I. Studies on the structure and function of Acacia confuca tryp- sin inhibitor by site-specific mutagenesis II.Studies on the structures and functions of isoabrins at gene level

• Main Authors: Chih-Hung Hung, 洪志宏
• Other Authors: Jung-Yaw Lin
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/11554345988055063105

博士 === 國立臺灣大學 === 生化學研究所 === 81 === PART I:相思樹胰蛋白脢抑制劑(ACTI)由A鏈及B鏈以一對雙硫鍵連結所組 成 ,A鏈含136個氨基酸,B鏈含39個氨基酸,A分子量共19.4KDa,屬於 Kunitz-typ trypsin抑制劑.合成可能的相思樹胰蛋白脢抑制劑A鏈的氨端 和B鏈的C端氨基酸的Oligonucleotides當作Primers,萃取尚未成熟種子的 cDNA當作模板,利用PCR的技術,選殖了ACTI的cDNA序列,轉譯成176個氨基 酸,發現比蛋白序列多出一個氨基酸,Ser137,其位於A鏈與B鏈連結的位置, 成熟的ACTI需藉一特異的酵素進行Post-translation processing將它切 除。將ACTI的Coding region崁入pGEX-2T質體,置於大腸桿菌中表現,沒有 經過Post-translation processing的融合蛋白質和重組ACTI皆具有可抑 制胰蛋白脢抑制劑活性。 Lys64可能是活性位置,所以利用Site- Specific mutagenesis將Lys64突變成Ile和Arg,K64I其活性完全喪失,但 K64R其活性幾乎不變,顯示Lys64-Ile 65是其活性位置,屬於離氨酸型的胰 蛋白脢抑制劑,且活性位置上離氨酸和精氨酸可互換而不影響其活性. ACTI含有兩對雙硫鍵,分別破壞其雙硫鍵的形成,結果位於A鏈內這對雙硫 鍵(Cys40-Cys86)對ACTI的活性影響很小,但連結A鏈與B鏈的這對雙硫鍵 Cys133-Cys141)破壞後,整個分子結構改變且活性完全喪失. PART II:雞 母珠毒蛋白是由A鏈及B鏈以一對雙硫鍵連結所組成,B鏈具有辨識細胞表面 受體的能力,A鏈具有N-glycosidase的活性,當進入細胞內,A鏈能水解真核 細胞核醣體上28S rRNA第4324位置上Adenine 的醣甘犍,而抑制蛋白質生 合成.雞母珠毒蛋白具有多種Isoforms,對蛋白質生合成抑制能力不相同. 利用PCR技術,選殖了三株異雞母珠毒蛋白cDNAs,依據氨基酸序列,物理化 學,和免疫反應,推測這三株異雞母珠毒蛋白是Abrin-a, Abrin-b和Abrin- d.將異雞母珠毒蛋白的A鏈嵌入pGEX-2T質體,置於大腸桿菌中表現,分析對 無細胞蛋白質生合成抑制活性或N-glycosidase活性,對無細胞蛋白質生合 成抑制活性分析結果,重組Abrin-a和Abrin-d的A鏈活性與原始Abrin-a A 鏈相似,但重組Abrin-b A鏈活性約低3倍,而N-glycosidase活性,重組 Abrin-b A鏈比另外兩種重組Abrin A鏈約低10倍.將Abrin-a A鏈可能是活 性位置的Glu164突變成Ala，Arg167突變成Leu或兩者皆突變,結果 ,在抑 制蛋白質生合成活性分析,E164A與R167L分別降低了25倍和625倍,而 E164 A.R167L更降低了1250倍,而N-glycisidase分析,與抑制蛋白質生合成之結 果相似.所以Glu164可能僅是穩定酵素與受質結和的結構，而Arg167直接 與催化作用有關. Part I:ACTI(Acacia confusa trypsin inhibitor)is one of the Kunitz -type inhibitors.The native form is composed of two different po- lypeptide: the A and B-chains, 136 amino acids and 39 amino acids residues,respectively.The molecular weight of ACTI is 19.4 kDa. The deduced amino acid sequence agreed with that determined by the peptide analysis except an extra amino acid residue,Ser, was found at the junction of A and B chain,which was removed by post- translation processing with specific protease(s). A recombinant plasmid containing the coding regions for ACTI has been construct ed and expressed in Escherichia coli cells,as a fusion protein be tween ACTI and glutathione S-transferase(GST).Both the reACTI and fusion protein have a strong inhibitory effect on trypsin activi- ty without post-translational proteolysis.We converted the Lys64 residue into Ile or Arg residue by using site-specific mutagene- sis to identify the active site of ACTI.The reACTIK64 I mutant lo- st its inhibitory effect on trypsin activity but the reACTIK64R mutant almost did not change its activity.It indicates that Lys64 is located at the reactive site of ACTI, and ACTI is one of lysine -type trypsin inhibitors. Lys or Arg at position of active site can be replaced each other,and it still maintains its fully inhi- bitory effect on trypsin activity.ACTI contains two disulfide bo- nds;one is an intradisulfide bond(Cys40-Cys86)locating in A chain and another is an interdisulfide bond(Cys133-Cys141)linking A and B chains. The disulfide bond was removed by converting Cys to Gly by site- specific mutagenesis. The results demonstrated that the trypsin inhibitory activity of reACTIC40G mutant was almost iden- tical to that of native ACTI,wheras the reACTIC133G mutant lost its trypsin inhibitory activity completely caused by the change of molecular conformation.It suggests that the interdisulfide bo- nd(Cys133-Cys141)is indispensible for the trypsin inhibitory act- ivity.