化膿性鏈球菌是一種常見的人類病原菌，會藉由許多不同的生存機制來躲避宿主的免疫清除，並導致疾病。化膿性鏈球菌免疫球蛋白G分解?(IdeS)為一種半胱胺酸?，它會專一性的水解免疫球蛋白G重鏈之絞鏈區，使免疫球蛋白G瓦解，進而幫助菌體不受到免疫攻擊而可在宿主體內生長與感染。IdeS有兩種差異型存在，稱之為IdeS-1與IdeS-2，被報導主要差異的區域在於112-205號胺基酸序列之間，它們會藉由不同的機制來抵抗宿主的免疫反應。為了探討IdeS-1與IdeS-2間在結構或是功能上之關連，我們將M1，M6，M12-IdeS與突變株R39I，T57I，C94S，Y257C，Y257S與K280E利用E. coli系統表現出來，並純化至精純。由膠體過濾層析法的分析顯示，IdeS-1與IdeS-2都會有寡聚體型態的產生。我們發現不同於以往論述的結果，M1，M6，M12-IdeS具有相似的IgG水解效力，其比活性值約為7,500 units/mg。相對地，Y257C與Y257S則顯示出比野生株降低約3倍左右的酵素活性，這樣的結果說明IdeS為一種具有高活性與高專一性的免疫球蛋白水解酵素。另外，我們發現M28-IdeS酵素活性較低的原因是源自於C257不能維持分子間交互作用而影響催化中心胺基酸的穩定性，而並非自我氧化的化學效應所造成。我們發現M12-IdeS對於以PNGaseF酵素去掉醣化的IgG水解效力降低了4倍以上，而Y257C則顯著降低了20倍以上的酵素活性，說明IgG表面的醣化區域在IdeS水解IgG的過程中，可能提供IdeS辨識的結合位點，以幫助催化過程的進行。本研究的結果顯示，酵素活性區域周邊之單一胺基酸變異會造成高專一性的IdeS活性下降，同時IdeS不同的差異間具有相似的酵素活性，然而，IdeS對於受質表面醣化區域的辨識，提供另一種蛋白質酵素與受質結合模式的可能性。

Streptococcus pyogenes (group A Streptococcus [GAS]), one of the most common human bacterial pathogens, has evolved diverse mechanisms that allow the bacteria to evade the immune system and cause disease. A secreted cysteine proteinase, an immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), specifically cleaves the heavy chain of IgG and enables the bacteria to colonize and circumvent human immune defense. IdeS contains 314-339 amino acid residues and only shares 10-15% identity and 20-35% similarity with other cysteine proteases. Two protein variants of IdeS, designated IdeS-1 and IdeS-2, have been reported based on differences in the amino acid sequences from 112 to 205. IdeS-1 has been reported to exert their inhibitory function through proteolytic cleavage of IgG, while IdeS-2 has low protease activity and binds to the FcRIII receptor by interfering phagocytic killing. In order to study functional difference between IdeS-1 and IdeS-2, R39I, T57I, C94S, Y257C, Y257S, H262A, and K280E mutant proteins were expressed in E. coli and purified to homogeneity. Based on the results of gel filtration analysis, all IdeS variants formed oligomers in solution but mainly exist as monomer (>95%). The endopeptidase activity analysis showed three IdeS variants (M1 IdeS-1, M6 IdeS-2, and M12 IdeS-2) have similar activity to hydrolyze human IgG with the specific activity of ~7,500 units/mg. The result demonstrates that three IdeS variants are highly active and selective cysteine protease with similar activity. In contrast, the Y257C and Y257S mutants exhibited ~3-fold less activity. Using 3D model structure of Y257C mutant, we found that the interaction between Y255, but not C255, residue and catalytic residue H262 resulted in low proteolytic activity in Y255 mutation. Using unglycosylated IgG as the substrate, we found that M1 IdeS-1, M12 IdeS-2, and M1 IdeS-1Y257C mutant exhibited 4.2-, 5.3-, and 16.7-folds less activity. This result suggests that the glycan of IgG may be recognized by IdeS. The detailed biochemical characterization of IdeS variants can provide new insights into the cleavage modulation of IgG by IdeS.

Agniswamy, J., Lei, B., Musser, J.M. & Sun, P.D. Insight of host immune evasion mediated by two variants of group A streptococcus Mac protein. J. Biol. Chem. 279:52789–52796, 2004.

Agniswamy, J., Nagiec, M.J., Liu, M., Schuck, P., Musser, J.M. & Sun, P.D.
Crystal structure of group A streptococcus Mac-1: insight into dimer-mediated specificity for recognition of human IgG. Structure. 14:225-235, 2006.

Akesson, P., Moritz, L., Truedsson, M., Christensson, B. & von Pawel-Rammingen, U. IdeS, a highly specific immunoglobulin G (IgG)- cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection. Infect. Immun. 74:497-503, 2006.

Berg, J. M., Tymoczko, J. L. & Stryer, L., Biochemistry. (5 th ed.). 2002.

Berge, A. & Bjorck, L. Streptococcal cysteine protease releases biologically active fragments of streptococcal surface proteins. J. Biol. Chem. 270: 9862-9867, 1995.

Bisno, A.L. & Collins, C.M. Molecular basis of group A streptococcal virulence.The Lancet Infectious Diseases 3: 191-200, 2003

Bisno A.L. Alternate complement pathway activation by group A streptococci : role of M protein. Infect. Immun. 26: 1172-1176, 1979.

Bjorck, L. & Kronvall, G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133:969–974, 1984.

Boraston, A.B., Bolam, D.N., Gilbert, H.J. & Davies, G.J. Carbohydrate -binding modules: fine-tuning polysaccharide recognition. Biochem. J. 382:769–781, 2004.

Burton, D.R. Immunoglobulin G: functional sites. Mol. Immunol. 22:161–206, 1985.

Carapetis, J.R., Steer, A.C., Mulholland, E.K. & Webber, M. The global burden of streptococcal disease. THE LANCET Infect. Dis. 5: 685-694, 2005.

Cleary, P.P. Streptococcus moves inward. Nat. Med. 12: 384-386, 2006.

Collin, M. & Olse’n, A. EndoS, a noval secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. EMBO. 20: 3046-3055, 2001.

Collin, M. & Olse’n, A. Extracellular enzymes with immunomodulating activities: variations on a theme in Streptococcus pyogenes. Infect Immun. 71:2983–2992, 2003.

Dale, J.B., Washburn, R.G., Marques, M.B. & Wessels, M.R. Hyaluronate capture and surface M protein in resistance to opsonization of group A streptococci. Infect. Immune. 64:1495-1501, 1996.

Degnan, B.A., Fontaine, M.C., Doebereiner, A.H., Lee, J.J., Mastroeni, P, Dougan, G, Goodacre, J.A. & Kehoe, M.A. Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make streptococcal acid glycoprotein. Infect Immun. 68:2441-2448, 2000.

DeAngelis, P.L., Yang, N. & Weigel, P.H. The Streptococcus pyogenes hyaluronidase synthetase: sequence comparison and conservation among various group A strains. Biochem. Biophy. Res. Commun. 199:1-10, 1994.

D’Costa, S. S. & Boyle, M. D. Interaction of a group A streptococcus within plasma results in assembly of a surface plasminogen activator that contributes to occupancy of surface plasmin-binding structure. Micro. Pathog. 24:341-349, 1998.

Diemand, A.V. & Scheib, H. iMolTalk: an interactive, internet-based protein structure analysis server. Nucleic Acids Res. 32(Web Server issue):W512-6, 2004.

Ezzeddine, S., & Al-Khalidi, U. A short note on the molecular weight determination by gel filtration in minutes. Mol Cell Biochem. 45: 127-128.1982.
File, T.M., Tan, J.S. & DiPersio, J.R. Group A streptococcal necrotizing fasciitis. Diagnosing and treating the "flesh-eating bacteria syndrome". Cleve Clin J Med. 65:241-249, 1998.

Goldsby, R.A., Kindt, T.J. & Osborne.B.A., Kuby immunology. (4th ed.). 2000.

Heath, D.G. & Cleary, P.P. Cloning and expression of the gene for an immunoglobulin G Fc receptor protein from a group A streptococcus. Infect. Immun. 55:1233–1238, 1987.

Hanski, E. & Caparon, M. Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc. Natl. Acad. Sci. U S A. 89:6172-6176, 1992.

Horstmann R.D., Sievertsen H.J., Knobloch, J & Fischetti, V.A. Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H. Proc. Natl. Acad. Sci. USA 85: 1657-1661,1998.

Ji, Y., McLandsborough, L., Kondagunta, A. and Cleary, P. P. C5 peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect. Immun. 64: 503-510, 1996.

Johnson, D.R., Stevens, D.L. & Kaplan, E.L. Epidemiologic analysis of group A streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis. J. Infect. Dis. 166: 374-382, 1992.

Kao, C.H., Chen, P.Y., Huang, F.L., Chen, C.W., Chi, C.S., Lin, Y.H., Shih, C.Y., Hu, B.S., Li, C.R., Ma, J.S., Lau, Y.J., Lu, K.C. & Yu HW. Clinical and genetic analysis of invasive group A streptococcal infections in central Taiwan. J. Microbiol Immunol Infect. 38: 105-111, 2005.

Kapur, V., Topouzis, S., Majesky, M.W., Li, L.L. Hanrick, M.R., Hamill, R.J., Patti, J.M. & Musser, J.M. A conserved Streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin. Microb. Pathog. 15: 327-346,1993.

Kaneko, Y., Nimmerjahn, F. & Ravetch, J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science. 313:670-673, 2006.

Kumar, S., Tamura. K., & Nei, M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics. 5:150-163, 2004.

Lannergard, J. & Guss, B. IdeE, an IgG-endopeptidase of Streptococcus equi ssp. equi.. FEMS Microbiol Lett. 262:230-235, 2006.

Lei, B., Mackie, S., Lukomski, S. & Musser, J.M. Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68:6807–6818, 2000.

Lei, B., Deleo, F.R., Hoe, N.P., Graham, M.R., Mackie, S.M., Cole, R., Liu, M., Hill, H.R., Low, D.E., Federle, M.J., Scott, J.R. & Musser, J.M. Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis. Nat. Med. 7:1298-1305, 2001.

Lei, B., DeLeo, F.R., Reid, S.D., Voyich, J.M., Magoun, L., Liu, M.,
Braughton, K.R., Ricklefs, S., Hoe, N.P. & Cole, R.L. Opsonophagocytosis- inhibiting mac protein of group A Streptococcus: identification and characteristics of two genetic complexes. Infect. Immun. 70:6880–6890, 2002.

Lei, B., Liu, M., Meyers, E.G., Manning, H.M., Nagiec, M.J. & Musser, J.M. Histidine and aspartic acid residues important for immunoglobulin G endopeptidase activity of the group A streptococcus opsonophagocytosis -inhibiting Mac protein. Infect. Immun. 71:2881-2884, 2003.

Madden, J.C., Ruiz, N. & Caparon, M. Cytolysin-mediated translocation (CMT): a functional equivalent of type II secretion in gram-positive bacteria, Cell. 104: 143-152, 2001.

Martin, B.R., Prescott, W.R., Zhu, M. Quantitation of rodent catalepsy by a computer-imaging technique. Pharmacol. Biochem. Behav. 43:381-6, 1992.

Musser, J.M., Hauser, A.R., Kim, M.H., Schlievert, P.M., Nelson, K. & Selander, R.K. Streptococcus pyogenes causing toxic shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc. Natl. Acad. Sci. USA 88:2668–2672, 1991.

Papageorgiou, A.C. & Acharya, K.R. Microbial superantigens: from structure to functions, Trends Microbiol. 8: 369-375, 2000.

Pinkney, M., Kapur, V., Smith, J., Weller, U., Palmer, M., Glanville, M., Messner, M., Musser, J.M., Bhakdi, S. & Kehoe, M.A. Different forms of streptolysin O produced by Streptococcus pyogenes and by Escherichia coli expressing recombinant toxin: cleavage by streptococcal cysteine protease. Infect. Immun. 63:2776-2779, 1995.

Robinson, J.H. & Kehoe, M.A. Group A streptococcal M proteins: virulence factors and protective antigens. Immunol Today 13:362-367, 1992.

Schwede, T., Kopp, J., Guex, N. & Peitsch, M.C. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 31:3381–3385, 2003.

Stevens, D.L., Tanner, M.H. & Winship, J. Severe group A streptococcal infections associated with a toxic shock like syndrome and scarlet fever toxin A. N. Engl. J. Med. 321:1-8, 1989.

Stevens, D.L. Invasive group A streptococcal disease. Infect Agents Dis. 5: 157-166, 1996.

Stevens, D.L. Streptococcal toxic shock syndrome associated necrotizing faciitis. Annu. Rev. Medicine. 51: 271-288, 2000.

Stevens, J., Corper, A.L., Basler, C.F., Taubenberger, J.K., Palese, P. & Wilson, I.A. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science. 303:1866-1870, 2004

Stockl, J., Majdic, O., Pickl, W.F., Rosenkranz, A., Prager, E., Gschwantler, E. & Knapp, W. Granulocyte activation via a binding site near the C-terminal region of complement receptor type 3-chain (CD11b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol -anchored FcIIIB (CD16) molecules. J. Immunol. 154:5452-5463, 1995.

Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680, 1994.

Tyrrell, G.J., Lovgren, M., Forwick, B., Hoe, N.P., Musser, J.M. & Talbot, J.A. M types of group A streptococci isolates submitted to the National Centre for Streptococcus (Canada) form 1993 to 1999. J. Clin. Microbiol. 40:4466-4471, 2002.

Vincents, B., von Pawel-Rammingen, U., Bjorck, L. & Abrahamson, M. Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry. 43:15540-15449, 2004.

von Pawel-Rammingen, U., Johansson, B.P. & Bjorck, L. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J. 21:1607-1615, 2002.

von Pawel-Rammingen, U., Johansson, B.P., Tapper, H. & Bjorck, L. Streptococcus pyogenes and phagocytic killing. Nat Med. 8:1044-1045, 2002.

von Pawel-Rammingen, U. & Bjorck, L. IdeS and SpeB: immunoglobulin G -degrading cysteine proteinases of Streptococcus pyogenes. Curr Opin Microbiol. 6:50-55, 2003.

Wenig, K., Chatwell, L., von Pawel-Rammingen, U., Bjorck, L., Huber, R. & Sondermann, P. Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG. Proc. Natl. Acad. Sci. USA 101:17371-17376, 2004.

Xia. X., & Xie. Z., DAMBE: Data analysis in molecular biology and evolution. Journal of Heredity. 92:371-373, 2001.

陳秋月，化膿性鏈球菌外毒素B的表現和特性之研究。國立成功大學生物化學研究所碩士論文，1999。

紀仁智，化膿性鏈球菌的免疫球蛋白G分解酶之表現與特性分析。國立成功大學生物化學研究所碩士論文，2005。

陳文怡，A 群鏈球菌免疫球蛋白G分解酶及篩選寡胜肽膜透酶相關蛋白之研究。國立成功大學醫學檢驗生物技術學系碩士論文，2006

王志傑，Streptopain 核磁共振之研究：C 端及催化圈環對於與抑制劑結合及蛋白酶活性所扮演的角色。國立成功大學生物化學研究所碩士論文，2006。