11S globular proteins from the cupin superfamily, are also known to be the culprits for causing millions of people to suffer from food allergies.1,2 Food allergies may be fatal, especially allergies to peanut and tree nuts.3,4 Most type I food allergies are triggered by immunoglobulin E (IgE) recognition of food allergens,5 and cross-reactivity among different foods6,7 poses additional risks to allergic patients. Currently, there is no cure for food allergies, and the food allergy problem is having a tremendous negative impact on the utilization and marketing of agricultural products. Tree nut allergies as a group are as prevalent as peanut allergy. However, there is little data concerning the prevalence of allergies to individual tree nuts. Studies on the identification and characterization of allergens also vary greatly from one tree nut to another. For example, while there are 8 food allergens defined in hazelnut (Corylus avellana), Car i 18 and Car i 49 are the only 2 allergens designated in pecan [Carya illinoinensis (Wangenh.) K. Koch] at the time this article was submitted. On the other hand, the annual consumption of pecan is 8 times greater than that of hazelnut.10 Additional studies of tree nut allergens and information on the prevalence of individual tree © XXXX American Chemical Society

nut allergies are required to minimize the negative effects of grouping tree nut allergies on the marketing of certain agricultural commodities such as pecan. Seed storage proteins have no known biological functions other than serving as a nutrient source for the young plant. The 7S globulins are called vicilins. They are present in both monocotyledonous and dicotyledonous species. We recently reported the crystal structure of Korean pine vicilin and revealed for the first time that vicilin is a copper protein in a large number of species.11 The copper coordination in vicilin is different from those in other copper proteins with known functions. Thus, it is of interest to characterize additional vicilins from other species, both for understanding the allergenicity of these food proteins and for investigating whether vicilins have a copper center related function that is yet to be discovered. Here, we report the identification of pecan vicilin (Cariv) as a food allergen and the structural characterization of this new allergen. Received: February 23, 2016 Revised: April 26, 2016 Accepted: April 29, 2016

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DOI: 10.1021/acs.jafc.6b00884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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together with the final refined structure. Molecular graphics were prepared using the programs rasmol,21 molscript22 Raster3D,23 and Pymol (http://pymol.org/). The structure of pecan vicilin has been submitted to the Protein Data Bank under accession number 5E1R.

MATERIALS AND METHODS

Patient Sera. Twenty-five sera (#1−5 and #8−27, corresponding to deidentified patient codes 5720, B5647, B5333, B5312, 5858, 2429, 5478, 6856, 5374, 5422, 6051, 5863, 6202, 5823, 2252, 2251, 5648, 2341, 6415, 5532, 2154, 6204, 6380, 5524, and 5426, respectively) were collected from patients with a history of IgE-mediated pecan allergy and with a positive oral food challenge to pecan. The patients were enrolled in the food allergy study at Stanford University under institutional review board approval with informed consent. Sera from 2 additional allergic subjects (#6 and #7) were acquired from PlasmaLab International (Everett, WA). IgE Recognition of Pecan Vicilin. Raw cultivar Pawnee nuts were collected from verified inventories at the USDA ARS Pecan Breeding & Genetics program, Burleson County, Texas. Shelled pecans were roasted at 163 °C for 10 min. In addition, roasted pecans of different cultivars were imported from China. For protein extraction, raw and roasted pecan kernels were ground in 5 g batches with 50 mL of 10 M urea and 30 mL of hexane. The ground pecan samples were centrifuged at 20000g for 30 min, and the middle aqueous layer was saved as pecan protein extract. Expression and purification of recombinant pecan vicilin (rCariv) and recombinant structural core of pecan vicilin (rcCariv) were carried out with reported protocols.12 Pecan protein extract and purified recombinant proteins were heated in 1× SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol) containing 100 mM β-mercaptoethanol at 96 °C for 10 min and separated by electrophoresis on 4− 20% gels. Multiple SDS−PAGEs were performed for IgE binding analysis by Western blot and for protein detection by coomassie brilliant blue (CBB) staining. For Western blot, proteins bands in the SDS gels were transferred to PVDF membranes with a Tans-Blot SD semidry transfer cell (Bio-Rad, Hercules, CA). The membranes were blocked in TBST (25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) containing 5% nonfat dry milk for 30 min at room temperature. At the same time, incubation of 200 μL of individual serum in 5 mL of TBST with 1% nonfat milk and 20 μL of protein A immobilized with agarose (Pierce, Rockford, IL, USA) was started at 4 °C. After incubation for 1 h, protein A was removed from the serum solutions by centrifugation at 1000 rpm for 2 min. The membranes were then incubated with individual serum solutions at 4 °C overnight. After being washed three times with TBST for a total of 15 min, the membranes were incubated for 1 h at room temperature with an antihuman IgE secondary antibody conjugated to peroxidase (Sigma) diluted 5000 times with TBST containing 1% nonfat milk. The membranes were washed again 5 × 5 min with TBST. Indirect detection of the bound IgE was performed using ECL Western blotting substrate (Pierce, Rockford, IL, USA) and a ImageQuant LAS4000 imaging system. N-Terminal Sequencing. Ten molar urea pecan extract was subjected to SDS−PAGE using an 8−25% gradient gel and transferred to a PVDF membrane. The blot was stained with CBB, and the protein bands of interest were excised and submitted to a vender for Nterminal amino acid sequencing by Edman degradation. Structure Refinement. The crystallization and X-ray data collection were previously described.12 Briefly, X-ray diffraction data were collected on the HMMI-CAT 28-2ID beamline at the ALS (Advanced Light Source), Lawrence Berkeley National Laboratory. Data processing with HKL200013 and a structure solution by Phaser14,15 molecular-replacement calculations were reported previously.12 Structure refinement was carried out with Phenix-refine16 alternated with model building and model improvement using coot.17 The final structure was refined with all data to 2.65 Å resolution, and the final model was checked by Molprobity Validation18 and PROCHECK.19 The quality of the structure model was also checked with a shake-and-omit protocol by first introducing random errors up to 0.3 Å to the coordinates of the final refined structure using the program pdbset distributed with CCP4.20 For each region to be checked, the concerned region of the shaken structure was manually omitted and 5 cycles of restrained refinements were carried out using Phenix-refine.16 This was followed by inspecting the Fo − Fc map

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RESULTS AND DISCUSSION Identification of Pecan Vicilin as an Allergen. Patients from Stanford University whose sera were used in this study had an average age of 11.36 (range 5−21). The mean diameter of results of their skin prick tests was 5.2 mm (range 3−8). All these patients had a positive DBPCFC with pecan. All patients including those two whose sera were obtained from PlasmaLab International also had an allergy to walnut. Some of them also have an allergy to one or more of the following foods: almond, cashew, egg, hazelnut, milk, peanut, pistachio, and sesame. The clinical information on the patients is shown in Table 1. Table 1. Clinical Findings of Pecan-Allergic Patients patient no./age

age

SPTa (mm)

1 2 3 4 5 8 9 10 11 12

8 9 14 13 10 11 21 16 6 8

13 14 15

IgEb (kU/ L)

symptomsc

diagnostic challenge

known allergy to other foodd

4 3 7 5 4 8 4 5 5 6

A, GI R A,R A R A, GI GI U U R

DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC

5 7 11

7 5 5

U U U

DBPCFC DBPCFC DBPCFC

16 17 18 19 20 21 22 23 24 25 26 27 6

12 8 20 13 15 7 6 14 9 11 17 13 24

4 3 8 6 5 5 5 4 7 6 5 4

R U U R R GI A, U R A GI GI U

DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC DBPCFC

7

56

peanut, walnut hazelnut, walnut walnut walnut walnut walnut cashew walnut walnut walnut peanut, sesame, walnut walnut walnut almond, peanut, walnut walnut walnut walnut walnut egg, milk, walnut walnut walnut milk, walnut walnut walnut walnut walnut cashew, hazelnut, pistachio, walnut almond, hazelnut, walnut

43.6 60

a

SPT = skin prick testing. bImmunoCAP specific IgE. cAbbreviations for symptoms: A, asthma; GI, gastrointestinal symptoms; R, rhinitis; U, urticaria. DBPCFC = double-blind, placebo-controlled food challenge. dOther food allergies diagnosed by DBPCFC (for patients 1−5 and 8−27) or ImmunoCAP (for patients 6, 7).

Most food allergens belong to a very limited number of protein families. The nonspecific lipid transfer proteins, profilins, the 2S seed storage proteins, the pathogen resistance proteins-10, and the 11S and 7S seed storage proteins account for more than two-thirds of the known food allergens from plant sources. The US Food and Drug Administration listed 19 B

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Journal of Agricultural and Food Chemistry nuts in the tree nut group of allergen sources24 for the purpose of food labeling (although not all of them are nuts in the botanical sense). Of these tree nuts, 7 of the 2S orthologous proteins and 7 of the 11S orthologous proteins have been designated as food allergens by the World Health Organization and International Union of Immunological Societies (WHO/ IUIS) Allergen Nomenclature Subcommittee, while the 7S vicilins have been identified as allergens in 4 of these tree nuts (e.g., cashews, hazelnuts, pistachios, and walnuts). Pecan vicilin is very likely to be a food allergen because it belongs to the cupin superfamily which contains the 7S and 11S proteins. We sought to use Western blot experiments to assay whether pecan vicilin is a food allergen by testing their recognition by serum IgE of 27 patients with a history of pecan allergy and positive reaction to oral pecan challenge. In one set of Western blot experiments, raw pecan extract samples were separated by SDS gels along with rcCariv and analyzed by Western blot with 7 patient sera. Three sera (sera 5, 6, and 7) clearly recognized the rcCariv, while in a parallel negative control experiment using identical protocols without any serum as first antibody, no false positive recognition was observed (Figure 1A, right panel). Serum 5 also recognized two bands with slightly higher molecular mass than that of rcCariv in the urea pecan extract. These two bands were among the major bands observed when the urea pecan extract was separated with identical SDS gels and stained with CBB (Figure 1A, left panel). Similar band patterns resulted when roasted pecan extracts were analyzed by SDS−PAGE (data not shown). Sera 6 and 7 recognized a number of protein bands in the urea pecan extract including the two bands recognized by serum 5. These are designated as bands 1 and 2 as indicated by the arrow heads at the left side of the gel images. Expression of the structural core of Cariv was designed on the basis of sequence alignments and structural information on allergens in pine nut11 and peanut.25,26 The theoretical molecular mass of rcCariv is 49 kDa, and that of the peptide deduced from the open reading frame of the pecan vicilin is 95 kDa. We thought that band 1 or band 2 in the 10 M urea pecan extract recognized by sera 5, 6, and 7 might have resulted from the native pecan vicilin as vicilins from many other species (including peanut27 and pine nut28) have a signal peptide removed in the mature proteins. To test this speculation and to determine the cleavage site of the signal peptide, we subjected bands 1 and 2 to N-terminal peptide sequencing. The results of five cycles of N-terminal peptide sequencing of band 1 (RQDPQ) indicated that a long leader sequence of Cariv was removed post-translationally. The calculated molecular mass of the mature Cariv is 55.9 kDa. This is consistent with the band position in the SDS gel (note that the 56 kDa protein in the prestained protein ladder consistently traveled to the same position as the 50 kDa proteins in other commercial protein standards under these conditions, although the other proteins in this ladder seemed to travel to their expected positions). Thus, band 1 was identified as vicilin, and these data indicated that pecan vicilin is a food allergen. The image of the membrane blotted with sera 1−4 (data not shown) revealed only very weak signal in the 10 M urea extraction sample, and none of these sera recognized rcCariv, indicating that no strong recognition of denatured pecan vicilin or other pecan proteins can be detected with Western blot. Whether these patient sera recognize conformational IgE epitopes of pecan vicilin warrants future studies.

Figure 1. Western blot showing recognition of pecan vicilin by sera from patients with pecan allergy. (A) Recombinant pecan vicilin structural core (lane rVc) and 10 M urea pecan extract (lane E) were separated with 4−20% SDS gels and transferred to PVDF membranes. Individual serum as indicated above each panel was used as primary antibody. The control was performed with otherwise identical procedures except that no serum was used. The far left panel is the image of the pecan extract separated with a 4−20% SDS gel and stained with CBB. The band positions of the reference bands in the EZ-Run Prestained Rec Protein Ladder (BP36031, Fisher Scientific) are indicated on the left side of the gel image with their respective molecular masses. Two of the bands indicated by arrowheads 1 and 2 were subjected to N-terminal peptide sequencing. (B) Recombinant pecan vicilin (lane rV) and its structural core (lane rVc) along with urea pecan extract (lane E) were separated with 4−20% SDS gels, transferred to PVDF membranes, and blotted as described above.

In a second set of Western blot experiments, raw pecan extract samples were separated by SDS gels along with rCariv and rcCariv and analyzed by Western blot with 20 patient sera. (Figure 1B). Five sera (sera 8, 11, 14, 16, and 18) contained IgE antibodies specific to pecan vicilin as shown by their recognition of rCariv and a band with similar size in the pecan protein extracts lane. Among these, all but one of them (seram 18) also recognized rcCariv, indicating that the linear epitope(s) recognized by serum 18 reside in the N-terminal flexible region of the mature protein. Other sera in this test either only recognized other protein bands but not vicilin (e.g., Figure 1B, right panel) or did not recognize any protein bands (data not shown). In short, 8 out of 27 sera tested reacted to pecan vicilin. Twenty-four percent (6 out of 25) of the sera from patients who were confirmed to be allergic to pecan by double-blind, placebo-controlled food challenge at Stanford C

DOI: 10.1021/acs.jafc.6b00884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry University recognized pecan vicilin. This revealed for the first time that pecan vicilin is a food allergen. The Structure of Pecan Vicilin. The refined structure gave R/Rfree values of 0.2020/0.2541 for all data to 2.65 Å (Table 2

molecules in the asymmetric unit, 8−10 residues at the Nterminal and 22−23 residues at the C-terminal could not be located in the electron density map and were not included in the refined structure. In addition, two flexible loops of 15−17 and 21−23 residues, respectively, were not in the structure due to the lack of electron density peaks. The final structure also included six 2-methyl-2,4-pentanediol, 21 water molecules, and 6 copper ligands. The assignment of the copper to the metal sites was based on sequence and structure alignment of pecan and Korean pine vicilins11 As expected, the most readily identifiable structure feature of pecan vicilin is the N-terminal and C-terminal domains related by a pseudodyad axis, each containing a cupin fold with a small protruded region composed of short helixes. Three vicilin molecules form a doughnut shaped trimer through head-to-tail association with their helical regions interlocked (Figure 3).

Table 2. X-ray Crystallographic Statistics and Refinement space group a, b, c (Å); α, β, γ (deg) resolution range (Å) R/Rfree (%) no. of reflections working set test set no. of residues/protein atoms no. of water molecules no. of ligand atoms Cu MPD av B factors (Å2) B-overall (from Wilson plot) (Å2) Molprobity scores all-atom clash score rotamer outliers (%) Ramachandran favored (%) Ramachandran outliers (%) rmsd from ideal geometrya bond lengths (Å) bond angles (deg)

P21212 42.69, 151.61, 342.78; 90.00, 90.00, 90.00 46.60−2.65 20.20/25.41 59078 2000 2143/16981 21 6 48 46.0 43.8 7.65 0.9 95.21 0.57

0.003 0.809

a

Empirical ideal geometry parameters are those derived by Engh and Huber.29

Figure 3. Trimeric structure of pecan vicilin with copper centers. The trimeric rcCariv is shown in a ribbon diagram with the monomers colored in red, blue, and green, respectively. In panels A and B, the trimer is shown in two orthogonal views. The copper atoms in all three monomers are shown in space-filling mode and colored in bronze.

This is similar to the structure of the 7S seed storage proteins from pine nut (4LEJ),11 peanut (PDB: 3S7I and 3SMH),25,26 soybean (PDB: 1UIK, 1IPK),30,31 adzuki bean (PDB: 2EAA),32 mung bean (PDB: 2CV6),33 kidney bean (PDB: 2PHL),34 and jack bean (PDB: 2CAU).35 Among these, only pine nut and pecan vicilin were copper proteins. All the other structures were vicilins from legumes and they did not contain a copper ligand, and the copper coordinating residues in pine nut and pecan vicilin were not conserved in vicilins from legumes based on multiple sequence alignments.11 Copper Center in Pecan Vicilin. The copper center of pecan vicilin consists of a cysteine (C652), 2 histidines (H654 and H698), and a tyrosine (Y379). The sulfur of C652, the ND1 of H654, and the NE2 of H698 form a trigonal planar structure with the copper ion in the center, but slightly away from the plane. The OH of Y379 provides the fourth copper coordination (Figure 4). This is similar to the copper center in Korean pine vicilin,11 but there are small variations in the geometry of the copper coordination. Although the coordination residues are not the same as in a typical type II copper center where four histidines bind to the copper from four corners of a pyramid, these vicilin copper centers are likely representatives of a divergent type II copper center. Proteins with diverse functions (including superoxide dismutase, amine oxidases, nitrite reductase, etc.) can utilize the property of type II copper centers to exert their activities.36 Whether vicilins

Figure 2. Structure of pecan vicilin. The crystallographic asymmetric unit contained two trimeric biological units which are represented by a ribbon diagram. In one of the biological units (left), the strands, helixes, and random coils are shown in magenta, cyan, and brown, respectively. In the other biological unit (right), chain A is shown in light gray, chain B in pink, and chain C in a rainbow coloring scheme with the N-terminus in blue and the C-terminus in red.

and Figure 2). The rmsd from ideal empirical values29 were 0.003 Å for bond lengths and 0.809° for bond angles, with no bond length outliers or bond angles deviating more than 6° from the “ideal” small molecule values.29 On the Ramachandran plot calculated with Molprobity Validation,18 in the final refined structure, there were six vicilin molecules in the crystallographic asymmetric unit with 2143 protein residues located in the electron density map. In the individual chains of the six protein D

DOI: 10.1021/acs.jafc.6b00884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(2) Garcia-Menaya, J. M.; Gonzalo-Garijo, M. A.; Moneo, I.; Fernandez, B.; Garcia-Gonzalez, F.; Moreno, F. A 17-kDa allergen detected in pine nuts. Allergy 2000, 55, 291−293. (3) Sampson, H. A.; Mendelson, L.; Rosen, J. P. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N. Engl. J. Med. 1992, 327, 380−384. (4) Yunginger, J. W.; Sweeney, K. G.; Sturner, W. Q.; Giannandrea, L. A.; Teigland, J. D.; Bray, M.; Benson, P. A.; York, J. A.; Biedrzycki, L.; Squillace, D. L.; et al. Fatal food-induced anaphylaxis. JAMA, J. Am. Med. Assoc. 1988, 260, 1450−1452. (5) Asero, R.; Ballmer-Weber, B. K.; Beyer, K.; Conti, A.; Dubakiene, R.; Fernandez-Rivas, M.; Hoffmann-Sommergruber, K.; Lidholm, J.; Mustakov, T.; Oude Elberink, J. N.; Pumphrey, R. S.; Stahl Skov, P.; van Ree, R.; Vlieg-Boerstra, B. J.; Hiller, R.; Hourihane, J. O.; Kowalski, M.; Papadopoulos, N. G.; Wal, J. M.; Mills, E. N.; Vieths, S. IgE-mediated food allergy diagnosis: Current status and new perspectives. Mol. Nutr. Food Res. 2007, 51, 135−147. (6) Beardslee, T. A.; Zeece, M. G.; Sarath, G.; Markwell, J. P. Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h 3. Int. Arch. Allergy Immunol. 2000, 123, 299−307. (7) de Leon, M. P.; Drew, A. C.; Glaspole, I. N.; Suphioglu, C.; O’Hehir, R. E.; Rolland, J. M. IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens. Mol. Immunol. 2007, 44, 463−471. (8) Sharma, G. M.; Irsigler, A.; Dhanarajan, P.; Ayuso, R.; Bardina, L.; Sampson, H. A.; Roux, K. H.; Sathe, S. K. Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan. J. Agric. Food Chem. 2011, 59, 4130−4139. (9) Sharma, G. M.; Irsigler, A.; Dhanarajan, P.; Ayuso, R.; Bardina, L.; Sampson, H. A.; Roux, K. H.; Sathe, S. K. Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan. J. Agric. Food Chem. 2011, 59, 9542−9552. (10) USDA. Economic Research Service. Fruit and tree nut data: yearbook tables. http://www.ers.usda.gov/data-products/fruit-and-treenut-data/yearbook-tables.aspx (Accessed: Sept 20, 2015). (11) Jin, T.; Wang, Y.; Chen, Y. W.; Fu, T. J.; Kothary, M. H.; McHugh, T. H.; Zhang, Y. Crystal structure of the Korean pine (Pinus koraiensis) 7S seed storage protein with copper ligands. J. Agric. Food Chem. 2014, 62, 222−228. (12) Lee, B.; Zhang, R.; Du, W. X.; Grauke, L. J.; McHugh, T. H.; Zhang, Y. Z. Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2014, 70, 1049−1052. (13) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 1997, 276, 307−326. (14) McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2005, 61, 458−464. (15) Storoni, L. C.; McCoy, A. J.; Read, R. J. Likelihood-enhanced fast rotation functions. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 432−438. (16) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; GrosseKunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213−221. (17) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (18) Lovell, S. C.; Davis, I. W.; Arendall, W. B., 3rd; de Bakker, P. I.; Word, J. M.; Prisant, M. G.; Richardson, J. S.; Richardson, D. C. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins: Struct., Funct., Genet. 2003, 50, 437−450. (19) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 1993, 26, 283−291.

Figure 4. Copper coordination in pecan vicilin. (A) A stereo view of a ball-and-stick representation of copper coordination in rcCariv is shown. Atom colors are gray, red, blue, yellow, and bronze for C, O, N, S, and Cu, respectively. The “bonding” between the copper ion and its coordinating atoms is shown as dotted lines. (B) The coordination geometry parameters about the copper. The orientation of the copper center and the coloring of the atoms are the same as those in panel A. The bond lengths between the copper ion and its coordinating atoms are shown with black numbers. The angles (in degrees) formed by two “bonds” connecting the copper ion and two of its coordinating atoms are shown in blue if Y379 is not involved in the “bonds”, otherwise in red.

from any tree nuts have a copper center dependent function warrants further investigations.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 510 559 5981. Fax: 510 559 5818. E-mail: yuzhu.zhang@ ars.usda.gov. Present Address §

B.L.: Division of Basic Science, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Funding

This research was supported by funds from the U.S. Department of Agriculture-Agricultural Research Service and by the Sean N. Parker Center for Allergy and Asthma Research at Stanford University. The Berkeley Center for Structural Biology was supported in part by the National Institutes of Health, National Institute of General Medical Sciences. The Advanced Light Source was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy, under Contract No. DE-AC02− 05CH11231. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

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ABBREVIATIONS USED Cariv, pecan (Carya illinoinensis) vicilin; CBB, coomassie brilliant blue; rCariv/rV, recombinant Cariv; rcCariv/rVc, recombinant structural core of Cariv

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REFERENCES

(1) Año,́ M. A.; Maselli, J. P.; Sanz, M. L.; Fernandez-Benitez, M. Allergy to pine nut. Allergol. Immunopathol. 2002, 30, 104−108. E

DOI: 10.1021/acs.jafc.6b00884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.6b00884 J. Agric. Food Chem. XXXX, XXX, XXX−XXX