酶解大豆分离蛋白的抗原活性变化及其抗原表位分析
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摘要
研究酶解过程中大豆蛋白分子成分和抗原性变化规律及酶解物中是否存在抗原表位序列。用碱性蛋白酶水解大豆分离蛋白后,首先分别用电泳和酶联免疫法分析蛋白质分子组分和抗原活性变化,结果发现,酶解过程中新生成23 ku和21 ku抗酶解肽链直到酶解120min时仍未消失,同时酶解物中剩余28%抗原活性。进一步通过胰蛋白酶对这两个肽链胶内酶解并用基质辅助激光解析串联飞行时间质谱仪分析后,将所得肽段氨基酸序列分别与抗原数据库中抗原表位序列和大豆分离蛋白氨基酸序列进行匹配解析,结果发现,在21 ku组分中含有两个完整的序列抗原表位(LQRFNQRSPQLQNLR和SEDKPFNLRSRDPIYSNKLGKFFEITPEKN),且均来自β-伴大豆球蛋白中的α-亚基,21 ku组分抗原剩余率为15.7%;在23 ku肽链中含有抗原表位的部分氨基酸序列,但未发现含有完整的抗原表位序列,23 ku组分抗原剩余率为2.1%,该组分中是否含有新的未知抗原表位或是否与糖链有关尚待深入研究。上述研究结果揭示了大豆蛋白酶解物中仍残留抗原性的部分原因。
The aim of this study was to investigate the changes in the molecular composition and antigenicity of soybean protein during its enzymatic hydrolysis and whether there could be antigenic epitope sequences in the hydrolysate. The enzyme used was alcalase. The changes in molecular composition and antigenicity were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and competitive enzyme-linked immunosorbent assay(ELISA),respectively. Some new peptides(i.e., 23 and 21 ku) were found in the hydrolysate and did not disappear even after 120 min of hydrolysis. Meanwhile the hydrolysate still retained 28% of the original antigenicity. In order to determine whether the new peptide chains were related to the antigenicity, the proteomic bands in the SDS-PAGE gel were hydrolyzed by trypsin and then analyzed by matrix-assisted laser desorption/ionization time of ?ight mass spectrometry(MALDI-TOF-MS). The amino acid sequences of the peptide fragments obtained were matched with the amino acid sequences in the antigen database and the soybean protein isolate database, respectively. The results showed that there were two complete sequence epitopes(LQRFNQRSPQLQNLR and SEDKPFNLRSRDPIYSNKLGKFFEITPEKN) in the 21 ku component, both of which came from the α subunit in β-conglycinin. The residual antigenicity of this peptide fragment was 15.7%. The amino acid sequences of the 23 ku peptide chain were matched partially with those of epitopes but no complete epitopes were found. However, its residual antigenicity was 2.1%. These findings may partially account for the antigenicity of soybean protein hydrolysates.
The aim of this study was to investigate the changes in the molecular composition and antigenicity of soybean protein during its enzymatic hydrolysis and whether there could be antigenic epitope sequences in the hydrolysate. The enzyme used was alcalase. The changes in molecular composition and antigenicity were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and competitive enzyme-linked immunosorbent assay(ELISA),respectively. Some new peptides(i.e., 23 and 21 ku) were found in the hydrolysate and did not disappear even after 120 min of hydrolysis. Meanwhile the hydrolysate still retained 28% of the original antigenicity. In order to determine whether the new peptide chains were related to the antigenicity, the proteomic bands in the SDS-PAGE gel were hydrolyzed by trypsin and then analyzed by matrix-assisted laser desorption/ionization time of ?ight mass spectrometry(MALDI-TOF-MS). The amino acid sequences of the peptide fragments obtained were matched with the amino acid sequences in the antigen database and the soybean protein isolate database, respectively. The results showed that there were two complete sequence epitopes(LQRFNQRSPQLQNLR and SEDKPFNLRSRDPIYSNKLGKFFEITPEKN) in the 21 ku component, both of which came from the α subunit in β-conglycinin. The residual antigenicity of this peptide fragment was 15.7%. The amino acid sequences of the 23 ku peptide chain were matched partially with those of epitopes but no complete epitopes were found. However, its residual antigenicity was 2.1%. These findings may partially account for the antigenicity of soybean protein hydrolysates.
引文
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[10]刘晓毅,薛文通,胡小苹,等.酶解法专一性去除大豆7S球蛋白中的α-亚基[J].食品科技,2005,30(8):16-18.DOI:10.13684/j.cnki.spkj.2005.08.005.
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[13]王俊,潘丽,赵元,等.大豆抗原蛋白β-伴大豆球蛋白抗酶解肽的分离纯化及其免疫活性鉴定[J].中国畜牧杂志,2012,48(23):53-56.
[14]王锦欣,秦贵信,龙国徽,等.β-伴大豆球蛋白酶解物的抗消化性及免疫活性变化的研究[J].中国畜牧杂志,2014,50(9):45-49.DOI:10.3969/j.issn.0258-7033.2014.09.011.
[15] KEUM E H, LEE S I. Effect of enzymatic hydrolysis of 7S globulin, a soybean protein, on its allergenicity and identification of its allergenic hydrolyzed fragments using SDS-PAGE[J]. Food Science and Biotechnology, 2006, 15(1): 128-132.
[16] LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227: 680-685.
[17] ASADI M, REZAEI BORJLOU S, FARAHANI H F, et al. Carbohydrate moieties on the in vitro, immunoreactivity of soy β-conglycinin[J]. Food Research International, 2009, 42(7): 819-825. DOI:10.1016/j.foodres.2009.03.003.
[18] PICARIELLO G, AMIGO-BENAVENT M, DEL CASTILLO M D, et al. Structural characterization of the N-glycosylation of individual soybean β-conglycinin subunits[J]. Journal of Chromatography A, 2013,1313(9):96-102.DOI:10.1016/j.chroma.2013.09.014.
[19] AMIGO-BENAVENT M, CLEMENTE A, FERRANTI P, et al. Digestibility and immunoreactivity of soybean β-conglycinin and its deglycosylated form[J]. Food Chemistry, 2011, 129(4): 1598-1605. DOI:10.1016/j.foodchem.2011.06.015.
[20]单晓红,孙秀兰,管露,等.大豆主要过敏原Gly m Bd 30K抗原决定簇表征预测研究[J].分析科学学报,2012,28(6):771-774.
[21]黄婷,布冠好,陈复生.酶解改性对大豆蛋白抗原性的影响研究[J].河南工业大学学报(自然科学版),2016,37(4):11-17.DOI:10.16433/j.cnki.issn1673-2383.2016.04.003.
[22]闫慧丽,席俊,贺梦雪,等.大豆主要过敏原Gly m Bd 28K的同源建模和B细胞表位的预测[J].河南工业大学学报(自然科学版),2016,37(4):41-45.DOI:10.16433/j.cnki.issn1673-2383.2016.04.008.
[23] BURKS A W, BROOKS J R, SAMPSON H A. Allergenicity of major component proteins of soybean determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting in children with atopic dermatitis and positive soy challenges[J]. Allergy Clincal Immunology, 1988, 81(6):1135-1142.DOI:10.1016/0091-6749(88)90881-0.
[24] PEDERSEN H S, DJURTOFT R. Antigenic and allergenic properties of acidic and basic peptide chains from glycinin[J]. Food and Agricultural Immunology, 1989, 1(2): 101-109. DOI:10.1080/09540108909354680.
[25] ADACHI M, KANAMORI J, MASUDA T, et al. Crystal structure of soybean 11S globulin: glycinin A3B4,homohexamer[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(12):7395-7400.DOI:10.1073/pnas.0832158100.
[26] LI L, WANG C, QIANG S, et al. Mass spectrometric analysis of N-glycoforms of soybean allergenic glycoproteins separated by SDS-PAGE[J]. Journal of Agricultural & Food Chemistry, 2016, 64(39): 7367-7376.DOI:10.1021/acs.jafc.6b02773.
[27] F?TISCH K, ALTMANN F, HAUSTEIN D, et al. Involvement of carbohydrate epitopes in the Ig E response of celery-allergic patients[J]. International Archives of Allergy & Immunology, 1999, 120(1): 30-42. DOI:10.1159/000024217.
[28] VAN R R. Carbohydrate epitopes and their relevance for the diagnosis and treatment of allergic diseases[J]. International Archives of Allergy and Immunology, 2002, 129(3): 189-197. DOI:10.1159/000066770.
[29]李玲梅,王承健,韩健利,等.β-伴大豆球蛋白的N-糖基化位点及其N-糖链的质谱分析[J].高等学校化学学报,2018,39(3):427-434.DOI:10.7503/cjcu20170584.
[30] AMIGOBENAVENT M, CLEMENTE A, CASTILLO M D D. Digestibility and immunoreactivity of soybean β-conglycinin and its deglycosylated form[J]. Food Chemistry, 2011, 129(4): 1598-1605. DOI:10.1016/j.foodchem.2011.06.015.
[31] MIRYAM A, VASILEOS I A, PASQUALE E, et al. Carbohydrate moieties on the in vitro, immunoreactivity of soy β-conglycinin[J]. Food Research International, 2009, 42(7): 819-825. DOI:10.1016/j.foodres.2009.03.003.
[2] NATARAJAN S, KHAN F H, SONG Q, et al. Characterization of soybean storage and allergen proteins affected by environmental and genetic factors[J]. Journal of Agricultural and Food Chemistry, 2016, 64(6):1433-1445.DOI:10.1021/acs.jafc.5b05172.
[3] MEINLSCHMIDT P, UEBERHAM E, LEHMANN J, et al. Immunoreactivity, sensory and physicochemical properties of fermented soy protein isolate[J]. Food Chemistry, 2016, 205(15): 229-238.DOI:10.1016/j.foodchem.2016.03.016.
[4] KRISHNAN H B, KIM W S, ANG S C, et al. All three subunits of soybean β-conglycinin are potential food allergens[J]. Journal of Agricultural and Food Chemistry, 2011, 57(3): 938-943. DOI:10.1021/jf802451g.
[5] SUN P, LI D F, LI Z J, et al. Effects of glycinin on IgE-mediated increase of mast cell numbers and histamine release in the small intestine[J]. The Journal of Nutritional Biochemistry, 2008, 19(9): 627-633.DOI:10.1016/j.jnutbio.2007.08.007.
[6] LIU X, FENG J, XU Z R, et al. Oral allergy syndrome and anaphylactic reactions in BALB mice caused by soybean glycinin and beta-conglycinin[J]. Clinical and Experimental Allergy, 2008, 38(2): 350-356.DOI:10.1111/j.1365-2222.2007.02893.
[7]吴淑清,王顺余,黄玉珍.热处理方法对大豆中抗营养因子的钝化作用[J].长春大学学报,2009,19(4):64-65.DOI:10.3969/j.issn.1009-3907-B.2009.02.023.
[8]黄婷,布冠好,陈复生.碱性蛋白酶对β-伴大豆球蛋白抗原性的影响[J].粮食与油脂,2018,31(2):54-59.
[9] WANG Z C, LI L J, YUAN D Q, et al. Reduction of the allergenic protein in soybean meal by enzymatic hydrolysis[J]. Food and Agricultural Immunology, 2014, 25(3): 301-310. DOI:10.1080/09540105.2013.782268.
[10]刘晓毅,薛文通,胡小苹,等.酶解法专一性去除大豆7S球蛋白中的α-亚基[J].食品科技,2005,30(8):16-18.DOI:10.13684/j.cnki.spkj.2005.08.005.
[11] SHUTOV A, RUDAKOVA A, RUDAKOV S, et al. Limited proteolysis regulates massive degradation of glycinin, storage 11S globulin from soybean seeds: an in vitro model[J]. Journal of Plant Physiology, 2012, 169(13):1227-1233.DOI:10.1016/j.jplph.2012.06.004.
[12]王章存,李乐静,赵学伟,等.碱性蛋白酶水解对豆粕中大豆抗原蛋白的影响[J].中国粮油学报,2013,28(2):6-9.DOI:10.3969/j.issn.1003-0174.2013.02.002.
[13]王俊,潘丽,赵元,等.大豆抗原蛋白β-伴大豆球蛋白抗酶解肽的分离纯化及其免疫活性鉴定[J].中国畜牧杂志,2012,48(23):53-56.
[14]王锦欣,秦贵信,龙国徽,等.β-伴大豆球蛋白酶解物的抗消化性及免疫活性变化的研究[J].中国畜牧杂志,2014,50(9):45-49.DOI:10.3969/j.issn.0258-7033.2014.09.011.
[15] KEUM E H, LEE S I. Effect of enzymatic hydrolysis of 7S globulin, a soybean protein, on its allergenicity and identification of its allergenic hydrolyzed fragments using SDS-PAGE[J]. Food Science and Biotechnology, 2006, 15(1): 128-132.
[16] LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227: 680-685.
[17] ASADI M, REZAEI BORJLOU S, FARAHANI H F, et al. Carbohydrate moieties on the in vitro, immunoreactivity of soy β-conglycinin[J]. Food Research International, 2009, 42(7): 819-825. DOI:10.1016/j.foodres.2009.03.003.
[18] PICARIELLO G, AMIGO-BENAVENT M, DEL CASTILLO M D, et al. Structural characterization of the N-glycosylation of individual soybean β-conglycinin subunits[J]. Journal of Chromatography A, 2013,1313(9):96-102.DOI:10.1016/j.chroma.2013.09.014.
[19] AMIGO-BENAVENT M, CLEMENTE A, FERRANTI P, et al. Digestibility and immunoreactivity of soybean β-conglycinin and its deglycosylated form[J]. Food Chemistry, 2011, 129(4): 1598-1605. DOI:10.1016/j.foodchem.2011.06.015.
[20]单晓红,孙秀兰,管露,等.大豆主要过敏原Gly m Bd 30K抗原决定簇表征预测研究[J].分析科学学报,2012,28(6):771-774.
[21]黄婷,布冠好,陈复生.酶解改性对大豆蛋白抗原性的影响研究[J].河南工业大学学报(自然科学版),2016,37(4):11-17.DOI:10.16433/j.cnki.issn1673-2383.2016.04.003.
[22]闫慧丽,席俊,贺梦雪,等.大豆主要过敏原Gly m Bd 28K的同源建模和B细胞表位的预测[J].河南工业大学学报(自然科学版),2016,37(4):41-45.DOI:10.16433/j.cnki.issn1673-2383.2016.04.008.
[23] BURKS A W, BROOKS J R, SAMPSON H A. Allergenicity of major component proteins of soybean determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting in children with atopic dermatitis and positive soy challenges[J]. Allergy Clincal Immunology, 1988, 81(6):1135-1142.DOI:10.1016/0091-6749(88)90881-0.
[24] PEDERSEN H S, DJURTOFT R. Antigenic and allergenic properties of acidic and basic peptide chains from glycinin[J]. Food and Agricultural Immunology, 1989, 1(2): 101-109. DOI:10.1080/09540108909354680.
[25] ADACHI M, KANAMORI J, MASUDA T, et al. Crystal structure of soybean 11S globulin: glycinin A3B4,homohexamer[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(12):7395-7400.DOI:10.1073/pnas.0832158100.
[26] LI L, WANG C, QIANG S, et al. Mass spectrometric analysis of N-glycoforms of soybean allergenic glycoproteins separated by SDS-PAGE[J]. Journal of Agricultural & Food Chemistry, 2016, 64(39): 7367-7376.DOI:10.1021/acs.jafc.6b02773.
[27] F?TISCH K, ALTMANN F, HAUSTEIN D, et al. Involvement of carbohydrate epitopes in the Ig E response of celery-allergic patients[J]. International Archives of Allergy & Immunology, 1999, 120(1): 30-42. DOI:10.1159/000024217.
[28] VAN R R. Carbohydrate epitopes and their relevance for the diagnosis and treatment of allergic diseases[J]. International Archives of Allergy and Immunology, 2002, 129(3): 189-197. DOI:10.1159/000066770.
[29]李玲梅,王承健,韩健利,等.β-伴大豆球蛋白的N-糖基化位点及其N-糖链的质谱分析[J].高等学校化学学报,2018,39(3):427-434.DOI:10.7503/cjcu20170584.
[30] AMIGOBENAVENT M, CLEMENTE A, CASTILLO M D D. Digestibility and immunoreactivity of soybean β-conglycinin and its deglycosylated form[J]. Food Chemistry, 2011, 129(4): 1598-1605. DOI:10.1016/j.foodchem.2011.06.015.
[31] MIRYAM A, VASILEOS I A, PASQUALE E, et al. Carbohydrate moieties on the in vitro, immunoreactivity of soy β-conglycinin[J]. Food Research International, 2009, 42(7): 819-825. DOI:10.1016/j.foodres.2009.03.003.