人肽基脯氨酰顺反异构酶cyclophilin的纯化及蛋白质交联的初步研究
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摘要
蛋白质分子间的交联是普遍存在的现象。它是多种生理和病理过程的共同特征。然而，由蛋白质的单体或功能形式转变为交联的二聚体或多聚体的分子机理还不太清楚。溶菌酶、核糖核酸酶A和蛋白质二硫键异构酶的体外实验显示，蛋白质交联可经三步完成：1)蛋白质构象包括二级结构改变；2)形成分子间二硫键；3)形成分子间异肽键。为了进一步探索蛋白质交联途径的三步假说的可行性，以及交联能否在异源肽链间发生，本文以人肽基脯氨酰顺反异构酶cyclophilin为模型展开了深入地研究。用RT-PCR方法从中国人胚肝中克隆了hPPI cyclophilin cDNA基因，并构建pTrcHisC-PPI重组质粒在大肠杆菌中高效表达，应用(His)_6亲和标签的固定化金属亲和层析技术纯化出了hPPI cyclophilin蛋白。将hPPI cyclophilin和lysozyme蛋白进行热变性交联实验，结果显示：在热变性的临界温度时，同源和异源的肽链间都有交联的二聚体和多聚体形成；而低温条件下，却没有交联出现；在热变性样品中加入二硫键还原剂β-巯基乙醇后，也检测不到任何交联。很明显，只有当蛋白质的二级结构改变到一定程度，才会发生交联；而且二硫键交联的形成对异肽键交联的形成起促进作用。这些都为蛋白质交联途径的三步假说提供了一些证据。
Protein interchain cross-linking is a common phenomenon. It has been implicated in several physiological and pathological processes. However the molecular mechanisms for converting monomeric/functional forms of proteins into cross-linked dimer/oligomer forms are not well understood. Experiments with lysozyme, ribonuclease A and protein disulfide isomerase in vitro show protein cross-linking can be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links. For further exploring if the three-step hypothesis of protein cross-linking is right, and if cross-linking can happen hi heterogeneous peptides, the investigation is extended with human peptidylprolyl-cis-trans-isomerase cyclophilin (hPPI cyclophilin). hPPI cyclophilin cDNA was cloned by RT-PCR from Chinese human fetal liver, and inserted into plasmid pTrcHisC for overexpression in E. coli. Then it was purified by Immobilized Metal Affinity
Chromatography with the help of 6*His affinity tag. At last, hPPI cyclophilin and lysozyme were mixed during thermal unfolding. The observation revealed that heterodimer/oligomer formation was as common as homodimer/oligomer formation in the midpoint temperature of thermal denaturation. However no cross-linked dimer/oligomer appeared in low temperature. If the thermal unfolding solution contained the reducing agent P -Mercaptoethanol, no cross-linking was detected. It was apparent that cross-linking can't happen until the protein secondary structure had changed to a certain extent, and that preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links. All these results confirmed the three-step hypothesis of protein cross-linking.
Protein interchain cross-linking is a common phenomenon. It has been implicated in several physiological and pathological processes. However the molecular mechanisms for converting monomeric/functional forms of proteins into cross-linked dimer/oligomer forms are not well understood. Experiments with lysozyme, ribonuclease A and protein disulfide isomerase in vitro show protein cross-linking can be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links. For further exploring if the three-step hypothesis of protein cross-linking is right, and if cross-linking can happen hi heterogeneous peptides, the investigation is extended with human peptidylprolyl-cis-trans-isomerase cyclophilin (hPPI cyclophilin). hPPI cyclophilin cDNA was cloned by RT-PCR from Chinese human fetal liver, and inserted into plasmid pTrcHisC for overexpression in E. coli. Then it was purified by Immobilized Metal Affinity
Chromatography with the help of 6*His affinity tag. At last, hPPI cyclophilin and lysozyme were mixed during thermal unfolding. The observation revealed that heterodimer/oligomer formation was as common as homodimer/oligomer formation in the midpoint temperature of thermal denaturation. However no cross-linked dimer/oligomer appeared in low temperature. If the thermal unfolding solution contained the reducing agent P -Mercaptoethanol, no cross-linking was detected. It was apparent that cross-linking can't happen until the protein secondary structure had changed to a certain extent, and that preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links. All these results confirmed the three-step hypothesis of protein cross-linking.
引文
1 Takahashi, N., Takahashi, Y., and Putnam, F. W. Primary structure of blood coagulation factor ⅩⅢa (fibrinoligase, transglutaminase) from human placenta. Proc.Natl. Acad. Sci. 1986, 83:8019-8023
2 Chen, J. S., and Mehta, K. Tissue transglutaminase: an enzyme with a split personality. Int. J.Biochem. Cell Biol. 1999, 31: 817-836
3 Rice, R. H., and Creen, H. J. Relation of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte. J Cell Biol. 1978, 76:705-711
4 Baden, H.P. Common transglutaminase substrates shared by hair, epidermis and nail and their function. J. Dermatol. Sci. 1994, S20-26
5 倪新华，江波，王璋．谷氨酰胺转胺酶在小麦粉制品中的应用．食品工业．2002(5)：6-8
6 John Leader. Protein crosslinking-the chemistry of cataracts and croissants. Marsden fund newsletter. 2002
7 Lorand, L., Hsu, L. K., Siefring, G. E. Jr., and Rafferty, N. S. Lens transglutaminase and cataract formation. Proc. Natl. Acad. Sci. 1981, 78:1356-1360
8 Ajit sadana, Tuan Vo-Dinh. Biomedical implication of folding and misfolding. J. Biotechnol. Appl. Biochem, 2001, 33:7-16
9 Mendoza JA, Rogers E, Lorimer GH, et al. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J. Biol. Chem. 1991,266:13044-13049.
10 Bellotti V, Mangione P, Stoppini M. Biological activity and pathological implications of misfolded protein. J. Cell Mol Life Sci. 1999, 55: 977-991.
11 Jen Ls, Hart A J, Relvas JB, et al. Alzheimer's peptide Kills cells of retina in vivo. J.Nature. 1998, 392: 140-141.
12 Gao Y, Mehta K. Interchain disulfide bonds promote protein cross-linking during protein folding. J biochem. 2001, 129(1): 179-183.
13 Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., Prusiner, S. B. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. 1993, 90: 10962-10966.
14 Perutz, M. F., Johnson, T., Suzuki, M., Finch, J. T. Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl. Acad. Sci. 1994, 91: 5355-5358.
15 张守印，谢春雷，王克强，张玉彬．牛海绵状脑病研究进展．吉林畜牧兽医．2002(7)：10-11．
16 Safar J, Roller PP, Gajdusek DC, Gibbs CJ. Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Science. 1993, 2: 2206-2216.
17 Huang Z, Prusiner SB, Cohen FE. Structures of prion proteins and conformational models for prion diseases. Curr Top Microbiol Immunol. 1996, 207: 49-67.
18 蒋明，沈涛，徐辉碧，刘长林．金属离子对蛋白质的折叠、识别、自组装及功能的影响．化学进展．2002，14(4)：263-272．
19 Green, H. Human genetic diseases due to codon reiteration: relationship to an evolutionary mechanism. Cell. 1993, 74: 955-956.
20 Lorand, L. Neurodegenerative diseases and transglutaminase. Proc. Natl. Acad. Sci.1996, 93: 14310-14313.
21 Chen X, Chen YH, Anderson VE. Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry. Anal Biochem. 1999, 273(2): 192-203.
22 Sollid, L. M. Coeliac disease: dissecting a complex inflammatory disorder. Nature Reviews Immunology. 2002, 2: 647-655.
23 L. Mangiarini et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell.1996, 87:493-506.
24 Violante V, Luongo A, Pepe I, Annunziata S, Gentile V. Transglutaminase-dependent formation of protein aggregates as possible biochemical mechanism for polyglutamine disease. Brain Res Bull. 2001, 56(3-4): 169-172.
25 Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol. 1995, 7: 215-223.
26 Ciechanover A, Schwartz AL. The ubiquitin-proteasome pathway: the complexity and myriad functions of proteins death. Proc Natl Acad Sci. 1998, 95(6): 27-30.
27 Puig, A., Primm, T.P., Surendran, R., Lee, J.C., Ballard, K.D., Orkiszewski, R.S., Makarov, V., and Gilbert, H.F. A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. J. Biol. Chem. 1997, 272, 32988-32994
28 万平．蛋白质二硫键异构酶分子间交联与其酶活性的关系．首都师范大学硕士学位论文．2001
29 邹承鲁．第二遗传密码——新生肽链及蛋白质折叠的研究．湖南：科技出版社，1982
30 Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 2002, 295:1852-1858
31 Hartl FU. Molecular chaperones in cellular protein folding. J. Nature. 1996, 381:571-580.
32 Landry. The Process of Protein Folding. Biochemistry, 1999,601
33 王志珍．新生肽链折叠的新概念．生命科学．1996，8(5)：5-10
34 徐明波，孟文华，马贤凯．脯氨酰顺-反异构酶的纯化及对重组蛋白质折叠反应的催化性能．生物化学与生物物理进展．1994，21(3)：247-251
35 Fischer G, Bang H, Mech C. Nachweis einer enzymkatalyse fur die cis-trans-isomerisierung der peptidbindung in prolinhaltigen peptiden. Biomed. Biochim. Acta.1984, 43:1101-1111
36 Jackson, S.E. and Fersht, A.R. Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding. Biochemistry. 1991, 30:10436-10443
37 Hayano T, et al. Two distinct form of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells. Biochemistry. 1991, 30:3041-3048
38 Yu HS, Kong HH, Chung DI. Cloning and characterization of Giardia intestinalis cyclophilin. Korean J Parasitol. 2002, 40(3): 131-138.
39 Berriman M, Fairlamb AH. Detailed characterization of a cyclophilin from the human malaria. Biochem J. 1998, 334 (Pt 2): 437-445
40 Gaston M.U. Pfluegl, Joerg Kallen, Tilman Schirmer, Johan N. Jansonius, Mauro G.M. Zurini and Malcolm D. Walkinshaw. X-ray structure of a decameric cyclophilin-cyclosporin crystla complex. Nature. 1993, 361: 91-94
41 Liu J, et al. Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis. Proc. Natl. Acad. Sci. 1990, 87:2304-2308
42 Haendler B, Hofer-Warbinek R, Hofer E. Complementary DNA for human T-cell cyclophilin. EMBO J. 1987, 6:947-950
43 Quemeneur E, et al. Engineering cyclophilin into a proline-specific endopeptidase. Nature. 1998, 391:301-304
44 李福胜，贡惠宇，李玉富，张智清，侯云德．表达产物可用IMAC快速纯化的原核表达载体的组建与应用．生物工程学报，1997，13(1)：42-46
45 TALON Metal Affinity Resins User Manual. 2000, 22-24
46 高音，杨志伟，华振玲，高虹，万平，黎红晔．人蛋白质二硫键异构酶cDNA基因的克隆及在大肠杆菌中的表达．生物工程学报，1999，15：349-354
47 高音，赵琦，万平，何红伟，杨志伟，华振玲．甜味蛋白thaumatin cDNA基因的合成与筛选．首都师范大学学报．2000，21：71-74
48 何红伟．蛋白质二硫键异构酶分子间异肽键交联的研究．首都师范大学硕士学位论文．2002
49 Labhardt, A.M. Secondary structure in ribonuclease: Equilibrium folding transitions seen by amide circular dichroism. J. Mol. Biol. 1982, 157:331-355
50 W.H. Freeman and Company. Protein folding. Library of Congress Cataloging-in-Publication Data. 1992:230-233
51 Kopito R R, Ron D. Conformational disease. Nat Cell Biol. 2000, 2:E207-E209
52 Carrell, R. W., and Lomas, D. A. Conformational disease. Lancet, 1997, 350(9071):134-138
53 陈岚，许彩民，潘华珍．蛋白质构象病提示的疾病防治新思路．生命的化学，2002，2，103-106
2 Chen, J. S., and Mehta, K. Tissue transglutaminase: an enzyme with a split personality. Int. J.Biochem. Cell Biol. 1999, 31: 817-836
3 Rice, R. H., and Creen, H. J. Relation of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte. J Cell Biol. 1978, 76:705-711
4 Baden, H.P. Common transglutaminase substrates shared by hair, epidermis and nail and their function. J. Dermatol. Sci. 1994, S20-26
5 倪新华，江波，王璋．谷氨酰胺转胺酶在小麦粉制品中的应用．食品工业．2002(5)：6-8
6 John Leader. Protein crosslinking-the chemistry of cataracts and croissants. Marsden fund newsletter. 2002
7 Lorand, L., Hsu, L. K., Siefring, G. E. Jr., and Rafferty, N. S. Lens transglutaminase and cataract formation. Proc. Natl. Acad. Sci. 1981, 78:1356-1360
8 Ajit sadana, Tuan Vo-Dinh. Biomedical implication of folding and misfolding. J. Biotechnol. Appl. Biochem, 2001, 33:7-16
9 Mendoza JA, Rogers E, Lorimer GH, et al. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J. Biol. Chem. 1991,266:13044-13049.
10 Bellotti V, Mangione P, Stoppini M. Biological activity and pathological implications of misfolded protein. J. Cell Mol Life Sci. 1999, 55: 977-991.
11 Jen Ls, Hart A J, Relvas JB, et al. Alzheimer's peptide Kills cells of retina in vivo. J.Nature. 1998, 392: 140-141.
12 Gao Y, Mehta K. Interchain disulfide bonds promote protein cross-linking during protein folding. J biochem. 2001, 129(1): 179-183.
13 Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., Prusiner, S. B. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. 1993, 90: 10962-10966.
14 Perutz, M. F., Johnson, T., Suzuki, M., Finch, J. T. Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl. Acad. Sci. 1994, 91: 5355-5358.
15 张守印，谢春雷，王克强，张玉彬．牛海绵状脑病研究进展．吉林畜牧兽医．2002(7)：10-11．
16 Safar J, Roller PP, Gajdusek DC, Gibbs CJ. Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Science. 1993, 2: 2206-2216.
17 Huang Z, Prusiner SB, Cohen FE. Structures of prion proteins and conformational models for prion diseases. Curr Top Microbiol Immunol. 1996, 207: 49-67.
18 蒋明，沈涛，徐辉碧，刘长林．金属离子对蛋白质的折叠、识别、自组装及功能的影响．化学进展．2002，14(4)：263-272．
19 Green, H. Human genetic diseases due to codon reiteration: relationship to an evolutionary mechanism. Cell. 1993, 74: 955-956.
20 Lorand, L. Neurodegenerative diseases and transglutaminase. Proc. Natl. Acad. Sci.1996, 93: 14310-14313.
21 Chen X, Chen YH, Anderson VE. Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry. Anal Biochem. 1999, 273(2): 192-203.
22 Sollid, L. M. Coeliac disease: dissecting a complex inflammatory disorder. Nature Reviews Immunology. 2002, 2: 647-655.
23 L. Mangiarini et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell.1996, 87:493-506.
24 Violante V, Luongo A, Pepe I, Annunziata S, Gentile V. Transglutaminase-dependent formation of protein aggregates as possible biochemical mechanism for polyglutamine disease. Brain Res Bull. 2001, 56(3-4): 169-172.
25 Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol. 1995, 7: 215-223.
26 Ciechanover A, Schwartz AL. The ubiquitin-proteasome pathway: the complexity and myriad functions of proteins death. Proc Natl Acad Sci. 1998, 95(6): 27-30.
27 Puig, A., Primm, T.P., Surendran, R., Lee, J.C., Ballard, K.D., Orkiszewski, R.S., Makarov, V., and Gilbert, H.F. A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. J. Biol. Chem. 1997, 272, 32988-32994
28 万平．蛋白质二硫键异构酶分子间交联与其酶活性的关系．首都师范大学硕士学位论文．2001
29 邹承鲁．第二遗传密码——新生肽链及蛋白质折叠的研究．湖南：科技出版社，1982
30 Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 2002, 295:1852-1858
31 Hartl FU. Molecular chaperones in cellular protein folding. J. Nature. 1996, 381:571-580.
32 Landry. The Process of Protein Folding. Biochemistry, 1999,601
33 王志珍．新生肽链折叠的新概念．生命科学．1996，8(5)：5-10
34 徐明波，孟文华，马贤凯．脯氨酰顺-反异构酶的纯化及对重组蛋白质折叠反应的催化性能．生物化学与生物物理进展．1994，21(3)：247-251
35 Fischer G, Bang H, Mech C. Nachweis einer enzymkatalyse fur die cis-trans-isomerisierung der peptidbindung in prolinhaltigen peptiden. Biomed. Biochim. Acta.1984, 43:1101-1111
36 Jackson, S.E. and Fersht, A.R. Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding. Biochemistry. 1991, 30:10436-10443
37 Hayano T, et al. Two distinct form of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells. Biochemistry. 1991, 30:3041-3048
38 Yu HS, Kong HH, Chung DI. Cloning and characterization of Giardia intestinalis cyclophilin. Korean J Parasitol. 2002, 40(3): 131-138.
39 Berriman M, Fairlamb AH. Detailed characterization of a cyclophilin from the human malaria. Biochem J. 1998, 334 (Pt 2): 437-445
40 Gaston M.U. Pfluegl, Joerg Kallen, Tilman Schirmer, Johan N. Jansonius, Mauro G.M. Zurini and Malcolm D. Walkinshaw. X-ray structure of a decameric cyclophilin-cyclosporin crystla complex. Nature. 1993, 361: 91-94
41 Liu J, et al. Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis. Proc. Natl. Acad. Sci. 1990, 87:2304-2308
42 Haendler B, Hofer-Warbinek R, Hofer E. Complementary DNA for human T-cell cyclophilin. EMBO J. 1987, 6:947-950
43 Quemeneur E, et al. Engineering cyclophilin into a proline-specific endopeptidase. Nature. 1998, 391:301-304
44 李福胜，贡惠宇，李玉富，张智清，侯云德．表达产物可用IMAC快速纯化的原核表达载体的组建与应用．生物工程学报，1997，13(1)：42-46
45 TALON Metal Affinity Resins User Manual. 2000, 22-24
46 高音，杨志伟，华振玲，高虹，万平，黎红晔．人蛋白质二硫键异构酶cDNA基因的克隆及在大肠杆菌中的表达．生物工程学报，1999，15：349-354
47 高音，赵琦，万平，何红伟，杨志伟，华振玲．甜味蛋白thaumatin cDNA基因的合成与筛选．首都师范大学学报．2000，21：71-74
48 何红伟．蛋白质二硫键异构酶分子间异肽键交联的研究．首都师范大学硕士学位论文．2002
49 Labhardt, A.M. Secondary structure in ribonuclease: Equilibrium folding transitions seen by amide circular dichroism. J. Mol. Biol. 1982, 157:331-355
50 W.H. Freeman and Company. Protein folding. Library of Congress Cataloging-in-Publication Data. 1992:230-233
51 Kopito R R, Ron D. Conformational disease. Nat Cell Biol. 2000, 2:E207-E209
52 Carrell, R. W., and Lomas, D. A. Conformational disease. Lancet, 1997, 350(9071):134-138
53 陈岚，许彩民，潘华珍．蛋白质构象病提示的疾病防治新思路．生命的化学，2002，2，103-106