Soybean P34 Probable Thiol Protease Probably Has Proteolytic

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Soybean P34 probable thiol protease probably has proteolytic activity on oleosins Luping Zhao, Xiangzhen Kong, Caimeng Zhang, Yufei Hua, and Yeming Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02190 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on July 1, 2017

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Journal of Agricultural and Food Chemistry

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Soybean P34 probable thiol protease probably has proteolytic activity on oleosins

2 Luping Zhao, Xiangzhen Kong, Caimeng Zhang, Yufei Hua, Yeming Chen*

3 4 5

State Key Laboratory of Food Science and Technology, Jiangnan University, School of Food

6

Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122,

7

PR China

8 9

Corresponding Author:

10

Yeming Chen

11

Telephone/Fax: 86-510-85329091. E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship

13

with the protease of 24 kDa oleosin of soybean oil bodies. In this study, nine days germinated

14

soybean was used to separate bio-processed P34 (P32) from bio-processed 30K (28K).

15

Interestingly, P32 existed as dimer, whereas 28K as monomer; P32-rich sample had proteolytic

16

activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas 28K-rich sample

17

showed low proteolytic activity; P32-rich sample contained one thiol protease. After mixing with

18

purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24

19

kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two

20

hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was

21

hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that

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P32 was the protease.

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Keywords: oil bodies; oleosins; thiol protease; P34 probable thiol protease; Gly m Bd 30K

2


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INTRODUCTION

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In traditional soymilk processing, soybean cell microstructure is disrupted by grinding and various

26

components (i.e., proteins, oil bodies, and enzymes) are released into aqueous phase. At the same

27

time, many proteins and enzymes, including β-conglycinin, glycinin, P34 probable thiol protease

28

(P34), Gly m Bd 30K (30K), β-amylase, phospholipidase D, and lipoxygenase, are bound to oil

29

bodies (OBs),

30

phospholipids and oleosins.3 Some biochemical reactions occur on OBs before thermal treatment,

31

i.e., oleosins are hydrolyzed by endogenous protease,1 and phospholipids are hydrolyzed by

32

phospholipidase D.2 As a result, some “wounds” are formed on the surface of OBs. In nature, the

33

oleosins provide the OBs with physical and chemical protection against environmental stresses,

34

such as moisture, temperature ﬂuctuations, and the presence of oxidative reagents.4–6 As a result,

35

the damaged OBs would decrease the physical and oxidative stability of soybean products, which

36

should be inhibited during the aqueous processing of soybean.

1,2

which possess a triglycerides (TAGs) core and a surface composed of

37

In soybean OBs, 24 kDa oleosin isoforms A and B and one 18 kDa oleosin are the three major

38

oleosins, followed by two intermediate 16 kDa oleosin isoforms, and some minor ones.7 By

39

extraction at neutral pH, crude OBs were obtained.1 By incubation at pH 4.0–10.0 and 20–50 °C,

40

oleosins (24 and 18 kDa) were hydrolyzed.1 To date, no researches have ever confirmed the

41

protease responsible for soybean oleosin hydrolysis, but one research showed a high possibility

42

that P34 and 30K were the proteases of oleosins.1

43

Initially, P34 was wrongly considered as one kind of oleosin due to its strong interaction with

44

OBs.8 Then it was found that P34, having considerable sequence similarity to the thiol proteases of

45

papain family, was stored in protein storage vacuoles as P34 dimer and disulfide bond (SS) linked 3



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form with α’/α subunit of β-conglycinin (α’/α-SS-P34).9,10 Structurally, the thiol proteases of the

47

papain family possessed highly conserved catalytic triad (Cys-His-Asn) and three SS.11 Compared

48

with these thiol proteases, a Gly37 replaced the conserved catalytic Cys in P34; of the three SS,

49

two were present in P34 (Cys68-Cys108 and Cys171-Cys224, Figure 1). However, one of the

50

three SS in other thiol proteases existed as Cys34 and Asn77 in P34, so it was suggested that the

51

Cys34 near Gly37 might be a free thiol and acted as the active-site residue.9,12 30K, an allergenic

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protein, was first identified by Ogawa et al.,13,14 and considered as similar protein to P34 owing to

53

their similar amino acid compositions. However, the Arg46, Ser75, Gln80, and Phe203 of P34

54

were replaced by the Ser46, Cys75, His80, and Glu203 of 30K (Figure 1). Several studies reported

55

that there were two spots for P34 and 30K on the two-dimensional electrophoresis gel, revealing

56

that P34 and 30K were two similar proteins with some differences.1,15,16

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In this study, the relationship between P34/30K and protease of oleosins was further examined.

58

As stated above, P34 existed in two forms in protein storage vacuoles, whereas no researches had

59

ever examined the existing states of 30K. During germination, storage proteins (such as

60

β-conglycinin and glycinin) were hydrolyzed, and P34 was processed into one protein (P32) with

61

the removal of a hydrophilic decapeptide (KKMKKEQYSC) at the N-terminus of P34.17

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Correspondingly, it was suggested that 30K was also processed into one protein (28K) with the

63

removal of a hydrophilic decapeptide (KKMKKEQYSC) at the N-terminus. It was reported that

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P32 and 28K were bound to degraded OBs after the disruption of germinated soybean.1 Therefore,

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the degraded OBs from germinated soybean were used to obtain P32-rich and 28K-rich samples,

66

and their proteolytic activities on oleosins were examined. Then the active-site residue, cleavage

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site of 24 kDa oleosin, and the interaction between P32/28K and oleosins were examined. 4


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MATERIALS AND METHODS Materials. Soybean (Heinong 64), harvested in 2015, was purchased from Northeast Soybean

70

Research

Institute

(Harbin,

China)

and

71

4-(2-aminoethyl)benzenesulfonyl

72

(EDTA-2Na), and pepstatin A were purchased from Sigma-Aldrich Trading Co., Ltd.

73

(Shanghai, China). N-[N-(L-3-transcarboxyirane-2-carbonyl)-L-leucyl]-agmatine (E-64) was

74

obtained from Abcam (Shanghai, China). All other reagents were of analytical grade.

75

Precision Plus Protein All Blue (Bio-Rad, USA) was used as protein marker.

fluoride

stored

at

hydrochloride

4

°C

until

(AEBSF),

it

was

disodium

used.

EDTA

76

Isolation and Purification of OBs (Substrate). This was performed by an aqueous

77

flotation-centrifugation method by Chen and Ono.18 Soybeans (20 g) were soaked in de-ionized

78

(DI) water at 4 °C for 18 h. The soaked soybeans were ground in fresh DI water (pre-cooled in a

79

4 °C refrigerator, seed/DI water, 1/9, w/w) with a blender (18 000 rpm, MJ-60BE01B, Midea) for

80

2 min. The homogenate was filtered through four layers of gauze to get raw soymilk. Sucrose

81

(20%, w/w) was added into raw soymilk and mixed well. The mixture was adjusted to pH 11.0

82

with 2 M NaOH, and separated into floating, supernatant, and precipitate fractions by

83

centrifugation (25000g for 30 min at 4 °C). The floating fraction was collected and washed three

84

times with 20% (w/w) sucrose solution at pH 11.0 before being centrifuged at 25000g for 30 min

85

at 4 °C. The floating fraction (pH 11-OB) was collected, dispersed into DI water (wet weight/DI

86

water, 1/5), and adjusted to pH 6.5.

87

Soybean Germination. Soybeans (250 g) were washed, and soaked in tap water for 9 h at room

88

temperature. A food-grade plastic container, without cover, was used for germination with natural

89

light, and there were many holes on its bottom and walls. The tap water was poured off, and the 5



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soybeans were uniformly placed on the container bottom. The boiled tap water (room temperature)

91

was sprayed on the soybean for 2 min, and a preservative film (with artificially small holes) was

92

used to cover the container for nine days germination. Everyday the soybeans were sprayed with

93

boiled tap water (room temperature) four times (at 8:00, 12:00, 16:00, and 20:00). During

94

germination, the room temperature was in a range of 20–30 °C.

95

Supernatants and OBs from Germinated Soybean Cotyledon. The cotyledon was harvested

96

from nine days germinated soybean, ground in pre-cooled (4 °C) DI water (1/2, w/w) for 2 min,

97

and filtered to obtain water extract. The obtained OBs, named as bio-degraded OBs, were

98

extracted by using the flotation-centrifugation method with six cycles (Figure 2), and supernatants

99

1–6 were obtained. The solution conditions were pH 7.5 (adjusted by 1 M NaOH) and 20% (w/w)

100

sucrose, and centrifugation (25000g for 30 min) was conducted at 4 °C. All procedures above

101

were conducted at 4 °C or lower temperature. Bio-degraded OBs were dispersed into pre-cooled

102

DI water (wet weight/DI water, 1/5) in an ice water bath, and pH was adjusted to 6.5 by 0.1 M

103

HCl. Supernatants 1–6 were concentrated by ultrafiltration tubes (Merck Millipore, 3 kDa;

104

Ireland), and 20 mM Tris-HCl buffer (pH 7.0) was added into the ultrafiltration tubes in order to

105

remove the sucrose. The protein concentrations in concentrated supernatants 2–6 were determined

106

by bicinchoninic acid method.

107

Effects of Supernatants 3–6 on pH 11-OB. Concentrated supernatants 3–6 were respectively

108

mixed with pH 11-OB, and incubated at pH 6.5 and 35 °C. After 0, 1, 3, and 12 h incubation, 0.5

109

mL of suspension was collected and mixed with Tricine–SDS–PAGE sample buffer to make the

110

SDS/protein mass ratio of 1.52/1, which was also conducted in other samples containing OBs.

111

This condition was the best at solubilizing the intrinsic and extrinsic proteins from OBs.19 In 6


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addition, the protein concentration in final sample was 2 mg/mL.

113

Effects of Various Reagents on the Proteolytic Activity of Supernatant 6. Concentrated

114

supernatant 6 was incubated with reagents in final concentrations of 2–4 mM AEBSF, 5–10 mM

115

EDTA-2Na, 0.5 mM E-64, 1–2 mM DTT, 1–2 mM NEM, and 1–2 mM IAA for 30 min at 37 °C,

116

and 2–4 µM pepstatin A for 60 min at 4 °C. Then, pH 11-OB was added into the above solutions,

117

and incubated at pH 6.5 and 35 °C. After 3 h, sample (0.2 mL) was collected and mixed with

118

Tricine–SDS–PAGE sample buffer. In each case, a control evaluation of the proteolytic activity

119

was done without inhibitor.

120

Tricine–SDS–PAGE. Sample preparation: sample was divided into three parts, which were

121

treated as follows: 1) no treatment, 2) in a boiling water bath for 5 min, 3) in a boiling water bath

122

for 5 min in presence of 2% (v/v) β-mercaptoethanol (β-ME). Then these samples were treated by

123

Tricine–SDS–PAGE, and respectively named as nonreducing, heating, and reducing

124

Tricine–SDS–PAGE.

125

Tricine–SDS–PAGE was conducted according to the method by Schagger with a 16%

126

acrylamide separating gel and a 4% acrylamide stacking gel.20 Sample (10 µL) was loaded into

127

sample well, electrophoresed at a constant voltage of 30 mV until all of protein sample entered

128

into stacking gel, then at a constant voltage of 100 mV until the end. The gel was stained with

129

Coomassie Brilliant Blue G-250, and band intensities and apparent molecular weights (MWs)

130

were analyzed by Image Lab Software (Bio-Rad, Hercules, CA).

131

Diagonal Electrophoresis. Sample was treated by nonreducing Tricine–SDS–PAGE. After

132

electrophoresis, the lane was cut from the gel and treated in two ways, in a boiling water bath for 5

133

min in presence and absence of β-ME (2%, v/v). Then the treated lane was used for the second 7



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dimensional electrophoresis of diagonal electrophoresis, and the gel was stained by Coomassie

135

Brilliant Blue G-250. They were named as nonreducing-reducing and nonreducing-heating

136

Tricine–SDS–PAGE, respectively.

137

Two-Dimensional Electrophoresis. Trichloroacetic acid at a final concentration of 10% (w/v)

138

was added into concentrated supernatants 3 and 6, and the mixtures were put at -20 °C for 5 min

139

and 4 °C for 15 min. After centrifugation (15000g for 15 min at 4 °C), the precipitate was

140

collected, mixed with acetone (pre-cooled in a -20°C refrigerator) with stirring for 10 min at 4 °C,

141

and centrifuged. This step was repeated three times. Finally, acetone was evaporated by nitrogen

142

flow, and the protein samples were obtained. The two-dimensional electrophoresis was performed

143

according to the Bio-Rad manufacturer’s instructions. Please check the details in the reference by

144

Chen et al.1

145 146

MALDI-TOF/TOF MS. This was totally conducted according to the method by Zhao et al.7

Please check the details in this reference.

147

Isolation and Purification of Hydrolyzed Products (HPs) of 24 kDa Oleosin. Concentrated

148

supernatant 6 was mixed with pH 11-OB, and incubated at pH 6.5 and 35 °C for 1 h. Then the

149

mixture was adjusted to pH 4.5, and treated by centrifugation (25000g for 30 min at 4 °C). The

150

supernatant was collected by syringe, and concentrated by using ultrafiltration tube above.

151

Protein fractionation by a C8 column. The HPLC equipment consisted of an Agilent 1100

152

series chromatograph (Agilent Technologies, Santa Clara, CA), equipped with a column oven

153

(G1311C), a quaternary pump (G1216A), and a UV detector (G1321B). An analytical C8

154

column [OC20S05-1546WT, 5 µm particle size, 200 Å, 150 × 4.6 mm (inside diameter),

155

YMC HPLC column] was used for the separation. Gradient elution was performed at a flow 8


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156

rate of 1 mL/min using two solvents: solvent A [0.1% (v/v) trifluoroacetic acid in water] and

157

solvent B [0.085% (v/v) trifluoroacetic acid in acetonitrile]. The gradient was as follows: 95%

158

A with 5% B from 0 to 5 min, 40% A with 60% B from 5 to 60 min, 100% B from 60 to

159

65min, and 95% A with 5% B from 65 to 70 min. The column temperature was kept at 34 °C,

160

and the detection was performed at 214 nm. The injection volume of the above supernatant

161

was 20 µL. Two major peaks (23.7–24.1 min, HP2a; 24.9–25.2 min, HP2b) were observed,

162

and respectively collected. The experiment was repeated twenty five times, and HP2a and

163

HP2b were respectively combined. The solvent was evaporated by nitrogen blow.

164

Protein fractionation by a C18 column. The HPLC equipment system was the same as above.

165

An analytical C18 column [ZORBAX Eclips XDB, 5 µm particle size, 80 Å, 250 × 4.6 mm

166

(inside diameter), Agilent Technologies HPLC columns] was used for the separation. Gradient

167

elution was performed at a flow rate of 0.8 mL/min using solvent C [0.06% (v/v)

168

trifluoroacetic acid in water] and solvent D [0.05% (v/v) trifluoroacetic acid in acetonitrile].

169

The gradient was as follows: 100% C from 0 to 3 min, linearly increased to 100% D from 3 to

170

20 min, 100% D from 20 to 25 min. The column temperature was kept at 34 °C, and the

171

detection was performed at 214 nm. HP2a was dissolved into solvent C, and 10 µL was

172

injected into the HPLC system. One major peak (13.3–14.5 min, HP2c) was observed, and

173

collected. The experiment was repeated twenty five times, and HP2c was combined. The

174

solvent was evaporated by nitrogen blow.

175

N-terminal Amino Acid Sequence Analysis. The amino acid sequence was determined by the

176

Edman method, using PPSQ-31A Automated Protein/Peptide Sequencer (Shimadzu) according to

177

the manufacturer’s instructions. Fifteen microliters of HP2b or HP2c (more than 50 pmol) was 9



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178

added to a polyvinylidene difluoride membrane and were dried by nitrogen blow for the analysis

179

of amino acid sequence (six amino acid residues at the N-terminus).

180

MWs of HP2b and HP2c. This was analyzed by ultra high performance liquid chromatography

181

coupled with a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (UHPLC-MS/MS). For

182

UHPLC, an ACQUITY UPLC Protein BEH C4 column [1.7 µm particle size, 300 Å, 150 × 2.1

183

mm (inside diameter)] was used. Methanol was used as mobile phase with a flow rate of 0.2

184

mL/min. The column temperature was kept at 35 °C, and the injection volume was 2 µL. The

185

mass spectrometer spectra were obtained using a Thermo Scientific Q Exactive hybrid

186

quadrupole-orbitrap mass spectrometer. Parameters of ion source were set as follows: ion

187

source, electrospray ion; scan type, full MS; scan range, 150.0 to 2000.0 m/z; polarity,

188

positive; spray voltage, 3.80 kV; capillary temperature, 350 °C; auxiliary gas heater

189

temperature, 425 °C; sheath gas flow rate, 50 mL/min; auxiliary gas flow rate, 13 mL/min;

190

sweep gas flow rate, 3 mL/min.

191

RESULTS

192

Protein Compositions of Supernatants 2–6 from Bio-degraded OBs of Nine Days

193

Germinated Soybean. Figure 3A shows the protein profile of ungerminated soybean OBs

194

extracted at pH 6.8, and it was found that P34 and 30K were processed into smaller proteins (P32

195

and 28K) after nine days germination (Figure 3B), in agreement with the results by Herman et al.17

196

Figure 3B shows that P32 and/or 28K were the major proteins in supernatants 2–6. On

197

nonreducing Tricine–SDS–PAGE gel (Figure 3C), supernatants 2–3 showed two major bands at 26

198

and 21 kDa, and supernatant 6 had one major band at 48 kDa; supernatants 4–5 possessed three

199

major bands at 48, 26, and 21 kDa. Figure 3D shows that supernatant 3 had one protein spot with 10


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200

isoelectric point (pI) at pH 4.3, whereas supernatant 6 had one with pI at pH 4.45 (Figure 3E).

201

Supernatant 4 was treated by nonreducing-reducing Tricine–SDS–PAGE (Figure 3F). Interestingly,

202

26 and 21 kDa bands had the same MWs (approximately 28 kDa) in the reducing condition, and

203

48 kDa band consisted of two proteins. According to the amino acid sequences of P34 and 30K

204

(Figure 1), it was concluded that 28K was more acidic than P32, which could be supported by the

205

fact that 28K was more easily removed from bio-degraded OBs than P32 by pH 7.5 washing

206

(Figures 2 and 3). Therefore, it was clarified that 26 and 21 kDa bands were 28K, and 48 kDa

207

band included P32 dimer and basic 7S globulin (Bg7S, supplementary information Table S1).

208

Bg7S consists of α and β chains, which are linked by one interchain SS.21 Together with the results

209

in Figures 1 and 3F, it was suggested that 28K had two forms in supernatants 2–5, one (21 kDa)

210

had three intrachain SS (28K3SS), and the other (26 kDa) had two intrachain SS (28K2SS). These

211

results showed that P32 tended to exist as dimer, whereas 28K as monomer.

212

Proteolytic Activity of Supernatants 3–6. Supernatants 3–6, which were adjusted to the same

213

protein concentration, were respectively mixed with pH 11-OB, and incubated at 35 °C and pH 6.5.

214

It was found that the protease in supernatants 3–6 preferentially hydrolyzed 24 kDa oleosin, and

215

three major HPs (HP1–3; apparent MWs: 14, 10, and 8 kDa) were observed in Figures 4A and B.

216

Interestingly, supernatant 6 had the highest proteolytic activity, followed by supernatants 5, 4, and

217

3. As shown in Figure 3, supernatant 6 contained the most P32 dimer and Bg7S. Bg7S adopts a

218

pepsin fold, but it lacks proteolytic activity due to the fact that one aspartate corresponding to the

219

catalytic residue of pepsin is replaced by Ser265 in Bg7S.21 When crude OBs extracted from

220

ungerminated soybean were incubated, the same HPs were detected on Tricine–SDS–PAGE gel,1

221

indicating that crude OBs and supernatant 6 should contain the same or similar proteases. At 11



222

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present stage, P34 was confirmed in crude OBs and processed P34 (P32) in supernatant 6.

223

Nonreducing Tricine–SDS–PAGE results showed that P32 dimers in supernatant 6 were

224

dissociated and bound to 24 kDa oleosin when supernatant 6 was mixed with pH 11-OB (Figure

225

4E); similarly, 28K3SS interacted with 24 kDa oleosin when supernatant 3 was mixed with pH

226

11-OB, but 28K2SS did not. Further, band intensity of 28K3SS-24 kDa oleosin slowly decreased

227

with prolonged incubation time (Figure 4C), whereas that of P32-24 kDa oleosin quickly

228

decreased (Figure 4D).

229

Cleavage Site of 24 kDa Oleosin. To further understand the properties of the endogenous

230

protease, the cleavage site of 24 kDa oleosin was examined. Figures 4A and B show that HP1 and

231

HP2 were observed after 1 h incubation, and HP3 appeared after 3 h incubation. To avoid the

232

effects of HP3 on purification, 1 h incubated mixture of supernatant 6 and pH 11-OB was used to

233

purify HP2. It was found that HP2 released from OBs into aqueous phase, which could be

234

separated from OBs by centrifugation. The aqueous phase was treated by a C8 column to obtain

235

HP2a and HP2b (Figure 5A), and HP2a was further treated by a C18 column to obtain HP2c

236

(Figure 5B). The purity of HP2a, HP2b, and HP2c was approximately 87, 97, and 95%,

237

respectively (Figure 5C). 24 kDa oleosin has two isoforms A and B, so HP2b and HP2c should be

238

respectively from the two isoforms. The N-terminal amino acid sequence analysis showed that the

239

six amino acid residues at the terminus of HP2b and HP2c were the same (TKEVGQ), meaning

240

that the cleavage site of isoform A was Lys161-Thr162, and that of isoform B was Lys160-Thr161.

241

Further, it was clarified that HP2 was from the C-terminal domain of 24 kDa oleosin, which

242

consists of N-terminal domain, hydrophobic central domain, and C-terminal domain.3 According

243

to the amino acid sequences of isoforms A and B (UniProt database), the released HPs from 12


Page 13 of 34


244

isoform A and B have theoretical MWs of 6942 and 6786 Da, respectively. To confirm whether the

245

N-terminal amino acid sequence results were correct, HP2b and HP2c were examined by

246

UHPLC-MS/MS, which showed that their MWs were 6786 and 6942 Da, respectively

247

(supplementary information Figure S1). Therefore, the amino acid sequence results were correct,

248

and HP2c originated from isoform A, while HP2b from isoform B. With prolonged incubation

249

time, it was found that the band intensity of HP2 first increased and then decreased, indicating that

250

HP2 was further hydrolyzed and there was another cleavage site on HP2. It was found that

251

Lys168-Thr169 and Lys167-Thr168 existed in isoform A and B, respectively. It was considered

252

that the two peptide bonds might be cleaved with prolonged incubation time, which could be

253

supported by the appearance and band intensity increase of HP3. These results showed that the

254

protease had cleavage site specificity for 24 kDa oleosin, and HP1–3 respectively had MWs of

255

approximately 17, 7, and 6 kDa, which were different from their corresponding apparent MWs (14,

256

10, and 8 kDa) on Tricine–SDS–PAGE gel.

257

Effects of Supernatant 6 on 24 kDa Oleosin at Different pHs and Temperatures. It was

258

found that 35–50 °C was the optimal temperature range, and pH 6.0–6.5 was the optimal pH range

259

for the hydrolysis of 24 kDa oleosin (Supplementary information Figure S2). In one previous

260

study, crude OBs were extracted from ungerminated soybean at neutral pH, and it was found that

261

the optimal conditions for the hydrolysis of 24 kDa oleosin were also pH 6.0–6.5 and 35–50 °C.1

262

Coincidently, the above crude OBs contained P34, and the mixture of supernatant 6 and pH 11-OB

263

contained P32.

264

Effects of Protease Inhibitors on the Proteolytic Activity of Supernatant 6. Figure 6A shows

265

that E-64 could obviously decrease the proteolytic activity, whereas EDTA obviously enhanced it 13



Page 14 of 34

266

(Figure 6B). Pepstatin A and AEBSF weakly affected the proteolytic activity (Figure 6B). In

267

addition, it was found that 1 mM DTT could obviously enhance the proteolytic activity, while

268

NEM and IAA could decrease it (Figure 7). These results clearly indicated that protease in

269

supernatant 6 was one thiol protease. And the protease in crude OBs from ungerminated soybean

270

was also one thiol protease (data not shown).

271

Interaction between 24 kDa Oleosin and P32 during Incubation. P32-24 kDa oleosin could

272

be observed on the nonreducing Tricine–SDS–PAGE gel (Figures 4D and 8C), which was

273

separated into two spots on the nonreducing-reducing Tricine–SDS–PAGE gel (Figure 4E).

274

Therefore, 24 kDa oleosin existed as monomer and P32-24 kDa oleosin in the mixture of

275

supernatant 6 and pH 11-OB. It was considered that their band intensities as function of incubation

276

time could be used to respectively represent their hydrolysis rates (Figure 8C), and the band

277

intensity as function of incubation time on the reducing Tricine–SDS–PAGE gel exhibit the

278

hydrolysis rate of total 24 kDa oleosin (Figure 8A). By incubation, band intensity of P32-24 kDa

279

oleosin slowly decreased from 0 to 4 h, whereas that of 24 kDa oleosin monomer decreased more

280

quickly than P32-24 kDa oleosin (Figures 8C and F). It was predictable that the hydrolysis rate of

281

total 24 kDa oleosin was between 24 kDa oleosin monomer and P32-24 kDa oleosin (Figures 8A,

282

C, and F). After 4 h incubation, hydrolysis rate of P32-24 kDa oleosin obviously increased.

283

Interestingly, the 48 kDa band intensity gradually increased with prolonged incubation time, so 8 h

284

incubated mixture was treated by nonreducing-reducing Tricine–SDS–PAGE. It was found that 48

285

kDa band contained Bg7S, P32 dimer, and P32-17 kDa HP (Figure 8D).

286

To examine whether the interaction in P32 dimer, P32-24 kDa oleosin, and P32-17 kDa HP was

287

SS, heating Tricine–SDS–PAGE (Figure 8B) and nonreducing-heating Tricine–SDS–PAGE 14


Page 15 of 34


288

(Figure 8E) were designed. Figure 8B shows that the interaction between P32 and 24 kDa oleosin

289

was destroyed by heating, whereas the interchain SS in Bg7S was not. Together with the fact that

290

24 kDa oleosin does not contain cysteine residue (UniProt database), the interaction in P32-24

291

kDa oleosin was not SS. Figure 8E shows that Bg7S was located on the diagonal line, and P32

292

dimer and P32-17 kDa HP were resolved into P32 monomer and 17 kDa HP under diagonal line.

293

Similarly, the interaction in P32-17 kDa HP was also not SS. Due to the fact that P32 dimer was

294

dissociated and bound to 24 kDa oleosin (Figure 8C), it was hard to consider that the interaction in

295

P32 dimer was SS.

296

DISCUSSION

297

P34 and P32. P32-rich sample showed proteolytic activity specifically on the Lys-Thr of 24

298

kDa oleosin to form two HPs (approximately 17 and 7 kDa). Further, it was confirmed that the

299

protease in P32-rich sample was one thiol protease; P32 existed as dimer, but the interaction in

300

P32 dimer was not SS. Therefore, Cys24 (marked by red pentagram in Figure 1) supplied a free

301

thiol for P32. To date, just one P34-like protein (SPE31) was found in the seeds of a legume plant

302

(Pachyrizhus erosus), and it was suggested that SPE31 and P34 formed a unique subfamily within

303

the papain family.22 It is noteworthy that the proteolytic activity of SPE31 has been confirmed, 22,23

304

so it is considered that P34/P32 should also have proteolytic activity.

305

Interaction between P32 and 24 kDa Oleosin. P32 dimers were dissociated and bound to 24

306

kDa oleosin after supernatant 6 was mixed with pH 11-OB. The sample was treated in three ways

307

before Tricine–SDS–PAGE analysis, 1) no further treatment, 2) heating, and 3) heating in

308

presence of β-ME. In the first way, P32-24 kDa oleosin as itself could be resolved into

309

Tricine–SDS–PAGE gel, whereas it was separated into P32 and 24 kDa oleosin monomers in the 15



Page 16 of 34

310

second and third ways. These results showed that P32 had high affinity for 24 kDa oleosin, and the

311

P32-24 kDa oleosin was considerably stable. It looked like that the P32-24 kDa oleosin interaction

312

was similar to the protease-inhibitor interaction. Generally, protease inhibitor was thought to

313

inhibit the proteolytic activity of protease by forming protease-inhibitor complex. However, it was

314

reported that trypsin could hydrolyze its inhibitor, although the hydrolysis rate was ∼1011 slower

315

for inhibitor than for substrate.24 CrmA is a “cross-class” serpin family inhibitor of the

316

proapoptotic serine protease, granzyme B, as well as thiol proteases of the caspase family. It was

317

reported that CrmA could be bound to granzyme B to form CrmA-granzyme B complex, which

318

could resist to heating in presence of SDS and DTT, and could be resolved into SDS–PAGE gel;

319

crmA also could be bound to caspase-1 to form crmA-caspase-1 complex, which could be

320

observed in native PAGE gel; interestingly, crmA was hydrolyzed by both granzyme B and

321

caspase-1, and peptide cleavage was specific.25

322

Generally, the catalytic mechanism of thiol protease is expressed as follows.26–28 The catalytic

323

Cys of thiol protease performed a nucleophilc attack to covalently (sulfur-carbon bond) link the

324

protease to the substrate to form protease-substrate Michaelis complex, which was stabilized by

325

oxyanion hole.27,28 The oxyanion then collapsed, cleaving the amide bond and releasing the

326

C-terminal end of the substrate, leaving the acylated protease formed via thioester bond between

327

protease and remaining substrate fragment. At last, the thioester bond was hydrolyzed to produce

328

the remaining substrate fragment and free protease. The catalytic mechanism of serine protease is

329

similar to that of thiol protease. It was reported that the serine protease-substrate complex and

330

acylated protease were relatively stable species though they were not detected by any direct

331

experimental techniques.29 In this study, both P32-24 kDa oleosin (corresponding to 16


Page 17 of 34


332

protease-substrate complex) and P32-17 kDa HP (corresponding to acylated protease) were

333

detected by nonreducing Tricine–SDS–PAGE analysis. Together with the facts that P34-like

334

protein (SPE31) had proteolytic activity and the protease in supernatant 6 was one thiol protease,

335

it was suggested that P32 was the protease. This could be used to explain the behaviors of P32-24

336

kDa oleosin in Figures 8C and F. After supernatant 6 was mixed with pH 11-OB, P32 dimers were

337

dissociated and bound to 24 kDa oleosin. By incubation, the Lys-Thr bond of 24 kDa oleosin was

338

cleaved to release one product (approximately 7 kDa), and P32-17 kDa HP was formed. Then

339

P32-17 kDa HP was hydrolyzed to produce 17 kDa HP and regenerating the free P32, which was

340

bound to the next 24 kDa oleosin. This procedure repeated again and again till that 24 kDa oleosin

341

was all consumed. As a result, band intensity of P32-24 kDa oleosin quickly decreased afterwards,

342

and P32 monomers were changed back to P32 dimers again.

343

Based on the results above, one hypothesis was proposed as follows. It is known that protease

344

inhibitor is bound to protease in a substrate-like manner. Protease can hydrolyze its substrates as

345

well as its inhibitors, although the former hydrolysis rate is greatly faster than the latter one. To

346

some extent, these inhibitors also can be considered as the substrates of protease, and substrates

347

and inhibitors can be considered as two extreme cases for protease. Therefore, it is reasonable to

348

consider that there are some proteins possessing properties between substrates and inhibitors, and

349

24 kDa oleosin may be one of them.

350

30K and 28K. 30K was proved to be one major allergenic protein in soybean, and no

351

researches reported that 30K had proteolytic activity. In this study, it was found that 30K was

352

processed into 28K after nine days germination, and it was successfully separated from P32.

353

28K-rich sample had very low proteolytic activity compared to P32-rich sample. In 28K-rich 17



Page 18 of 34

354

sample, 28K existed as two different monomers, one had two intrachain SS, and the other

355

possessed three. As a result, 28K2SS had two free thiols, and 28K3SS contained no free thiols

356

(Figure 1). Therefore, it was suggested that the low proteolytic activity of 28K-rich sample might

357

originate from 28K2SS, and 28K3SS did not possess proteolytic activity.

358

In this study, P32 and 28K were isolated from bio-degraded OBs of nine days germinated

359

soybean, and P32 was successfully separated from 28K. P32-rich sample contained one thiol

360

protease, which specifically cleaved the Lys-Thr bond of 24 kDa oleosin; P32 existed as dimer.

361

However, the interaction in P32 dimer was not SS, so each P32 in dimer had one free thiol. After

362

mixing with pH 11-OB, P32 dimers were dissociated and bound to 24 kDa oleosin, and the

363

interaction between P32 and 24 kDa oleosin was not SS. As a result, 24 kDa oleosin existed as

364

monomer and P32-24 kDa oleosin in the mixture above. By incubation, P32-24 kDa oleosin

365

slowly decreased in the beginning, but quickly after 24 kDa oleosin monomers were all

366

hydrolyzed. After 8 h incubation, it was found that some P32 were changed back to P32 dimers,

367

and some P32 were detected as P32-17 kDa HP. The interaction between P32 and 17 kDa HP was

368

also not SS. Based on the knowledge about the protease-inhibitor and protease-substrate

369

interactions, it was suggested that P32 was the thiol protease, and 24 kDa oleosin possessed the

370

properties between inhibitors and substrates. 28K existed as two different monomers, one had

371

three intrachain SS, and the other possessed two. It was suggested that the latter had low

372

proteolytic activity. In all, this study was meaningful for 1) supplying one method for separating

373

28K from P32, 2) confirming that the protease of soybean oleosins was one thiol protease, 3)

374

confirming that the thiol protease specifically cleaved the Lys-Thr bond of 24 kDa oleosin, 4)

375

finding that P32-24 kDa oleosin interaction was partially similar to protease-inhibitor interaction, 18


Page 19 of 34


376

and was also partially similar to protease-substrate interaction.

377

Funding

378

This study was supported by the National Great Project of Scientific and Technical Supporting

379

Programs of China (No. 2012BAD34B04-1), and Hi-tech Research and Development Program of

380

China (No. 2013AA102204-3).

381

Notes

382

The authors declare no competing financial interest.

383 384

Supporting Information Available:

385

Identification of one protein band and two protein spots in Figure 3 by MALDI-TOF/TOF MS

386

(Table S1), molecular weights of two hydrolyzed products examined by UHPLC-MS/MS (Figure

387

S1), the effects of temperature and pH on the proteolytic activity of P32-rich sample (Figure S2).

388 389

REFERENCES

390

(1) Chen, Y.; Zhao, L.; Cao, Y.; Kong, X.; Hua, Y. Oleosins (24 and 18 kDa) are hydrolyzed not

391

only in extracted soybean oil bodies but also in soybean germination. J. Agric. Food Chem. 2014,

392

62, 956–965.

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(2) Simpson, T. D.; Nakamura, L. K. Phospholipid degradation in membranes of isolated soybean

394

lipid bodies. J. Am. Oil Chem. Soc. 1989, 66, 1093–1096.

395

(3) Huang, A. H. C. Oil bodies and oleosins in seeds. Annu. Rev. Plant Physiol. Plant Mol. Biol.

396

1992, 43, 177–200.

397

(4) Leprince, O.; van Aelst, A. C.; Pritchard, H. W.; Murphy, D. J. Oleosins prevent oil-body 19



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coalescence during seed imbibitions as suggested by a low-temperature scanning electron

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microscope study of desiccation-tolerant and -sensitive oilseeds. Planta 1998, 204, 109–119.

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(5) Shimada, T. L.; Shimada, T.; Takahashi, H.; Fukao, Y.; Hara-Nishimura, I. A novel role for

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oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana. Plant J. 2008, 55, 798–809.

402

(6) Iwanaga, D.; Gray, D. A.; Fisk, I. D.; Decker, E. A.; Weiss, L.; McClements, D. J. Extraction

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and characterization of oil bodies from soy beans: A natural source of pre-emulsified soybean oil.

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J. Agric. Food Chem. 2007, 55, 8711–8716.

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(7) Zhao, L.; Chen, Y.; Cao, Y.; Kong, X.; Yufei, H. The integral and extrinsic bioactive proteins in

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the aqueous extracted soybean oil bodies. J. Agric. Food Chem. 2013, 61, 9727–9733.

407

(8) Herman, E. M. Immunogold-localization and synthesis of an oil-body membrane protein in

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developing soybean seeds. Planta 1987, 172, 336–345.

409

(9) Kalinski, A.; Weisemann, J. M.; Matthews, B. F.; Herman, E. M. Molecular cloning of a

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protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain

411

family. J. Biol. Chem. 1990, 265, 13843–13848.

412

(10) Wadahama, H.; Iwasaki, K.; Matsusaki, M.; Nishizawa, N.; Ishimoto, M.; Arisaka, F.; Takagi,

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K.; Urade, R. Accumulation of β-conglycinin in soybean cotyledon through the formation of

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disulfide bonds between α'- and α-subunits. Plant Physiol. 2012, 158, 1395–1405.

415

(11) Wenig, K.; Chatwell, L.; von Pawel-Rammingen, U.; Bjorck, L.; Huber, R.; Sondermann, P.

416

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for

417

IgG. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17371–17376.

418

(12) Kalinski, A.; Melroy, D. L.; Dwivedi, R. S.; Herman, E. M. A soybean vacuolar protein (P34)

419

related to thiol protease is synthesized as a glycoprotein precursor during seed maturation. J. Biol. 20


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420

Chem. 1992, 267, 12068–12076.

421

(13) Ogawa, T.; Bando, N.; Tsuji, H.; Okajima, H.; Nishikawa, K.; Sasaoka, K. Investigation of

422

the IgE-binding proteins in soybean by immunoblotting with the sera of the soybean sensitive

423

patients with atopic dermatitis. J. Nutr. Sci. Vitaminol. 1991, 37, 555–565.

424

(14) Ogawa, T.; Tsuji, H.; Bando, N.; Kitamura, K.; Zhu, Y.; Hirano, H.; Nishikawa, K.

425

Identification of the soybean allergenic protein, Gly m Bd 30K, with the soybean seed 34-kDa

426

oil-body-associated protein. Biosci. Biotech. Biochem. 1993, 57, 1030–1033.

427

(15) Herman, E. M.; Helm, R. M.; Jung, R.; Kinney, A. J. Genetic modification removes an

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immunodominant allergen from soybean. Plant Physiol. 2003, 132, 36–43.

429

(16) Natarajan, S.; Xu, C.; Bae, H.; Caperna, T. J.; Garrett, W. M. Proteomic analysis of allergen

430

and antinutritional proteins in wild and cultivated soybean seeds. J. Plant Biochem. Biotech. 2006,

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15, 103–108.

432

(17) Herman, E. M.; Melroy, D. L.; Buckhout, T. J. Apparent processing of a soybean oil body

433

protein accompanies the onset of oil mobilization. Plant Physiol. 1990, 94, 341–349.

434

(18) Chen, Y.; Ono, T. Simple extraction method of non-allergenic intact soybean oil bodies that

435

are thermally stable in an aqueous medium. J. Agric. Food Chem. 2010, 58, 7402–7407.

436

(19) Ying, Y.; Zhao, L.; Kong, L.; Kong, X.; Hua, Y.; Chen, Y. Solubilization of proteins in

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extracted oil bodies by SDS: A simple and efficient protein sample preparation method for

438

Tricine–SDS–PAGE. Food Chem. 2015, 181, 179–185.

439

(20) Schagger, H. Tricine–SDS–PAGE. Nat. Protoc. 2006, 1, 16–22.

440

(21) Yoshizawa, T.; Shimizu, T.; Yamabe, M.; Taichi, M.; Nishiuchi, Y.; Shichijo, N.; Unzai, S.;

441

Hirano, H.; Sato, M.; Hashimoto, H. Crystal structure of basic 7S globulin, a xyloglucan-specific 21



Page 22 of 34

442

endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity

443

against endo-β-glucanase. FEBS J. 2011, 278, 1944–1954.

444

(22) Zhang, M.; Wei, Z.; Chang, S.; Teng, M.; Gong, W. Crystal structure of a papain-fold protein

445

without the catalytic residue: a novel member in the cysteine proteinase family. J. Mol. Biol. 2006,

446

358, 97–105.

447

(23) Chang, S.; Song, X.; Yan, M.; Zhou. Z.; Wu, F.; Gong, W. Purification, characterization and

448

preliminary crystallographic studies of a cysteine protease from Pachyrrhizus erosus seeds. Acta

449

Cryst. D. 2004, 60, 187–189.

450

(24) Perakyla, M.; Kollman, P. A. Why does trypsin cleave BPTI so slowly? J. Am. Chem. Soc.

451

2000, 122, 3436–3444.

452

(25) Swanson, R.; Raghavendra, M. P.; Zhang, W.; Froelich, C.; Gettins, P. G.; Olson, S. T. Serine

453

and cysteine proteases are translocated to similar extents upon formation of covalent complexes

454

with serpins. Fluorescence perturbation and fluorescence resonance energy transfer mapping of

455

the protease binding site in CrmA complexes with granzyme B. J. Biol. Chem. 2007, 282,

456

2305–2313.

457

(26) Buller, A. R.; Townsend, C. A. Intrinsic evolutionary constraints on protease structure,

458

enzyme acylation, and the identity of the catalytic triad. Proc. Natl. Acad. Sci. U. S. A. 2013, 110,

459

653–661.

460

(27) Ma, S.; Devi-Kesavan, L.; Gao, J. Molecular dynamics simulations of the catalytic pathway

461

of a cysteine protease: A combined QM/MM study of human Cathepsin K. J. Am. Chem. Soc.

462

2007, 129, 13633–13645.

463

(28) Ke, Z.; Zhou, Y.; Hu, P.; Wang, S.; Xie, D.; Zhang, Y. Active site cysteine is protonated in the 22


Page 23 of 34


464

PAD4 Michaelis complex: evidence from Born-Oppenheimer Ab Initio QM/MM molecular

465

dynamics simulations. J. Phys. Chem. B 2009, 113, 12750–12758.

466

(29) Ishida, T.; Kato, S. Theoretical perspectives on the reaction mechanism of serine proteases:

467

the reaction free energy profiles of the acylation process. J. Am. Chem. Soc. 2003, 125,

468

12035–12048.

469

23



Page 24 of 34

470

Figure captions

471

Figure 1. Amino acid sequences of P34 probable thiol protease and Gly m Bd 30K (UniProt

472

database). It is known that two disulfide bonds are formed between Cys68 and Cys108 and

473

between Cys171 and Cys224. There are two other Cys residues (Cys10 and Cys34) in P34, and

474

three (Cys10, Cys34, and Cys75) in 30K. The decapeptide (KKMKKEQYSC) at the N-terminus

475

of P34/30K is removed during soybean germination.

476

Figure 2. Preparation of supernatants 1–6 from nine days germinated soybean.

477

Figure 3. (A) Protein profile of crude OBs extracted from ungerminated soybean at pH 6.8. Lane

478

1, marker; lane 2, crude OBs. (B) Reducing Tricine–SDS–PAGE profiles of supernatants 2–6.

479

Lane 1, marker; lanes 2–6, supernatants 2–6. (C) Nonreducing Tricine–SDS–PAGE profiles of

480

supernatants 2–6. Lane 1, marker; lanes 2–6, supernatants 2–6. (D) Two-dimensional

481

electrophoresis profile of supernatant 3. (E) Two-dimensional electrophoresis profile of

482

supernatant 6. (F) Nonreducing-reducing Tricine–SDS–PAGE profile of supernatant 4.

483

Figure 4. (A, B) Hydrolysis of oleosins in mixture of pH 11-OB and supernatant 3/4/5/6 (reducing

484

condition). (C, D) Hydrolysis of oleosins in mixture of pH 11-OB and supernatant 3/4/5/6

485

(nonreducing condition). (E) Nonreducing-reducing Tricine–SDS–PAGE profile of mixture of pH

486

11-OB and supernatant 6 (0 h incubation). The incubation was conducted at 35 °C and pH 6.5.

487

Figure 5. (A) Fractionation of the supernatant from 1 h incubated (35 °C, pH 6.5) mixture of

488

supernatant 6 and pH 11-OB by a C8 column. Two protein fractions (HP2a and HP2b) were

489

obtained. (B) Fractionation of the HP2a by a C18 column. One protein fraction (HP2c) was

490

obtained. The absorbance was given in milliabsorbance units (mAU). (C) Reducing

491

Tricine–SDS–PAGE profiles of HP2a, HP2b, and HP2c. Lane 1, marker; lane 2, HP2a; lane 3, 24


Page 25 of 34


492

HP2b; lane 4, HP2c.

493

Figure 6. The effects of protease inhibitors (E-64, pepstatin A, EDTA, and AEBSF) on proteolytic

494

activity of supernatant 6.

495

Figure 7. The effects of DTT, NEM, and IAA on the proteolytic activity of supernatant 6. C,

496

control, incubated for 0 h without any reagents.

497

Figure 8. Reducing (A), heating (B), and nonreducing (C) Tricine–SDS–PAGE profiles of

498

incubated mixture of supernatant 6 and pH 11-OB. Nonreducing-reducing (D) and

499

nonreducing-heating (E) Tricine–SDS–PAGE profiles of 8 h incubated mixture of supernatant 6

500

and pH 11-OB. (F) Band intensities of P32-24 kDa oleosin, 24 kDa oleosin monomer, and total 24

501

kDa oleosin in panels A and C as function of incubation time. The incubation was conducted at

502

35 °C and pH 6.5.

25



Page 26 of 34

Figure 1.

26


Page 27 of 34


Figure 2.

27



kDa 100 75

Lipoxygenase kDa 100 αʼ+α 75 γ-conglycinin 50 β A3 37 A

50 37 25

26 kDa (28K2SS) 25

20

24 kDa oleosin B

20

20 21 kDa (28K3SS)

15

15

18 kDa oleosin 16 kDa oleosin 1 16 kDa oleosin 2

15

P32 dimer + Bg7S (1)

48 kDa

37

25

P34 + 30 K

Lipoxygenase

kDa 100 75 50

Lipoxygenase

Page 28 of 34

10 10

10

1

2

1

2

3

(A)

4

5

6 1

2

3

(B)

75 50

28K

37

P32

25

20

6

kDa

75 50 37

25

5

(C)

kDa

kDa 75 50 37

4

28K

20

25 P32 + α chain (2)

15 10

15

20

10

15

β chain (3)

10

3

4

5

6

(D)

7

8

9

10

3

4

5

6

7

8

9

10

(E)

(F)

Figure 3.

28


Page 29 of 34


S3 + pH 11-OB

kDa 100 75 50 37

S4 + pH 11-OB

25

25

20

20

15

15

10

10

Incubation time (h)

S3

0

1

3

12

S4 0

1

3

12

24 kDa oleosin 18 kDa oleosin 16kDa oleosin + HP1 HP2 HP3

S5

0

1

3

(A) S4 + pH 11-OB

S6

0

1

3

12

S6 + pH 11-OB

S5 + pH 11-OB kDa 75

28K3SS-24 kDa oleosin

50

37

12

(B)

S3 + pH 11-OB kDa 75 50

S6 + pH 11-OB

S5 + pH 11-OB

kDa 100 75 50 37

37

28K2SS

25

25

20 15

20

P32-24 kDa oleosin P32-HP1 + Bg7S

steroleosin Bg 7S

caleosin

24 kDa oleosin 18 kDa oleosin 16 kDa oleosin + HP1

15 28K3SS

10

Incubation time (h)

HP2 HP3

10

pH 11-OB S3

0

1 12 S4

0

1

3

pH 11-OB S5

0

(C)

P32

1

3

S6

0

1

3

(D)

α chain 24 kDa oleosin 18 kDa oleosin β chain

16 kDa oleosin + HP1

(E) Figure 4.

29



HP2a

Page 30 of 34

HP2c

HP2b

(A)

(B) kDa 75 50 37

25 20 15

10

1

2

3

4

(C) Figure 5.

30


Page 31 of 34


kDa 75

kDa

50

75 50

37

37

25

24 kDa oleosin

20

25

24 kDa oleosin

20

15

15

10

10

Incubation time (h) 0

3

6 9 Control

3 6 9 + 0.5 mM E-64

Inhibitor concentration

0 mM 0

Incubation time (h)

4

3

3

3

0

5

10

+ EDTA (mM) 3 3 3

0

2

4

+ AEBSF (mM) 3 3 3

90 0h

3h

6h

9h

100 80 60 40 20

Percentage of residual 24 kDa oleosin (%)

120 Percentage of residual 24 kDa oleosin (%)

2

+ Pepstatin A (µM)

80

0

2 4 µM

0 2

0

4 mM

70 60 50

10 mM 5

40 30 20 10 0

0 Control

E64 E-64

Pepstatin A

(A)

EDTA

AEBSF

(B)

Figure 6.

31



Page 32 of 34

kDa 75 50

kDa 75 50

kDa 75 50

37

37

25

25

20

20

15

15

10

10

37 25 20

24 kDa oleosin

15

C 1 3 6 1 3 6 + DTT 0 mM 1 mM

C 1 3 6 1 3 6 + NEM 0 mM 1 mM

1 3 6 2 mM

120

1 mM DTT 100

2 mM DTT

80 60 40 20 0


0 mM DTT

120 Percentage of residual 24 kDa oleosin (%)

Incubation time (h)

1

3 6 2 mM

C 1

0 mM NEM 1 mM NEM

100

2 mM NEM 80 60 40 20

1

2

3

4

Incubation time (h)

(A)

5

6

1

3

6

0 mM

2 mM

0 mM IAA

120

1 mM IAA 100

2 mM IAA

80 60 40 20 0

0 0

3 6 1 3 6

1 mM

+ IAA


10

0

1

2 3 4 5 Incubation time (h)

6

(B)

0

1

2

3

4

5

6

Incubation time (h)

(C)

Figure 7.

32


Page 33 of 34


kDa P32-24 kDa oleosin

75 50 37

P32-HP1 + Bg7S Bg7S

25 20

24 kDa oleosin

15

18 kDa oleosin 16 kDa oleosin + HP1

10

HP2 HP3

Incubation time (h) 0

1

2

3

4

5

6

0

1

(A)

2

3

4

5

6

0

1

2

(B)

α chain β chain

P32 18 kDa oleosin 16 kDa oleosin + HP1

HP1

HP1

18 kDa oleosin 16 kDa oleosin + HP1

Percentage of residual band intensity (%)

P32

4

5

6

(C) Total 24 kDa oleosin 24 kDa oleosin monomer P32-24 kDa oleosin

120 Bg7S

3

100 80 60 40 20 0 0

1

2

3

4

5

6

Incubation time (h)

(D)

(E)

(F)

Figure 8.

33



Page 34 of 34

TOC Graphics

34


Soybean P34 Probable Thiol Protease Probably Has Proteolytic

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