超抗原SED免疫识别机制的研究
|
摘要
超抗原是一种具有强大免疫刺激功能的新型蛋白质抗原。业已证明，超抗原参与多种疾病的发病过程。对超抗原免疫识别机制的深入研究，可望进一步为相关疾病的防治提供新的方法。金黄色葡萄球菌(金葡菌)超抗原家族由于涉及的疾病谱非常广泛，一直是比较活跃的研究领域。目前发现的金葡菌超抗原分子多达十余种。对金葡菌超抗原免疫识别机制研究较多的是SEA、SEB、SEC和TSST-1。已取得的研究进展提示我们，超抗原尽管具有相似的生物学活性，但它们的免疫识别机制存在明显的不同，因此，为了揭示金葡菌超抗原家族免疫识别的全貌，针对该家族其它超抗原分子的进一步研究是十分必要的。
SED是一种常见的毒性作用较强的金葡菌肠毒素，但对其免疫识别的研究相对较少，目前尚无涉及SED与TCR相互作用的研究。已有研究发现SEC3和SEB的TCR结合位位于分子两个结构域之间的沟槽内。将SED与SEB、SEC3的氨基酸序列进行比较分析，发现某些与TCR结合的活性位点比较保守。值得关注的是，这些保守的活性位点是否同样决定了SED的TCRVβ特异性?SED与其它肠毒素超抗原的TCR免疫识别位有何异同?
目前对SED与MHC的结合方式尚不明确。有研究表明，SEA具有两个MHC结合位，氨基末端的结合位与MHCⅡ的α链结合，羧基末端的结合位与MHCⅡ的β链结合，阻止SEA与MHCⅡα链结合可影响SEA对某些TCR V β ~+T细胞的活化。研究发现SED的某些MHC结合位点与SEA一致，那么二者与MHC的结合方式是否也相同?SED与MHCⅡα链亲和力的改变对其TCRVβ特异性有何影响?目前尚未见这方面的研究。
基于以上问题，我们对SED可能的活性位点进行预测，构建了一系列
超抗原SED免疫识别机制的研究
SED定点突变体，检测这些突变体的 MHC 11结合活性和 TCRV fi特异性，
寻找与TCRV 6结合的关键位点，进一步探讨SED与MHC和TCR的相互
作用方式。主要研究内容和结果包括以下几方面：
1．构建SED的原核表达系统，并对蛋白的表达和纯化条件进行优化，
用两种系统即人的PBMC和小鼠脾脏细胞，检测了纯化SED的促T淋巴细
胞增殖活性和TCRV 6特异性。获得了纯度较高，并具有超抗原活性的SED，
为构建SED突变体和后续的功能研究奠定了良好的基础。
2．对金葡菌超抗原家族的氨基酸序列进行对比分析，首次运用同源建
模的方法构建了SED的三维空间结构模型，比较SED与其它肠毒素超抗原
结构的差异，对可能的活性位点进行预测，最终确定SED的N23、F45、L59、
N6、192和 F203位氨基酸为突变位点。
3，以丙氨酸扫描突变方案，采用大引物PCR技术，在选定的突变位点
处成功引入预期突变，构建了SEDN23A、SEDF45A、SEDL59A、SEN6lA、SEDI92A
和 SEDFZ。3＾突变体。由于随机突变的发生，我们还构建了 SEDN23AIH26R双突
变体。并对突变体蛋白进行分析和纯化，获得了可用于后续功能实验的SED
突变体。
4．检测了SED突变体促人和小鼠T淋巴细胞的增殖活性，筛选出促增
殖活性降低的突变体；进而用竞争结合实验检测突变体与 MHC 11的结合活
性，并用流式细胞仪检测突变体TCRV p特异性变化。结果：①证实了F45
是胚D与 MHO结合的关键位点，首次发现％D对人 TCRV 6 5”、TCRV
p矿、TC RVRV p 12．广和小鼠TC RVRV p 8．U8．3”T细胞的活化增殖能力依赖于
N5位点与MHC 11的结合；②首次证明NZ 3位氨基酸是邪D与人TC RV p
5和小鼠 TCRV 6 8．2／8．3结合的关键位点；③与 SEDN23A相比，SE民23。6。
对人淋巴细胞的促增殖活性明显减低，二者的人TCRV p 5、TCRV p 8、TCRV
elZ＊特异性无显著差别，提示 26位氨基酸是 SED与人的其它 TCRV 6结
合的关键位点；④与SED相比，突变体SEDLS“、SEDNI小SEDI92＾和
SEDFZ仍＾对人和小鼠T淋巴细胞的增殖活性无显著变化，提示这些氨基酸残
基可能不是SED的关键活性位点。
Vlll
第三军医大学博士研究生论文
5．结合本研究和其它研究的结果，从以下几个方面进行了探索性研究：
①分析了 SED与 MHC的结合方式，对 SED－MHC的空间结构进行模拟，推
测 SED与 MHC 11的结合方式与 SEA相似，具有两个结合位，分别与 MHC
11a链和p链结合，交联两个MHC分子；②对SED的TCR结合位进行结
构－功能分析，进一步解释我们的研究结果；③通过TCRV p氨基酸序列的
对比分析，发现与同一种超抗原反应的一组TCRV 0具有特征性的保守序
列，这些保守序列很可能与超抗原的TCRV p特异性有关，这一发现为进一
步从TCR的角度探讨超抗原TCRV p特异性的本质提供了重要的线索；④
最后，对MHCEED－TCR复合物的结构模型进行预测，与MHC－eptide－TCR
的空间结构进行比较，对SED的免疫识别获得了更加全面的认识。
综上，本研究首次对SED与TCR结合的位点进行研究，发现N23和
The family of straphylococcal enterotoxins (SEs), including SEA-E, SEG, SEH and SEI, has been known to contribute to a broad spectrum
of diseases ranging from tissue infections to life-threating speticemia and toxic
shock syndromes, and from Kawasaki's syndrome to atopic dermatitis, even
multisystem vasculitis. In recent years, much attention has been paid to the immune
recognition of superantigen SEs. However, so far little has been known about the
immune recognition of SED, especially about the interaction between SED and TCR.
Comparing the sequences of SEs, we found that the members of SEs share the high
sequence homology, and some active sites are conservative. We wonder whether
these conservative sites determine the TCRV β specificity of SED, and whether
SED and other SEs share the same mode interacting with TCRV β .
It has been demonstrated that SED and SEA have the same sites binding to MHC Ⅱ.
Several amino acid residues of SED have been proved to be important in the
interaction between SED and MHC Ⅱ, but the mode of SED binding to MHC
Ⅱ is not yet clear. This study is designed to explore the above issues.
The main content and results of this study are as follows:
Firstly, the prokaryotic expression system of SED was constructed.
The conditions of expression and purification of SED protein were optimized.
Then we detected the mitogen activity and TCRV β
specificity of SED with human PBMC and mice plenocytes. The results showed that
the expressed SED have superantigenic activity.
Secondly, we compared the amino acid sequences of SEs and constructed the
three-dimension structure of SED by homology modeling method. On the basis of results of
comparing the amino acid sequences and structure of SED
IV
with other SEs, we chosen the N23, F45, L59, N61, 192 and F203 in SED as mutant residues.
Thirdly, The expected mutants were introduced into the SED DNA by megaprimer PCR. Then the expressed mutants were purified and analyzed with immunoblot. The results showed that these SED mutants could be used in the subsequent functional study.
Fourthly, we detected the mitogen activity of SED mutants and found that the mitogen activity of mutant SEDN23A, SEDN23A/H26R and SEDp45A decreased significantly. Furthermore, competition assay was used to detect the ability of mutant SEDN23A, SEDN23A/H26R and SEDF45A binding to MHCII. The TCRV ?specificity of these mutants was then determined with FACS. The results are as follows: (T)We confirmed that F45 of SED was an important residue binding to MHC II. ?The human TCRV 3 5,TCRV 3 8,TCRV 3 12.1 and mice TCRV 3 8.2/8.3 specificity of SED was dependent on the residue F45 binding to MHC II. ㏑esidue N23 played an important role on SED interacting with human TCRV 3 5 and mice TCRV 3 8.2/8.3. Residue H26 was probably an active site of SED binding to other human TCRV 3 , but was not appeared to be important on the interaction between SED and mice TCRV 3 . @The mitogen activity of ott mutants SEDt59A> SED^iA. SEDi92A and SEDp203Ahad no significant change, so L59, N61,192 and F203 seem not to be active sites of SED.
Finally, on the basis of structure-function analysis, we predicted the mode of interaction between SED and MHC II and modeled the structure of SED-MHCII. We suppose that SED shares the same mode interacting with MHC II a chain and 3 chain and cross-links two MHC II molecules to transduct active signal more efficiently. Furthermore, through comparing the amino acid sequences of TCRV 3 used by SED, SEA and SEE, we discovered that TCRV 3 segments used by different superantigens have characteristic conservative motifs. This result maybe can explain the TCRV 3 specificity of superantigens from a
SED
new respect and provide an important clue for further study. Finally the model of MHC-SED-TCR was constructed to make us obtain a better understanding about immune recognition of SED.
In conclusion, our results first indicate that N23 and H26 on SED are the important residues involve
SED是一种常见的毒性作用较强的金葡菌肠毒素，但对其免疫识别的研究相对较少，目前尚无涉及SED与TCR相互作用的研究。已有研究发现SEC3和SEB的TCR结合位位于分子两个结构域之间的沟槽内。将SED与SEB、SEC3的氨基酸序列进行比较分析，发现某些与TCR结合的活性位点比较保守。值得关注的是，这些保守的活性位点是否同样决定了SED的TCRVβ特异性?SED与其它肠毒素超抗原的TCR免疫识别位有何异同?
目前对SED与MHC的结合方式尚不明确。有研究表明，SEA具有两个MHC结合位，氨基末端的结合位与MHCⅡ的α链结合，羧基末端的结合位与MHCⅡ的β链结合，阻止SEA与MHCⅡα链结合可影响SEA对某些TCR V β ~+T细胞的活化。研究发现SED的某些MHC结合位点与SEA一致，那么二者与MHC的结合方式是否也相同?SED与MHCⅡα链亲和力的改变对其TCRVβ特异性有何影响?目前尚未见这方面的研究。
基于以上问题，我们对SED可能的活性位点进行预测，构建了一系列
超抗原SED免疫识别机制的研究
SED定点突变体，检测这些突变体的 MHC 11结合活性和 TCRV fi特异性，
寻找与TCRV 6结合的关键位点，进一步探讨SED与MHC和TCR的相互
作用方式。主要研究内容和结果包括以下几方面：
1．构建SED的原核表达系统，并对蛋白的表达和纯化条件进行优化，
用两种系统即人的PBMC和小鼠脾脏细胞，检测了纯化SED的促T淋巴细
胞增殖活性和TCRV 6特异性。获得了纯度较高，并具有超抗原活性的SED，
为构建SED突变体和后续的功能研究奠定了良好的基础。
2．对金葡菌超抗原家族的氨基酸序列进行对比分析，首次运用同源建
模的方法构建了SED的三维空间结构模型，比较SED与其它肠毒素超抗原
结构的差异，对可能的活性位点进行预测，最终确定SED的N23、F45、L59、
N6、192和 F203位氨基酸为突变位点。
3，以丙氨酸扫描突变方案，采用大引物PCR技术，在选定的突变位点
处成功引入预期突变，构建了SEDN23A、SEDF45A、SEDL59A、SEN6lA、SEDI92A
和 SEDFZ。3＾突变体。由于随机突变的发生，我们还构建了 SEDN23AIH26R双突
变体。并对突变体蛋白进行分析和纯化，获得了可用于后续功能实验的SED
突变体。
4．检测了SED突变体促人和小鼠T淋巴细胞的增殖活性，筛选出促增
殖活性降低的突变体；进而用竞争结合实验检测突变体与 MHC 11的结合活
性，并用流式细胞仪检测突变体TCRV p特异性变化。结果：①证实了F45
是胚D与 MHO结合的关键位点，首次发现％D对人 TCRV 6 5”、TCRV
p矿、TC RVRV p 12．广和小鼠TC RVRV p 8．U8．3”T细胞的活化增殖能力依赖于
N5位点与MHC 11的结合；②首次证明NZ 3位氨基酸是邪D与人TC RV p
5和小鼠 TCRV 6 8．2／8．3结合的关键位点；③与 SEDN23A相比，SE民23。6。
对人淋巴细胞的促增殖活性明显减低，二者的人TCRV p 5、TCRV p 8、TCRV
elZ＊特异性无显著差别，提示 26位氨基酸是 SED与人的其它 TCRV 6结
合的关键位点；④与SED相比，突变体SEDLS“、SEDNI小SEDI92＾和
SEDFZ仍＾对人和小鼠T淋巴细胞的增殖活性无显著变化，提示这些氨基酸残
基可能不是SED的关键活性位点。
Vlll
第三军医大学博士研究生论文
5．结合本研究和其它研究的结果，从以下几个方面进行了探索性研究：
①分析了 SED与 MHC的结合方式，对 SED－MHC的空间结构进行模拟，推
测 SED与 MHC 11的结合方式与 SEA相似，具有两个结合位，分别与 MHC
11a链和p链结合，交联两个MHC分子；②对SED的TCR结合位进行结
构－功能分析，进一步解释我们的研究结果；③通过TCRV p氨基酸序列的
对比分析，发现与同一种超抗原反应的一组TCRV 0具有特征性的保守序
列，这些保守序列很可能与超抗原的TCRV p特异性有关，这一发现为进一
步从TCR的角度探讨超抗原TCRV p特异性的本质提供了重要的线索；④
最后，对MHCEED－TCR复合物的结构模型进行预测，与MHC－eptide－TCR
的空间结构进行比较，对SED的免疫识别获得了更加全面的认识。
综上，本研究首次对SED与TCR结合的位点进行研究，发现N23和
The family of straphylococcal enterotoxins (SEs), including SEA-E, SEG, SEH and SEI, has been known to contribute to a broad spectrum
of diseases ranging from tissue infections to life-threating speticemia and toxic
shock syndromes, and from Kawasaki's syndrome to atopic dermatitis, even
multisystem vasculitis. In recent years, much attention has been paid to the immune
recognition of superantigen SEs. However, so far little has been known about the
immune recognition of SED, especially about the interaction between SED and TCR.
Comparing the sequences of SEs, we found that the members of SEs share the high
sequence homology, and some active sites are conservative. We wonder whether
these conservative sites determine the TCRV β specificity of SED, and whether
SED and other SEs share the same mode interacting with TCRV β .
It has been demonstrated that SED and SEA have the same sites binding to MHC Ⅱ.
Several amino acid residues of SED have been proved to be important in the
interaction between SED and MHC Ⅱ, but the mode of SED binding to MHC
Ⅱ is not yet clear. This study is designed to explore the above issues.
The main content and results of this study are as follows:
Firstly, the prokaryotic expression system of SED was constructed.
The conditions of expression and purification of SED protein were optimized.
Then we detected the mitogen activity and TCRV β
specificity of SED with human PBMC and mice plenocytes. The results showed that
the expressed SED have superantigenic activity.
Secondly, we compared the amino acid sequences of SEs and constructed the
three-dimension structure of SED by homology modeling method. On the basis of results of
comparing the amino acid sequences and structure of SED
IV
with other SEs, we chosen the N23, F45, L59, N61, 192 and F203 in SED as mutant residues.
Thirdly, The expected mutants were introduced into the SED DNA by megaprimer PCR. Then the expressed mutants were purified and analyzed with immunoblot. The results showed that these SED mutants could be used in the subsequent functional study.
Fourthly, we detected the mitogen activity of SED mutants and found that the mitogen activity of mutant SEDN23A, SEDN23A/H26R and SEDp45A decreased significantly. Furthermore, competition assay was used to detect the ability of mutant SEDN23A, SEDN23A/H26R and SEDF45A binding to MHCII. The TCRV ?specificity of these mutants was then determined with FACS. The results are as follows: (T)We confirmed that F45 of SED was an important residue binding to MHC II. ?The human TCRV 3 5,TCRV 3 8,TCRV 3 12.1 and mice TCRV 3 8.2/8.3 specificity of SED was dependent on the residue F45 binding to MHC II. ㏑esidue N23 played an important role on SED interacting with human TCRV 3 5 and mice TCRV 3 8.2/8.3. Residue H26 was probably an active site of SED binding to other human TCRV 3 , but was not appeared to be important on the interaction between SED and mice TCRV 3 . @The mitogen activity of ott mutants SEDt59A> SED^iA. SEDi92A and SEDp203Ahad no significant change, so L59, N61,192 and F203 seem not to be active sites of SED.
Finally, on the basis of structure-function analysis, we predicted the mode of interaction between SED and MHC II and modeled the structure of SED-MHCII. We suppose that SED shares the same mode interacting with MHC II a chain and 3 chain and cross-links two MHC II molecules to transduct active signal more efficiently. Furthermore, through comparing the amino acid sequences of TCRV 3 used by SED, SEA and SEE, we discovered that TCRV 3 segments used by different superantigens have characteristic conservative motifs. This result maybe can explain the TCRV 3 specificity of superantigens from a
SED
new respect and provide an important clue for further study. Finally the model of MHC-SED-TCR was constructed to make us obtain a better understanding about immune recognition of SED.
In conclusion, our results first indicate that N23 and H26 on SED are the important residues involve
引文
1. Du-C, Sriram-S. Induction of interleukin-12/p40 by superantigens in macrophages is mediated by activation of nuclear factor-kappaB. Cell-Immunol,2000, 199(1): 50-7.
2. Conrad B, Weldmann E, Trucco G, et al. Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetology. Nature, 1994,371:351.
3. Conrad B, Weissmahr RN, Boni J,et al. A human endogenous retroviral superantigen as candidate autoimmune gene in type Ⅰ diabetes. Cell, 1997,90:303.
4. Kim A, Jun HS,Wong L,et al. Human endogenous retrovirus with a high genomic sequence homology with IDDMK(1,2)22 is not specific for Type I (insulin-dependent) diabetic patients but ubiquitous. Diabetologia, 1999,42(4): 413-8.
5. Acha Orbea H, Mitchell DJ, Timmerman L, et al. Limited heterogeneity of T cell receptors from lymphocyte mediating autoimmune encephalomyelitis allows specific immune intervention. Cell, 1988, 54:263.
6. Brocke S, Gaur A, Piercy C, et al. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature, 1993,365:642.
7. Das-MR, Cohen-A,Zamvil-SS, et al. Prior exposure to superantigen can inhibit or exacerbate autoimmune encephalomyelitis: T-cell repertoire engaged by the autoantigen determines clinical outcome.J-Neuroimmunol, 1996,71 (1-2): 3-10.
8. Sawitzke A, Joyner D, Knudtson K, et al. Anti-MAM antibodies in rheumatic disease: evidence for a MAM-like superantigen in rheumatoid arthritis? J-Rheumatol, 2000,27(2): 358-64.
9. Kraft M, Filsinger S, Kramer KL, et al. Synovial fibroblasts as target cells for staphylococcal enterotoxin-induced T-cell cytotoxicity. Immunology, 1998,93(1): 20-5.
10. Fischer P, Uttenreuther-Fischer MM, Gaedicke G. Superantigens in the aetiology of Kawasaki disease. Lancet 1996;348(9021):202.
11. Leung DY, Giorno RC, Kazemi LV, et al. Evidence for superantigen involvement in cardiovascular injury due to Kawasaki syndrome. J Immunol, 1995,155(10):5018-21.
12. Leung DY, Meissner C, Fulton D, et al. The potential role of bacterial superantigens in the pathogenesis f Kawasaki syndrome. J Clin Immunol, 1995,15(6 Suppl):11S-17S.
13. Skov L, Baadsgaard O. Bacterial superantigens and inflammatory skin diseases. Clin-Exp-Dermatol, 2000,25(1): 57-61.
14. Davison S, Allen M, Harmer A, et al. Increased T-cell receptor vbeta2 chain expression in skin homing lymphocytes in psoriasis.Br-J-Dermatol,1999, 140(5): 845-8.
15. Yamamoto T; Katayama I; Nishioka K. Clinical analysis of staphylococcal superantigen hyper-reactive patients with psoriasis vulgaris. Eur J Dermatol, 1998,8(5): 325-9.
16. Strickland I, Hauk PJ, Trumble AE, et al. Evidence for superantigen involvement in skin homing of T cells in atopic dermatitis. J Invest Dermatol, 1999,112(2): 249-53.
2. Conrad B, Weldmann E, Trucco G, et al. Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetology. Nature, 1994,371:351.
3. Conrad B, Weissmahr RN, Boni J,et al. A human endogenous retroviral superantigen as candidate autoimmune gene in type Ⅰ diabetes. Cell, 1997,90:303.
4. Kim A, Jun HS,Wong L,et al. Human endogenous retrovirus with a high genomic sequence homology with IDDMK(1,2)22 is not specific for Type I (insulin-dependent) diabetic patients but ubiquitous. Diabetologia, 1999,42(4): 413-8.
5. Acha Orbea H, Mitchell DJ, Timmerman L, et al. Limited heterogeneity of T cell receptors from lymphocyte mediating autoimmune encephalomyelitis allows specific immune intervention. Cell, 1988, 54:263.
6. Brocke S, Gaur A, Piercy C, et al. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature, 1993,365:642.
7. Das-MR, Cohen-A,Zamvil-SS, et al. Prior exposure to superantigen can inhibit or exacerbate autoimmune encephalomyelitis: T-cell repertoire engaged by the autoantigen determines clinical outcome.J-Neuroimmunol, 1996,71 (1-2): 3-10.
8. Sawitzke A, Joyner D, Knudtson K, et al. Anti-MAM antibodies in rheumatic disease: evidence for a MAM-like superantigen in rheumatoid arthritis? J-Rheumatol, 2000,27(2): 358-64.
9. Kraft M, Filsinger S, Kramer KL, et al. Synovial fibroblasts as target cells for staphylococcal enterotoxin-induced T-cell cytotoxicity. Immunology, 1998,93(1): 20-5.
10. Fischer P, Uttenreuther-Fischer MM, Gaedicke G. Superantigens in the aetiology of Kawasaki disease. Lancet 1996;348(9021):202.
11. Leung DY, Giorno RC, Kazemi LV, et al. Evidence for superantigen involvement in cardiovascular injury due to Kawasaki syndrome. J Immunol, 1995,155(10):5018-21.
12. Leung DY, Meissner C, Fulton D, et al. The potential role of bacterial superantigens in the pathogenesis f Kawasaki syndrome. J Clin Immunol, 1995,15(6 Suppl):11S-17S.
13. Skov L, Baadsgaard O. Bacterial superantigens and inflammatory skin diseases. Clin-Exp-Dermatol, 2000,25(1): 57-61.
14. Davison S, Allen M, Harmer A, et al. Increased T-cell receptor vbeta2 chain expression in skin homing lymphocytes in psoriasis.Br-J-Dermatol,1999, 140(5): 845-8.
15. Yamamoto T; Katayama I; Nishioka K. Clinical analysis of staphylococcal superantigen hyper-reactive patients with psoriasis vulgaris. Eur J Dermatol, 1998,8(5): 325-9.
16. Strickland I, Hauk PJ, Trumble AE, et al. Evidence for superantigen involvement in skin homing of T cells in atopic dermatitis. J Invest Dermatol, 1999,112(2): 249-53.