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Agricultural and Environmental Chemistry

Identification and characterization of #-lathyrin, an abundant glycoprotein of grass pea (Lathyrus sativus L.), as a potential allergen Quanle Xu, Bo Song, Fengjuan Liu, Yaoyao Song, Peng Chen, Shanshan Liu, and Hari B. Krishnan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02314 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Journal of Agricultural and Food Chemistry

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Identification and characterization of β-lathyrin, an abundant glycoprotein of grass

2

pea (Lathyrus sativus L.), as a potential allergen

3 4

Quanle Xu1,2, Bo Song2,3, Fengjuan Liu1, Yaoyao Song1, Peng Chen1, Shanshan Liu3, and

5

Hari B. Krishnan2,4*

6 7

1

College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China

8

2

Plant Science Division, University of Missouri, Columbia, MO 65211

9

3

Key Laboratory of Soybean Biology at the Chinese Ministry of Education, Northeast

10

Agricultural University, Harbin, China

11

4

Plant Genetics Research, USDA-Agricultural Research Service, Columbia, MO 65211

12 13 14 15 16 17 18

Corresponding author’s address:

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*Dr. Hari B. Krishnan

20

Plant Genetics Research Unit

21

USDA-ARS, 108 Curtis Hall

22

University of Missouri, Columbia, MO 65211

23

E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Grass pea, a protein-rich, high-yielding and drought-tolerant legume, is

25

utilized as food and livestock feed in several tropical and subtropical regions of the

26

world. The abundant seed proteins of grass pea are salt-soluble globulins, which can be

27

separated into vicilins and legumins. In many other legumes, the members of vicilin seed

28

proteins have been identified as major allergens. However, very little information is

29

available on the allergens of grass pea. In this study, we have identified an abundant 47

30

kDa protein from grass pea which was recognized by IgE antibodies from sera drawn

31

from several peanut-allergic patients. The IgE-binding 47 kDa protein was partially

32

purified by affinity chromatography on a Con-A sepharose column. MALDI-TOF mass

33

spectrometry analysis of the 47 kDa grass pea protein revealed sequence homology to 47

34

kDa vicilin from pea and Len c 1 from lentil. Interestingly the grass pea vicilin was found

35

to be susceptible to pepsin digestion in vitro. We have also isolated a cDNA encoding the

36

grass pea 47 kDa vicilin (β-lathyrin) and the deduced amino acid sequence revealed

37

extensive homology to several known allergens including those from peanut and soybean.

38

A homology model structure of the grass pea β-lathyrin, generated using the X-ray

39

crystal structure of the soybean β-conglycinin β-subunit as a template, revealed potential

40

IgE-binding epitopes located on the surface of the molecule. The similarity in the three-

41

dimensional structure and the conservation of the antigenic epitopes on the molecular

42

surface of vicilin allergens explains for the IgE-binding cross-reactivity.

43 44

KEYWORDS: Allergen; β-lathyrin; grass pea; storage protein


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INTRODUCTION

46

Grass pea (L. sativus L.) is an important legume that is cultivated as food and forage crop

47

in North Africa, Near East, Western Asia and the Indian subcontinent.1-3 This under-

48

utilized legume can withstand harsh environmental conditions including drought and

49

flooding. On account of these characteristics grass pea is often grown in drought-prone

50

areas.3 Like other legumes, grass pea seeds are relatively rich in protein (about 20% of

51

the seed dry weight) and can serve as economic source of highly nutritious and balanced

52

source of human dietary protein. However, its use as a dietary protein source is limited

53

due to the presence of the neurotoxic nonprotein amino acid β-N-oxalyl-L-α,β-

54

diaminopropionic acid (β-ODAP). Prolonged consumption of grass pea as a sole protein

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source leads to paralysis of lower limbs, a condition referred to as lathyrism.4,5 The

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concerted efforts of international research organizations have resulted in the development

57

of improved varieties that contain lower levels of β-ODAP.6,7 Such improved grass pea

58

varieties are vital for reducing incidence of lathyrism among subsistence poor farmers in

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counties that regularly have harsh environmental conditions.

60

Unlike the major legumes, little is known about the seed storage proteins of grass pea.

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Though several early studies have been reported,8-11 it was only in the year 2000 a

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detailed biochemical investigation of grass pea seed proteins was initiated.12 It was

63

reported that the protein content of grass pea was about 20% of the seed dry weight.

64

Globulin and albumin fractions represented 60 and 30% of the total seed proteins,

65

respectively. The globulins were grouped into α-lathyrin, β-lathyrin, and γ-lathrin. The

66

α-lathyrin, the most abundant globulin of grass pea seeds, is represented by three proteins

67

that were further subjected to proteolytic processing resulting in larger and small



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subunits, similar to the 11S globulins of other legumes.12 The β-lathyrin (7S globulin) is

69

made up of several polypeptides of different molecular weights (14-66 kDa). At least

70

two of the subunits of β-lathyrin are glycosylated and one of them contains a disulfide

71

bond.12 Thus, the seed storage protein composition of grass pea is similar to that of other

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well characterized legumes, though slight changes in the sedimentation coefficients are

73

evident.12 Immunological studies further confirm the similarity between lupin conglutins

74

and grass pea lathyrins in subunit and polypeptide composition.12

75

Allergic reactions to legume seed protein (especially peanut, soybeans, lentil,

76

chickpea and pea) have been widely reported in the literature.13 In Mediterranean and

77

Asian countries, allergic reactions to the ingestion of lentil, chickpea, and pea are

78

prevalent. For example, 10% of the Spanish pediatric population was reported to be

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allergic to lentil proteins.14 The major allergen of lentil has been identified as vicilin (Len

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c 1), a member of 7S globulin.15 Interestingly, the 7S globulin members are also most

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dominant allergens in peanut and soybean.16-18 In contrast to other legumes, very little is

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known about the allergenic potential of grass pea seed proteins. In sensitized individuals,

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grass pea can induce symptoms of food allergy as well as occupational-induced allergies

84

such as bronchial asthma, rhinoconjunctivitis, facial oedema, and generalized urticaria.19-

85

23

86

present in the blood from grass pea allergic patients.21 Subsequently, a 24 kDa allergenic

87

protein from grass pea was purified and crystallized.23 This allergenic protein was

88

identified as a member of 2S albumin and showed 85% sequence homology with the 2S

89

albumin of Pisum sativum. Since grass pea is often the only crop that poor farmers are

90

able to depend on to survive in extremely drought-prone growing regions, it is critical to

Three polypeptides (sized 46, 28, and 21 kDa) were recognized by IgE antibodies


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identify and characterize potential allergens present in seeds of grass pea. Here, we have

92

identified and characterized a 47 kDa IgE-binding protein from grass pea.

93 94

MATERIALS AND METHODS

95

Seed materials. Dry seeds of L. sativus cv. LZ(2) were harvested in 2016 from an

96

experimental farm of Northwest A&F University, China. Seed of soybean (Glycine max

97

cv Williams 82) were obtained from Missouri Crop Improvement Association, Columbia,

98

MO. Seeds of Spanish peanut (Arachis hypogaea) was purchased from a local grocery

99

store, Columbia, MO. All seeds were stored at 4 °C until used.

100

Patient’s Sera. Sera from eight patients with documented allergy to peanut were

101

obtained from IBT Laboratories (Lenexa, KS). ImmunoCAP® FEIA assay, which has been

102

approved for diagnostic use by the U.S. Food and Drug Administration, revealed 175.0 - 50.9

103

kU/L peanut-specific IgE in the sera of these patients. Clinical symptoms of these

104

patients ranged from impaired breathing, itchy skin, nausea and runny nose. Four of eight

105

peanut allergic patients also contained IgE specific to soybean flour, as determined by the

106

ImmunoCAP® FEIA assay. Serum from a health individual with no known history of

107

allergy was also acquired from IBT Laboratories.

108

Protein

isolation

and

Sodium

Dedecyl

Sulfate−Polyacrylamide

Gel

109

Electrophoresis (SDS-PAGE). Dry seeds were ground to a fine powder in a mortar and

110

pestle. Then 20 mg of seed powder was transferred to a 2 mL microcentrifuge tube and 1

111

mL of sodium dodecyl sulfate (SDS)-sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS

112

(w/v), 10% glycerol (v/v), and 5% 2-mercaptoethanol (v/v)) was added. The contents of

113

each tube were vigorously vortexed for 10 min at room temperature followed by boiling



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for 5 min. Each sample was then subjected to centrifugation at 15,800g for 5 min. The

115

clear supernatant was collected and designated as total seed protein fraction. Seed

116

proteins were then resolved by SDS-PAGE gels using a Hoeffer SE 250 Mini-Vertical

117

electrophoresis apparatus (GE Healthcare, Pittsburg, PA, U.S.A.), and ~50 µg of seed

118

proteins were loaded onto a 13.5% acrylamide gel. Separation of proteins was achieved

119

with a constant 20 mA/gel until the tracking dye reached the bottom of the gel. Gels were

120

then removed from the cassette and stained overnight with Coomassie Blue R-250.

121

Immunoblot Analysis. Seed proteins were first resolved by SDS-PAGE and then

122

electrophoretically transferred to nitrocellulose membranes (Protran, Schleicher &

123

Schuell Inc., Keene, NH) as previously described.24 Membranes were blocked with 5%

124

non-fat milk in Tris-buffer saline (TBS, pH 7.3) for 1 h at room temperature. Following

125

this step, the nitrocellulose membranes were incubated in a 1:500 dilution of serum from

126

peanut allergic patients (IBT Laboratories, Lenexa, KS) overnight at room temperature

127

with gentle rocking. After washing (four times with TBS containing 0.05% Tween-20,

128

TBST, 10 min/each), each membrane was incubated for 2 h in a 1:5000 dilution of goat

129

anti-human IgE-horseradish peroxidase conjugate antibody (Biosource, Camerillo, CA).

130

IgE-binding polypeptides were then detected using an enhanced chemiluminescent

131

substrate (Super Signal West Pico trial kit; Pierce Biotechnology, Rockford, IL)

132

according to the manufacturer's protocol.

133

Con-A-Sepharose Affinity Chromatography. Five grams of dry grass pea seed was

134

ground to a fine powder using a mortar and pestle. To the seed powder, 50 mL of 50 mM

135

Tris-HCl, pH 7.5, 150 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride was added.

136

The mixture was then placed in an orbital shaker (160 rpm, 30 C) for 30 min. The slurry


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was clarified by centrifugation at 13,320g for 20 min. To the resulting clear supernatant

138

solid, (NH4)2SO4 was added gradually to achieve 70% saturation. The precipitated

139

proteins were then recovered by centrifugation as described previously. The resulting

140

protein pellet was resuspended in 20 mL of dialysis buffer (100 mm sodium phosphate

141

(pH 6.5), 1 mM MnCl2, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, and 0.5 M NaCl), and

142

the concentrated protein solution was dialysed overnight against the same buffer at room

143

temperature. The dialyzed protein solution was then loaded onto a column (1 x 16 cm) of

144

Concanavalin A-Sepharose (Sigma Chemicals, St. Louis, MO), and the column was

145

washed extensively with dialysis buffer and then subjected to a step gradient of 0 to 250

146

mM α-methyl manoside in dialysis buffer. Fractions of 5 mL were collected, and

147

alternative fractions were subjected to SDS-PAGE analysis.

148

MALDI-TOF Mass Spectrometry Analysis. Partially purified grass pea vicilin

149

fractions obtained from ConA-sepharose chromatography were subjected to SDS-PAGE.

150

The 47 kDa IgE-binding protein was excised from the acrylamide gel, washed in distilled

151

water and then destained in a 50% solution of acetonitrile (v/v) containing 25 mM

152

ammonium bicarbonate. After destaining, a 100% acetonitrile wash was performed, and

153

the 47 kDa protein was digested with 20 µL (10 µg/mL) of modified porcine trypsin in 25

154

mM ammonium bicarbonate (Promega, Madison, WI). The peptides resulting from

155

tryptic digestion were then analyzed by mass spectroscopy as previously described.24

156

Enzymatic Deglycosylation of Grass Pea Allergen. Deglycosylation of the grass

157

pea 47 kDa vicilin was carried out using GlycoPro Enzymatic Deglycosylation Kit

158

(Prozyme, Hayward, CA) per the manufacturer's instructions. The kit includes N-

159

Glycanase,

sialidase

A,

O-Glycanase,

β(1-4)


Glactosidase

and

β-N-


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Acetylglucosaminidase, which removes all N-linked glycans and simple O-linked glycans

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from glycoproteins. About 100 µg of ConA-sepharose purified vicilin fraction was

162

denatured and deglycosylated following the manufacturer’s protocol. The efficiency of

163

the deglycosylation was verified by a shift in protein mobility on a 10% SDS-PAGE gel.

164

Protease Digestion of Grass Pea Allergen. The stability of grass pea 47 kDa vicilin

165

to pepsin digestion was carried out as described previously.25,26 About 20 µg of the

166

ConA-sepharose purified vicilin fraction was digested with pepsin (Sigma), at one of

167

several time intervals at 37 °C in simulated gastric fluid (SGF): Aliquots of samples were

168

removed after 30 seconds, 1 min, 5 min, 30 min and 60 min. To these aliquots 30 µL of

169

Na2CO3 (200 mM), 34 µL of 6× SDS−sample treatment buffer (350 mM Tris-HCl, 10%

170

SDS, 30% glycerol, and 175 mM bromophenol blue, pH 6.8), and 10 µL of β-ME were

171

added, mixed, and then boiled for 5 min. Controls included pepsin in SGF without added

172

protein or protein samples in SGF that did not contain pepsin. The generation of

173

proteolytic fragments due to pepsin digestion was examined by SDS-PAGE and

174

immunoblot analysis.

175

Homology-Model of the 47 kDa vicilin from Grass Pea. Utilizing the three-

176

dimensional structure model of the homotrimeric soybean β-conglycinin β-subunit27 as a

177

template, a homology model of the grass pea 47 kDa vicilin was build. The homology

178

model was generated as a homotrimer and energy minimized using the Swiss-Model

179

website (http://swissmodel.expasy.org//SWISS-MODEL.html).28 The stereochemistry

180

and overall quality of the homology model were examined via the program Procheck.29

181

RESULTS AND DISCUSSION


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Immunoglobulin E cross-reactivity of grass pea seed proteins with sera from

183

peanut allergic persons. Allergy to peanuts is a major public health concern in the

184

United States. It has been estimated that about 2% of US children have peanut allergy.

185

The official allergen database (htpp:/www.allergen.org) has recognized twelve peanut

186

allergens. Two of them belong to the cupin (Ara h 1, Ara h 3), four to the prolamin (Ara h

187

2, Ara h 6, Ara h 7, Ara h 9), one to the profiling (Ara h 5), one to the Bet v 1 (Ara h 8),

188

two to the glycosyl transferease GT-C (Ara h 10, Ara h 11) and two to the scorpion-like

189

knottin (Ara h 12, Ara h 13) superfamilies.30 Interestingly, peanut allergic patients also

190

develop clinically relevant sensitization to several different legumes such as soybean,

191

pea, lupin and lentil.30 Allergic reactions to consumption of legumes such as lupin, lentil,

192

chickpea and pea are commonly reported in Mediterranean and Asian countries. Serum

193

from persons with peanut and/or soybean allergy have been used to demonstrate IgE

194

cross-reactivity with seed proteins from some of these legumes.30-33 To verify if the sera

195

from peanut allergy patients can cross-react with seed proteins of grass pea, we

196

performed immunoblot analysis utilizing serum from 8 different patients with peanut

197

allergies (Figure 1). As expected, peanut allergy sera from all the eight patients

198

recognized several allergens from the peanut protein extract. The sera also cross-reacted

199

with both soybean and grass pea proteins. Serum from 6/8 peanut allergic subjects

200

strongly reacted against grass pea proteins while no cross-reactivity was detected from

201

the sera from another two patients. In the case of grass pea, a protein with apparent

202

molecular weight of 47 kDa cross-reacted with sera from most of peanut allergic subjects.

203

In addition to 47 kDa immunodominant protein, Ig-E cross-reactivity against few other

204

proteins were also detected from grass pea protein extract (Figure 1).



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Identification of grass pea 47 kDa IgE binding protein as β -lathyrin. The molecular

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weight and relatively high abundance of grass pea 47 kDa IgE binding protein suggested

208

that this protein may belong to 7S vicilin family. Native vicilin are present as trimers with

209

molecular weight ranging from 150 – 210 kDa and are encoded by multigene families.34

210

Vicilins fall into two groups, the first made of subunits with molecular weights of

211

approximately 50 kDa, while the second group includes 60 – 70 kDa proteins.34 These

212

two group of proteins are largely homologous except that the first group bears a N-

213

terminal extension of variable length which results in higher molecular weights.34 Vicilin

214

are subjected to posttranslational modifications (glycosylation and proteolysis) leading to

215

variation in their subunit molecular weights. Most of these glycosylated proteins are

216

potential allergens and are resistant to digestion and food processing.35 In the case of

217

grass pea, it has been previously shown that some grass pea 7S globulins are

218

glycoproteins.12 Therefore, we utilized Con-A Sepharose affinity chromatography to

219

purify the 47 kDa IgE-binding protein from grass pea. When Tris-buffered saline (TBS)

220

extracted grass pea proteins were loaded on a Con-A column, several of the abundant

221

seed proteins did not bind to the Con-A column (Figure 2).

222

A 47 kDa protein, which was initially retained on the Con-A column, was eluted with

223

approximately 50 mM α-methylmanoside. SDS-PAGE analysis of 50 mM α-

224

methylmanoside eluted protein fraction revealed an abundant protein with an estimated

225

molecular weight of 47 kDa and several other less abundant proteins (Figure 2).

226

Antibodies raised against soybean β-subunit of β-conglycinin, a glycoprotein, strongly

227

reacted against the 47 kDa protein (Figure 2). Similarly, western blot analysis using


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pooled serum from peanut allergy patients also reacted strongly against the 47 kDa

229

protein. We excised the IgE-binding SDS-PAGE separated 47 kDa protein, digested it

230

with trypsin and subjected it to MALDI-TOF-MS (Table 1). Using Mascot, the

231

empirically determined mass-to-charge ratios of the peptides were then compared to

232

known peptides present in the National Center for Biotechnology Information

233

nonreduandant database. Three peptides from the 47 kDa protein gave statistically

234

significant protein scores for matches with Len c 1, a major allergen from lentil seeds

235

(Table 1). Similarly, the same three peptides gave significant MOWSE score matches

236

against a hypothetical protein from clover (Trifolium subterraneum) and pea vicilin

237

(Table 1). Blast analysis of this hypothetical protein revealed significant amino acid

238

sequence homology to vicilins from several legumes: Medicago truncatula, Cicer

239

arietinum, Pisum sativum, Glycine max as well as convicilins from Vicia narbonensis,

240

Pisum sativum and Lotus japonicas.

241

Our laboratory is also analyzing the expression profiles of grass pea genes by RNA

242

sequencing and have generated in-house grass pea transcriptome database. A search of

243

this database with Medicago truncatula vicilin sequences resulted in the identification of

244

a transcript encoding the 47 kDa β-lathyrin. The deduced amino acid sequence of

245

identified cDNA encodes a truncated protein with apparent molecular weight of 42 kDa.

246

Bioinformatic analysis indicated that the grass pea β-lathyrin belongs to the cupin

247

superfamily (Figure 3). Blast analysis revealed that grass pea β-lathyrin shares extensive

248

amino acid sequence homology with 47 kDa vicilin from Pisum sativum and Medicago

249

truncatula, a hypothetical protein from Medicago truncatula, allergen Len c 1 from Lens

250

culinaris and several other legume 7S globulin family members. (Supplementary Figure



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1). A search of the amino acid sequence of the 47 kDa grass pea β-lathyrin revealed exact

252

matches to several trypsin digested peptide sequences generated from the 47 kDa IgE-

253

binding peptide (Figure 3). Based on these analyses, the 47 kDa IgE binding protein from

254

grass pea has been identified as β-lathyrin.

255 256

Carbohydrate moiety of the grass pea β-lathyrin is not responsible for the

257

immunoreactivity.

258

Glycosylation is a common feature to many food allergens.35 It is believed that glycan

259

moieties

260

immunogenicity.36,37 Even though antibodies specific to carbohydrate determinants are

261

commonly found, they appear not to have clinical relevance.35 However, anaphylaxis

262

induced by carbohydrate epitopes has been reported.38 To evaluate the role of the

263

carbohydrate moiety in IgE-binding, partially purified grass pea β-lathyrin was subjected

264

to deglycosylation (Figure 4).

265

The product of enzymatic deglycosylation were then analyzed by SDS-PAGE which

266

revealed two prominent proteins of 47 and 45 kDa proteins. The 47 kDa and 45 kDa

267

proteins presumably represent the glycosylated and the deglycosylated form of β-lathyrin

268

(Figure 4). The removal of the carbohydrate moiety caused a decrease in molecular

269

weight of the β-lathyrin; protein corresponding to the same size was not detected in the

270

control lane (Figure 4). Western blot analysis, using pooled serum from peanut allergy

271

patients, revealed IgE binding to both native and deglycosylated proteins (Figure 4). This

272

observation indicates that the carbohydrate moiety of β-lathyrin may not be critical for

273

IgE-binding. Interestingly, antibodies raised against the soybean beta subunit of β-

may exert

a stabilizing effect

on

protein structure and


enhance

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conglycinin (which is also a glycoprotein) recognized the native but not the

275

deglycosylated β-lathyrin (Figure 4). The reason why soybean β-conglycinin specific

276

antibodies did not react against the deglycosylated β-lathyrin is not clear. It is likely that

277

that soybean β-conglycinin antibodies are mainly targeted to the core oligosaccharides

278

covalently attached to β-conglycinin and β-lathyrin. Soybean β-conglycinin contains N-

279

linked mannose-containing core oligosaccharides39 and the strong cross-reactivity of the

280

soybean β-conglycinin antibodies against the β-lathyrin indicate the presence of similar

281

core oligosaccharides bound to the grass pea β-lathyrin.

282

Grass pea β -lathyrin is susceptible to pepsin digestion. It is generally believed that

283

food allergens can survive the acidic gastric environment as they are resistant to pepsin

284

digestion.40 On account of this, digestion assays in simulated gastric fluid (SGF) are

285

routinely employed to predict the allergenic potential of food proteins. An examination of

286

the stability of 25 different food proteins to pepsin digestion demonstrated that allergens

287

have greater stability to protease digestion.26 To examine if grass pea β-lathyrin is also

288

stable to digestion, partially purified proteins were subjected to pepsin digestion in vitro

289

(Figure 5).

290

Incubation of the β-lathyrin with pepsin resulted in rapid breakdown of the mature β-

291

lathyrin and the appearance of proteolytic products within 30 seconds. By the end of 60

292

min the β-lathyrin was completely digested (Figure 5). Western blot analyses using

293

antibodies raised against soybean β-conglycinin antibodies or pooled serum from peanut

294

allergy patients failed to detect either mature convicilin or the breakdown products after 1

295

min pepsin digestion (Figure 5). Earlier studies have shown that soybean Kuntiz trypsin

296

inhibitor (KTi), a potent allergen capable of inducing food anaphylaxis,41 has



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demonstrated remarkable stability to both thermal and acid denaturation.42 For

298

comparison, we examined the stability of KTi to pepsin digestion under the experimental

299

conditions used to test the stability of β-lathyrin. Unlike grass pea β-lathyrin, soybean

300

KTi was stable even up to 60 min digestion using pepsin (Figure 5). However, resistance

301

to pepsin digestion alone can’t be used as criteria for predicting the allergenicity of a

302

protein. Previous studies have established that some well-known allergens are also

303

digested by pepsin within a few seconds.43,44

304

Identification of IgE-binding Epitopes on the Modeled Structure of Grass Pea β -

305

Lathyrin. A homology model structure of the grass pea β-lathyrin was generated using

306

the X-ray crystal structure of the soybean β-conglycinin β-subunit (SWISS-MODEL

307

Template Library ID: 1ipk.1.A) core domain (residues 49-397) as a template.45 The core

308

domain of the soybean β-conglycinin β-subunit and grass pea β-lathyrin share 59.4%

309

amino acid sequence identity over the region included in the X-ray structure. The Global

310

Model Quality Estimation (GMQE) and Qualitative Model Energy ANalysis (QMEAN)

311

scores were 0.75 and -1.38, respectively, which suggested a good quality of homology

312

model structure. Figure 6 shows the ribbon diagrams of the grass pea β-lathyrin. The

313

modeled structure of grass pea β-lathyrin is consistent with those of vicilin family

314

members.45 Grass pea β-lathyrin model structure is very similar to the structures of the 7S

315

seed storage proteins from soybean,45 peanut,46,47 adzuki bean,48 kidney bean,50 jack

316

bean,51 and pecan.52 IgE-binding epitopes within grass pea β-lathyrin was also identified

317

utilizing AlgPred server.53 This computational method identified a single IgE binding

318

epitope (ELHLLGFGIN), and predicted grass pea β-lathyrin as an allergen. Similarly, a

319

BLAST search of β-lathyrin against 2890 allergen-representative peptides also confirmed


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the

existence

of

an

allergenic-representative

321

(PHFNSKAMVIVVVNKGTGNLELVA) in β-lathyrin. The NetMHCⅡ 2. 2 Server was

322

used to measure the binding ability between β -lathyrin and MHC II, and results suggested

323

that β-laythrin contains nine potential IgE-binding epitopes. These potential epitopes,

324

named T1-T9, are highlighted by different colors in one the monomers (Figure 6).

325

Epitopes T2, T6, T7 and T8 were located in the conserved domain of Cupin superfamily

326

proteins (Fig 3). Epitope T8 is common in other legume allergens, such as Ara h 1, Gly m

327

5, Len c 1, Lup an 1, Pis s 2, and Vig r 2. In contrast T4 is only shared with Len c1.

328

Epitope T2 was specific for β-lathyrin and was not found in other species. The

329

conservation of highly similar IgE-binding epitopes among different legume allergens

330

may explain the IgE-cross-reactivity of sera from peanut sensitive patients to grass pea β-

331

lathyrin.

332

In conclusion, we have identified grass pea β-lathyrin as a potential allergenic protein.

333

Most of the IgE-binding epitopes of β-lathyrin identified in this study were found to be in

334

common to other legume allergens and located on the predicted protein surface. The

335

conservation of IgE-binding epitopes among several distinct legume allergens may be

336

responsible for the observed phenomenon of cross-reactivity. Additional studies, leading

337

to a better understanding of allergenicity potential of grass pea β-lathyrin, will aid in the

338

design and development of effective immunotherapy strategies for the diagnosis and

339

treatment of grass pea-related food allergy.

340 341

ACKNOWLEDGEMENTS


peptide


Page 16 of 31

Page # 16 342

This research was supported by the USDA-Agricultural Research Service, the Chinese

343

Universities Scientific Fund (2014YB040), China Postdoctoral Science Foundation

344

(2016M590975),

345

(2016BSHEDZZ119), P.R. China. Mention of a trademark, vendor, or proprietary

346

product does not constitute a guarantee or warranty of the product by the USDA and does

347

not imply its approval to the exclusion of other products or vendors that may also be

348

suitable.

Postdoctoral

Science

Foundation

of

Shaanxi

province

349 350

Supporting Information

351

Amino acid sequence alignment of grass pea β-lathyrin with several other legume 7S

352

globulin family members, and phylogenetic analysis of known food allergens.

353

REFERENCES

354

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Lathyrus sativus (grass pea) and its neurotoxin ODAP. Phytochemistry 2006, 67,

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protein of mung bean. Acta Crystallogr. Sect D Biol. Crystallogr. 2006, 62, 824–

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(53) Saha, S.; Raghava, G. P. S. (2006) AlgPred: prediction of allergenic proteins and mapping of IgE epitopes. Nucleic Acids Res. 2006, 34, W202–W209.

504 505 506 507

Figure captions

508

Figure 1. SDS-PAGE and IgE cross-reactivity of grass pea proteins. Panel A: Total seed

509

proteins extracted from seeds of peanut (lane 1); soybean (lane 2) and grass pea (lane 3)

510

were separated by 13.5 % SDS-PAGE gel. Resolved proteins were detected by staining

511

the gel with Coomassie Blue. Panel B – I: Western blot analysis showing cross-reactivity

512

of grass pea proteins with sera from patients with peanut allergy. Each panel was

513

incubated with sera from individual peanut-allergic patients. Cross-reacting proteins were

514

identified using goat anti-human IgE-horseradish peroxidase conjugate antibody followed



Page 22 of 31

Page # 22 515

by chemiluminescent detection. Lane M contains the molecular weight markers whose

516

sizes in kDa are shown on the left side of the figure.

517

Figure 2. Purification of 47 kDa IgE-binding grass pea protein. Panel A: Grass pea

518

protein were separated by 10 % SDS-PAGE and visualized by staining with Coomassie

519

Blue (Panel A) or electrophoretically transferred to nitrocellulose membrane and

520

incubated with pooled sera from several peanut-allergic patients (Panel B), or soybean β-

521

conglycinin antisera (Panel C). Immunoreactive proteins were identified using goat anti-

522

human IgE-horseradish peroxidase conjugate or anti-rabbit IgG-horseradish peroxidase

523

conjugate followed by chemiluminescent detection. Lane 1, TBS extracted proteins from

524

grass pea; lane 2, Con-A Sepharose unbound proteins; lane 3, 50 mM α-methylmanoside

525

eluted proteins. The numbers on the left side of the figure indicates the sizes of protein

526

standards in kDa.

527

Figure 3. Schematic representation of grass pea β-lathyrin coding sequences showing

528

Cupin-1 domains (Panel A) and amino acid regions that match with the trypsin digested

529

peptide sequences from the 47 kDa IgE-binding protein (Panel B).

530

Figure 4. SDS-PAGE/immunoblot analysis of enzymatically deglycosylated grass pea

531

β-lathyrin. Partially purified native (lane 1) and deglycosylated (lane 2) grass pea β-

532

lathyrin were separated by SDS-PAGE on a 10% gel and stained with Coomassie Blue

533

(Panel A), electrophoretically transferred to nitrocellulose membrane and challenged with

534

pooled sera from peanut allergic patients (Panel B), or soybean β-conglycinin antisera

535

(Panel C). Immunoreactive proteins were identified using goat anti-human IgE-

536

horseradish peroxidase conjugate or anti-rabbit IgG-horseradish peroxidase conjugate

537

followed by chemiluminescent detection. The numbers on the left side of the figure


Page 23 of 31


Page # 23 538

indicates the sizes of protein standards in kDa.

539

Figure 5. SDS-PAFE analysis of pepsin digested grass pea β-lathyrin and soybean

540

Kunitz trypsin inhibitor. Partially purified grass pea β-lathyrin (Panel A) and

541

commercially purchased Kuntiz trypsin inhibitor (Panel C) were digested with pepsin for

542

30 seconds, 1 min, 5 min, 30 min and 60 min and the products were fractionated by SDS-

543

PAGE on a 15% gel and stained with Coomassie Blue. Grass pea pepsin (Panel B)

544

digested β-lathyrin was electrophoretically transferred to nitrocellulose membrane and

545

challenged with pooled sera from peanut allergic patients. Immunoreactive proteins were

546

identified using goat anti-human IgE-horseradish peroxidase conjugate or anti-rabbit

547

IgG-horseradish peroxidase conjugate followed by chemiluminescent detection. The

548

numbers on the left side of the figure indicates the sizes of protein standards in kDa. Lane

549

M, protein markers; lane 1, pepsin in the absence of β-lathyrin, 0-time point; lane 2,

550

pepsin in the absence of β-lathyrin, 60 min time point; lane 3, β-lathyrin in the absence

551

pepsin, 0-time point; lane 4, β-lathyrin in the absence pepsin 60 min time point; lane 5, 6,

552

7, 8, and 9 contain pepsin digested protein samples that were removed at 30 seconds, 1

553

min, 5 min, 30 min and 60 min, respectively.

554

Figure 6. Homology-modeled structure of grass pea β-lathyrin. The trimeric grass pea β-

555

lathyrin is shown in a ribbon diagram with each monomer colored in red, blue and green

556

(Panel A). Potential IgE-binding epitopes of β -laythrin (Panel B). Panel C shows the

557

structural features corresponding to the 9 predicated antigenic epitopes, which are colored

558

following

the

color

code

shown


in

panel

B.


Page 24 of 31

Page # 24

Table 1. MALDI-TOF-MS identification of the grass pea 47 kDa protein.

Protein ID

MOWSE

Peptides Matched (Sequences) ---% Coverage

Allergen Len c 1.0101, partial

249

3 (3) ---10 %

255

3 (3) ---9%

GAU31893.1

(41-53) (245-256) (394-412)

205

3 (3) ---7%

VCLC PEA

(43-55) (66-74) (246-257)

(Lens culinarus) Hypothetical protein TSUD_270780

(Trifolium subterraneum) Vicilin OS

(Pisum sativum)

CAD87730.1

(16-28) (219-230) (368-386)

R.FQTLFENENGHIR.L K.SVSSESEPFNLR.N R.NFLAGEEDNVISQIQRPVK.E

71 80 98

R.FQTLFENENGHIR.L K.SVSSESEPFNLR.N R.NFLAGEEDNVISQIQRPVK.E

78 80 98

K.FQTLFENENGHIR.L K.IFENLQNYR.L K.SVSSESEPFNLR.S

78 48 80


(Position) Peptide Matched

Peptide Score

Accession

Page 25 of 31


Page # 25

Figure 1

M

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

1

2

97 — 97 —

66 —

66 — 45 — 45 — 31 — 31 — 21 — 21 — 14 — 7—

A

B

C

D

E


F

G

H

I

3


Page 26 of 31

Page # 26 Figure 2

M

1

2

3

1

2

3

1

2

3

97 — 66 —

45 —

31 —

21 —

A

B


C

Page 27 of 31


Page # 27

Figure 3

β-lathyrin Specific hits Superfamilies

YRLLEYKSKP HTLFLPQYTD ADFILVVLSG KAILTVLNSN DRNSFSLERG DTIKIPAGTI 70 80 90 100 110 120 AYLANRDDNE DLRVLDLAIP VNKPGQLQPF LLSGTQNQPS LLSGFSKKVL EAAFNTNYEE 130 140 150 160 170 180 IEKVLLEQQE QEPQHRRSLK DRRQEINEEN VIVKVSREQI EELSKNAKSS SKKSVSSESE 190 200 210 220 230 240 PFNLRSRNPI YSNKFGKFFE ITPEKNQQLQ DLDIFVNSVE IKEGSLLLPN YNSRAIVIVT 250 260 270 280 290 300 VNEGKGDFEL LGIRNENQRE ESDEEEEQEE ETSKQVQRYR AKLSPGDVFV IPAGHPVAIN 310 320 330 340 350 360 ASSNLNLIGF GINAENNQRN FLAGEEDNVI SQIQRPVKEL VFPGSSREVD KLLKNQRQSY 370 FANAQPLQRE



Page 28 of 31

Page # 28

Figure 4

M

1

2

1

2

1

2

97 —

66 —

45 —

31 —

A

B


C

Page 29 of 31


Page # 29

Figure 5

M 1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

7

8

9

M 1

2

3

4

5

97 — 66 — 45 —

31 —

21 —

14 — 7—

A

B


C

6

7

8

9


Page 30 of 31

Page # 30

Figure 6

A

90 °

B

C

T1:YRLLEYKSK; T2:LLSGFSKKV; T3:KVLLEQQEQ; T4:HRRSLKDRRQ; T5:IVKVSREQI; T6:LRSRNPIYSNKF; 7:TSKQVQRYRAKLS; T8:FLAGEEDNVISQIQRPV; T9:LLKNQRQSYFANA

180 °

180°


Page 31 of 31


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