MHC-Ⅰ类肽组装复合体组分重要性的定量分析(英文)
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摘要
TAP2、tapasin和calreticulin(CRT)是MHC-Ⅰ类内源性抗原加工和递呈途径肽组装复合体(PLC)中的3个重要蛋白质.虽然对于肽组装复合体及其单个蛋白组分在抗原加工递呈中的作用,国内外已有大量的研究,但是由于研究手段的缺乏,其在复合体中的相对重要性还未得到清楚和定量的分析.本文中我们建立了一种新型的抗原递送方法,利用猪链球菌溶血酶(SLY)对鼠纤维母细胞系K41及其CRT缺陷细胞系K42、tapasin缺陷细胞系90a及tapasin、TAP2双缺陷细胞系91a进行穿孔并定量递送入鸡卵清白蛋白(ovalbumin,OVA),使其被内源性加工处理后的多肽SIINFEKL与小鼠H2-Kb装配成复合物并被递呈至细胞表面.通过利用小鼠T杂交瘤细胞B3Z和单克隆抗体25D1.16识别并检测不同细胞表面SIINFEKL与小鼠H2-Kb复合物,我们发现CRT缺陷细胞系K42的抗原提呈能力相比91a及90a,较K41有着更大幅度的下降,说明CRT在提呈过程中可能有着较预期更重要的作用.本文是首个对抗原递呈途径蛋白相对重要性的研究,其研究结果对于进一步研究MHC-Ⅰ类抗原加工和递呈途径相关蛋白的作用提供了理论依据与研究手段.
TAP2, tapasin and calreticulin(CRT) are three crucial components of the peptide loading complex(PLC) in MHC class Ⅰantigen processing and presentation. While the functions of the proteins within the complex have been progressively defined, the hierarchical importance of the components has not been clearly and quantitatively analyzed. To compare the efficiency of MHC class Ⅰantigen processing in different cell lines, we established a novel suilysin-mediated antigen delivery method to quantitatively analyze antigen processing in a number of cell lines, including K41, the CRT-deficient line K42, the tapasin-deficient line 90 a and the tapasin/TAP2-deficient cell line 91 a. We ranked the importance of the components of MHC class Ⅰ antigen processing such as CRT,TAP and tapasin using OVA challenge as the antigen model. Surprisingly, CRT was shown to be the most important component of the PLC, even though TAP was speculated to be the most crucial protein involved in the pathway. This is the first report that ranks the components of the antigen-processing pathway. Further investigation is required for elucidation of the roles played by the individual proteins within the complex.
TAP2, tapasin and calreticulin(CRT) are three crucial components of the peptide loading complex(PLC) in MHC class Ⅰantigen processing and presentation. While the functions of the proteins within the complex have been progressively defined, the hierarchical importance of the components has not been clearly and quantitatively analyzed. To compare the efficiency of MHC class Ⅰantigen processing in different cell lines, we established a novel suilysin-mediated antigen delivery method to quantitatively analyze antigen processing in a number of cell lines, including K41, the CRT-deficient line K42, the tapasin-deficient line 90 a and the tapasin/TAP2-deficient cell line 91 a. We ranked the importance of the components of MHC class Ⅰ antigen processing such as CRT,TAP and tapasin using OVA challenge as the antigen model. Surprisingly, CRT was shown to be the most important component of the PLC, even though TAP was speculated to be the most crucial protein involved in the pathway. This is the first report that ranks the components of the antigen-processing pathway. Further investigation is required for elucidation of the roles played by the individual proteins within the complex.
引文
[1]Hughes E A,Cresswell P.The thiol oxidoreductase ERp57 is a component of the MHC class I peptide-loading complex.Curr Biol,1998,8(12):709-712
[2]Cresswell P,Bangia N,Dick T,et al.The nature of the MHC class I peptide loading complex.Immunol Rev,1999,172(1):21-28
[3]Townsend A,Bodmer H.Antigen recognition by class I-restricted T lymphocytes.Annu Rev Immunol,1989,7(7):601-624
[4]Williams A P,Peh C A,Purcell A W,et al.Optimization of the MHC classⅠpeptide cargo is dependent on tapasin.Immunity,2002,16(4):509-520
[5]Powis S J,Townsend A R,Deverson E V,et al.Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter.Nature,1991,354(6354):528-531
[6]Lankat-Buttgereit B,Tampe R.The transporter associated with antigen processing TAP:structure and function.FEBS Lett,1999,464(3):108-112
[7]Anderson K,Cresswell P,Gammon M,et al.Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis.J Exp Med,1991,174(2):489-492
[8]Gao B,Adhikari R,Howarth M,et al.Assembly and antigenpresenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin.Immunity,2002,16(1):99-109
[9]Farmery M R,Allen S,Allen A J,et al.The role of ERp57 in disulfide bond formation during the assembly of major histocompatibility complex classⅠin a synchronized semipermeabilized cell translation system.J Biol Chem,2000,275(20):14933-14938
[10]Peterson J R,Ora A,Van P N,et al.Transient,lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.Mol Biol Cell,1995,6(9):1173-1184
[11]Michalak M,Corbett E F,Mesaeli N,et al.Calreticulin:one protein,one gene,many functions.Biochem J,1999,344(pt2):281-292
[12]Sadasivan B,Lehner P J,Ortmann B et al.Roles for calreticulin and a novel glycoprotein,tapasin,in the interaction of MHC classⅠmolecules with TAP.Immunity,1996,5(2):103-114
[13]Grandea A G,Golovina T N,Hamilton S E,et al.Impaired assembly yet normal trafficking of MHC classⅠmolecules in Tapasin mutant mice.Immunity,2000,13(2):213-222
[14]Fu H,Ding J,Flutter B,et al.Investigation of endogenous antigen processing by delivery of an intact protein into cells.J Immunol Methods,2008,335(1-2):90-97
[15]Walev I,Bhakdi S C,Hofmann F,et al.Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.Proc Natl Acad Sci USA,2001,98(6):3185-3190
[16]Sanderson S,Shastri N.LacZ inducible,antigen/MHC-specific T cell hybrids.Int Immunol,1994,6(3):369-376
[17]Wu Y,Liu C,Sun M.et al.A specific cytotoxic T-lymphocyte epitope presentation system for antitumor immunity.Int J Cancer,2010,126(10):2373-2386
[18]Karttunen J,Sanderson S,Shastri N.Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens.Proc Natl Acad Sci USA,1992,89(13):6020-6024
[19]Porgador A,Yewdell J W,Deng Y,et al.Localization,quantitation,and in situ detection of specific peptide-MHC classⅠcomplexes using a monoclonal antibody.Immunity,1997,6(6):715-726
[20]Jones B,Janeway C A Jr.Cooperative interaction of B lymphocytes with antigen-specific helper T lymphocytes is MHC restricted.Nature,1981,292(5823):547-549
[21]Lewis J W,Sewell A,Price D,et al.HLA-A*0201 presents TAP-dependent peptide epitopes to cytotoxic T lymphocytes in the absence of tapasin.Eur J Immunol,1998,28(10):3214-3220
[22]Goldberg A L,Cascio P,Saric T,et al.The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.Mol Immunol,2002,39(3-4):147-164
[23]Brodsky F M,Guagliardi L E.The cell biology of antigen processing and presentation.Annu Rev Immunol,1991,9(9):707-744
[24]Ljunggren H G,Stam N J,Ohlén C,et al.Empty MHC classⅠmolecules come out in the cold.Nature,1990,346(6283):476-480
[25]Tan P,Kropshofer H,Mandelboim O,et al.Recruitment of MHC classⅠmolecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading.J Immunol,2002,168(4):1950-1960
[26]Panter M S,Jain A,Leonhardt R M,et al.Dynamics of major histocompatibility complex classⅠassociation with the human peptide-loading complex.J Biol Chem,2012,287(37):31172-31184
[27]Garstka M A,Fritzsche S,Lenart I,et al.Tapasin dependence of major histocompatibility complex classⅠmolecules correlates with their conformational flexibility.FASEB J,2011,25(11):3989-3998
[28]Wearsch P A,Cresswell P.Selective loading of high-affinity peptides onto major histocompatibility complex classⅠmolecules by the tapasin-ERp57 heterodimer.Nat Immunol,2007,8(8):873-881
[29]Praveen P V,Yaneva R,Kalbacher H,et al.Tapasin edits peptides on MHC classⅠmolecules by accelerating peptide exchange.Eur J Immunol,2010,40(1):214-224
[30]Karre K,Ljunggren H G,Piontek G,et al.Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy.Nature,1986,319(6055):675-678
[31]Gold L I,Eggleton P,Sweetwyne M T,et al.Calreticulin:non-endoplasmic reticulum functions in physiology and disease.FASEB J,2010,24(3):665-683
[32]Michalak M,Groenendyk J,Szabo E,et al.Calreticulin,a multi-process calcium-buffering chaperone of the endoplasmic reticulum.Biochem J,2009,417(3):651-666
[33]Cerundolo V,de la Salle H.Description of HLA classⅠ-and CD8-deficient patients:Insights into the function of cytotoxic T lymphocytes and NK cells in host defense.Semin Immunol,2006,18(6):330-336
[34]Fenton M,Bone N,Sinclair A J.The efficient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line.J Immunol Methods,1998,212(1):41-48
[35]Abarzúa P,LoSardo J E,Gubler M L,et al.Microinjection of monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the transcription activation function to mutant p53.Cancer Res,1995,55(16):3490-3494
[36]Lin J J,Feramisco J R.Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody.Cell,1981,24(1):185-193
[37]Bhakdi S,Bayley H,Valeva A,et al.Staphylococcal alpha-toxin,streptolysin-O,and Escherichia coli hemolysin:prototypes of pore-forming bacterial cytolysins.Arch Microbiol,1996,165(2):73-79
[38]Holasová S,Mojzísek M,Buncek M,et al.Cholesterol conjugated oligonucleotide and LNA:a comparison of cellular and nuclear uptake by Hep2 cells enhanced by streptolysin-O.Mol Cell Biochem,2005,276(1-2):61-69
[39]Bennink J R,Yewdell J W,Smith G L,et al.Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells.Nature,1984,311(5986):578-579
[40]Jacobs A A,Loeffen P L,van den Berg A J,et al.Identification,purification,and characterization of a thiol-activated hemolysin(suilysin)of Streptococcus suis.Infect Immun,1994,62(5):1742-1748
[41]Lv Q Y,Hao H J,Bi L L,et al.Purification and biological activities analysis of Streptococcus suis Serotype 2 suilysin.Chin J Cell Mol Immunol,2011,27(4):374-376
[42]Gottschalk M,Segura M.The pathogenesis of the meningitis caused by Streptococcus suis:the unresolved questions.Vet Microbiol,2000,76(3):259-272
[43]Xu L,Huang B,Du H,et al.Crystal structure of cytotoxin protein suilysin from Streptococcus suis.Protein Cell,2010,1(1):96-105
[44]Rossjohn J,Polekhina G,Feil S C,et al.Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins.J Mol Biol,2007,367(5):1227-1236
[45]Ramachandran R,Tweten R K,Johnson A E.Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment.Nat Struct Mol Biol,2004,11(8):697-705
[2]Cresswell P,Bangia N,Dick T,et al.The nature of the MHC class I peptide loading complex.Immunol Rev,1999,172(1):21-28
[3]Townsend A,Bodmer H.Antigen recognition by class I-restricted T lymphocytes.Annu Rev Immunol,1989,7(7):601-624
[4]Williams A P,Peh C A,Purcell A W,et al.Optimization of the MHC classⅠpeptide cargo is dependent on tapasin.Immunity,2002,16(4):509-520
[5]Powis S J,Townsend A R,Deverson E V,et al.Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter.Nature,1991,354(6354):528-531
[6]Lankat-Buttgereit B,Tampe R.The transporter associated with antigen processing TAP:structure and function.FEBS Lett,1999,464(3):108-112
[7]Anderson K,Cresswell P,Gammon M,et al.Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis.J Exp Med,1991,174(2):489-492
[8]Gao B,Adhikari R,Howarth M,et al.Assembly and antigenpresenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin.Immunity,2002,16(1):99-109
[9]Farmery M R,Allen S,Allen A J,et al.The role of ERp57 in disulfide bond formation during the assembly of major histocompatibility complex classⅠin a synchronized semipermeabilized cell translation system.J Biol Chem,2000,275(20):14933-14938
[10]Peterson J R,Ora A,Van P N,et al.Transient,lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.Mol Biol Cell,1995,6(9):1173-1184
[11]Michalak M,Corbett E F,Mesaeli N,et al.Calreticulin:one protein,one gene,many functions.Biochem J,1999,344(pt2):281-292
[12]Sadasivan B,Lehner P J,Ortmann B et al.Roles for calreticulin and a novel glycoprotein,tapasin,in the interaction of MHC classⅠmolecules with TAP.Immunity,1996,5(2):103-114
[13]Grandea A G,Golovina T N,Hamilton S E,et al.Impaired assembly yet normal trafficking of MHC classⅠmolecules in Tapasin mutant mice.Immunity,2000,13(2):213-222
[14]Fu H,Ding J,Flutter B,et al.Investigation of endogenous antigen processing by delivery of an intact protein into cells.J Immunol Methods,2008,335(1-2):90-97
[15]Walev I,Bhakdi S C,Hofmann F,et al.Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.Proc Natl Acad Sci USA,2001,98(6):3185-3190
[16]Sanderson S,Shastri N.LacZ inducible,antigen/MHC-specific T cell hybrids.Int Immunol,1994,6(3):369-376
[17]Wu Y,Liu C,Sun M.et al.A specific cytotoxic T-lymphocyte epitope presentation system for antitumor immunity.Int J Cancer,2010,126(10):2373-2386
[18]Karttunen J,Sanderson S,Shastri N.Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens.Proc Natl Acad Sci USA,1992,89(13):6020-6024
[19]Porgador A,Yewdell J W,Deng Y,et al.Localization,quantitation,and in situ detection of specific peptide-MHC classⅠcomplexes using a monoclonal antibody.Immunity,1997,6(6):715-726
[20]Jones B,Janeway C A Jr.Cooperative interaction of B lymphocytes with antigen-specific helper T lymphocytes is MHC restricted.Nature,1981,292(5823):547-549
[21]Lewis J W,Sewell A,Price D,et al.HLA-A*0201 presents TAP-dependent peptide epitopes to cytotoxic T lymphocytes in the absence of tapasin.Eur J Immunol,1998,28(10):3214-3220
[22]Goldberg A L,Cascio P,Saric T,et al.The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.Mol Immunol,2002,39(3-4):147-164
[23]Brodsky F M,Guagliardi L E.The cell biology of antigen processing and presentation.Annu Rev Immunol,1991,9(9):707-744
[24]Ljunggren H G,Stam N J,Ohlén C,et al.Empty MHC classⅠmolecules come out in the cold.Nature,1990,346(6283):476-480
[25]Tan P,Kropshofer H,Mandelboim O,et al.Recruitment of MHC classⅠmolecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading.J Immunol,2002,168(4):1950-1960
[26]Panter M S,Jain A,Leonhardt R M,et al.Dynamics of major histocompatibility complex classⅠassociation with the human peptide-loading complex.J Biol Chem,2012,287(37):31172-31184
[27]Garstka M A,Fritzsche S,Lenart I,et al.Tapasin dependence of major histocompatibility complex classⅠmolecules correlates with their conformational flexibility.FASEB J,2011,25(11):3989-3998
[28]Wearsch P A,Cresswell P.Selective loading of high-affinity peptides onto major histocompatibility complex classⅠmolecules by the tapasin-ERp57 heterodimer.Nat Immunol,2007,8(8):873-881
[29]Praveen P V,Yaneva R,Kalbacher H,et al.Tapasin edits peptides on MHC classⅠmolecules by accelerating peptide exchange.Eur J Immunol,2010,40(1):214-224
[30]Karre K,Ljunggren H G,Piontek G,et al.Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy.Nature,1986,319(6055):675-678
[31]Gold L I,Eggleton P,Sweetwyne M T,et al.Calreticulin:non-endoplasmic reticulum functions in physiology and disease.FASEB J,2010,24(3):665-683
[32]Michalak M,Groenendyk J,Szabo E,et al.Calreticulin,a multi-process calcium-buffering chaperone of the endoplasmic reticulum.Biochem J,2009,417(3):651-666
[33]Cerundolo V,de la Salle H.Description of HLA classⅠ-and CD8-deficient patients:Insights into the function of cytotoxic T lymphocytes and NK cells in host defense.Semin Immunol,2006,18(6):330-336
[34]Fenton M,Bone N,Sinclair A J.The efficient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line.J Immunol Methods,1998,212(1):41-48
[35]Abarzúa P,LoSardo J E,Gubler M L,et al.Microinjection of monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the transcription activation function to mutant p53.Cancer Res,1995,55(16):3490-3494
[36]Lin J J,Feramisco J R.Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody.Cell,1981,24(1):185-193
[37]Bhakdi S,Bayley H,Valeva A,et al.Staphylococcal alpha-toxin,streptolysin-O,and Escherichia coli hemolysin:prototypes of pore-forming bacterial cytolysins.Arch Microbiol,1996,165(2):73-79
[38]Holasová S,Mojzísek M,Buncek M,et al.Cholesterol conjugated oligonucleotide and LNA:a comparison of cellular and nuclear uptake by Hep2 cells enhanced by streptolysin-O.Mol Cell Biochem,2005,276(1-2):61-69
[39]Bennink J R,Yewdell J W,Smith G L,et al.Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells.Nature,1984,311(5986):578-579
[40]Jacobs A A,Loeffen P L,van den Berg A J,et al.Identification,purification,and characterization of a thiol-activated hemolysin(suilysin)of Streptococcus suis.Infect Immun,1994,62(5):1742-1748
[41]Lv Q Y,Hao H J,Bi L L,et al.Purification and biological activities analysis of Streptococcus suis Serotype 2 suilysin.Chin J Cell Mol Immunol,2011,27(4):374-376
[42]Gottschalk M,Segura M.The pathogenesis of the meningitis caused by Streptococcus suis:the unresolved questions.Vet Microbiol,2000,76(3):259-272
[43]Xu L,Huang B,Du H,et al.Crystal structure of cytotoxin protein suilysin from Streptococcus suis.Protein Cell,2010,1(1):96-105
[44]Rossjohn J,Polekhina G,Feil S C,et al.Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins.J Mol Biol,2007,367(5):1227-1236
[45]Ramachandran R,Tweten R K,Johnson A E.Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment.Nat Struct Mol Biol,2004,11(8):697-705