Study On Immune Recognition Of Superantigen SED

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 SEDIVwith 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 aSEDnew 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...