【作者】 陶虎；

【导师】 张友尚； 崔大敷；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2006， 博士

【摘要】 多肽及较小的寡肽,在激素调控、细胞免疫、信息传递等生命活动中日益显示重要的生物功能。为研究它们的作用机理,多肽合成是提供这些多肽的有效途径,至于N端、C端及侧链经过加工的天然多肽或含有非编码氨基酸多肽的制备,目前仍必须依靠多肽的化学合成或体外的酶促合成。本论文研究活性多肽类似物的设计、制备和功能,分为四个部分,采用多肽固相和液相合成的方法,分别合成了活性多肽绿豆胰蛋白酶抑制剂Lys片段长链16肽、天花粉胰蛋白酶抑制剂、癌胚抗原肽、甜味肽等一系列类似物,并测定了这些类似物的生物活性,有以下新的研究结果:1、furin酶抑制剂的设计、制备和功能;2、芳香氨基酸芳环之间的π-π共轭能部分补偿二硫键的作用;3、癌胚抗原肽Gly4被L-Pro替代可增加与HLA-A2分子的结合;4、肽键对甜味肽保持甜味是必须的。第一章是将绿豆胰蛋白酶抑制剂Lys片段改造为furin和kexin抑制剂。高活性、低分子量的furin酶抑制剂有可能发展成为新型的抗细菌内毒素和抗病毒感染的药物,我们以绿豆胰蛋白酶抑制剂Lys片段长链22肽为模板,设计了一系列能抑制furin和kexin活性的突变体,采用如下三种方法逐步优化:一、去除Lys片段长链三对二硫键中的一对,用Ser替换Cys,减少可能存在的二硫键错误配对;二、抑制剂的活性中心根据furin底物的专一性逐步改变,即P1、P2、P4和P6位分别变为Arg、Lys、Arg和Arg,同时为了避免P7 Asp和P6 Arg之间可能存在的相互作用,将P7位Asp替换为Ala;三、将两对二硫键形成的双环的外面5个氨基酸残基去除。最后所得的16肽对furin和kexin的抑制常数分别为:2.45×10-9 M和5.60×10-7 M。上述得到的突变体抑制剂为‘自杀性’抑制剂,若与酶长时期保温,P1与P’1间的肽键将被裂解,抑制活性也就丧失。进一步将P’1位Ser变为D-Ser或N-methyl Ser,P’1位为N-methyl Ser的突变体对furin的抑制活力为4.70×10-8 M,与酶保温3小时后仍保留80%以上的抑制活性,P’1位为D-Ser的突变体与酶保温3小时后抑制活性几乎不变,但它的抑制常数(Ki)大大增加。因此N-methyl Ser的突变体对furin有较强的抑制活性,也不易被降解,是一种较理想的furin抑制剂,为进一步研究发展为抗病毒和细菌外毒素的药物提供了实验依据。第二章是对天花粉胰蛋白酶抑制剂的两对二硫键的作用进行了研究,二硫键在蛋白质的折叠和功能中有重要的作用,芳香族氨基酸的芳环之间形成π-π共轭能否部分补偿二硫键的作用?我们将天花粉胰蛋白酶抑制剂(Trichosanthes trypsin inhibitor I, TTI)的Cys14- Cys26或Cys8- Cys20的二硫键分别替换为Tyr和Phe,C14F-C26Y的突变体抑制活性为M6A-TTI-I的1/140。作为阴性对照的C14S-C26S突变体没有活性( Ki > 0.4 mM), Tyr和Phe间的π-π共轭可部分地补偿了Cys14- Cys26二硫键的作用。C8Y-C20F和C8S-C20S突变体都没有胰蛋白酶抑制活性(Ki > 0.4 mM),Cys8- Cys20二硫键的作用不能被Tyr和Phe间的相互作用替代。说明蛋白质的某些二硫键作用可以被芳香族氨基酸的芳环之间形成的π-π共轭部分补偿,但是这种补偿作用在一定条件下才能发生。第三章是研究癌胚抗原肽CAP-1(YLSGANLNL)的1-5位氨基酸残基对其活性的影响。癌胚抗原肽是一种HLA-A2限制的抗原表位,由于中国人群中HLA-A2阳性率大于40%,而且90%的胃癌、结肠癌和直肠癌患者癌胚抗原的表达呈阳性,因此这个抗原表位有极高的应用价值,临床已使用自体树突状细胞递呈癌胚抗原肽CAP-1治疗癌胚抗原阳性的肿瘤患者。为了进一步研究CAP-1的各个位点的氨基酸残基与其活性的关系,我们合成了一些CAP-1的第1,2,3,4,5位氨基酸分别替换为其他的氨基酸的类似物,并测定这些类似物与HLA-A2分子的结合能力和产生杀伤性T细胞的能力。其中Gly4变为L-Pro的肽(YLSPANLNL)HLA-A2分子的结合能力比CAP-1高,但是产生杀伤性T细胞的能力比CAP-1略低,提示Gly4对CAP-1与HLA-A2分子的结合以及与T细胞受体结合都有影响,为进一步研究提供了方向。第四章是研究甜味肽的结构和功能,甜味肽(L-天冬氨酸-L-苯丙氨酸甲酯)的肽键替换为酯键,甜味消失。甜味肽X射线晶体结构研究表明肽键的“–NH”可与L-天冬氨酸侧链羧基的“O”形成氢键,肽键替换为酯键后,氢键消失。文献已报道甜味肽的天冬氨酸可被天冬酰胺替换,仍保持甜味。我们合成了甜味肽的类似物L-天冬酰氨酰-L-α-羟基苯丙酸甲酯,将甜味肽的天冬氨酸替换为天冬酰胺,肽键替换为酯键。这个类似物的酰胺的“–NH”可与酯键的“O”形成氢键,但是经测定它没有甜味,表明肽键不能替换为酯键。肽键的“C-N”键有双键的性质,C、O、N、H四个原子在同一平面,肽键替换为酯键破坏了肽键特有的构象,使其没有甜味。更多还原

【Abstract】 This study is design and preparation of active peptide analogues of mung bean trypsin inhibitor Lys fragment, Trichosanthes trypsin inhibitor I, carcinoembryonic antigen peptide and aspartame using solid and liquid phase peptide synthesis methods. Based on the activities of the synthtic analogues, the ralationship of structure and activity was discussed.Chapter one is template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor. Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity toward furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45×10^-9 M for furin and of 5.60×10^-7 M for kexin, was designed by three incremental approaches. First, two non-essential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide miss-folding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P₁, Lys at P₂, Arg at P₄ and Arg at P₆. In addition, the P₇ residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N and C terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P’1 cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P’1 residue Ser was mutated to D-Ser or N-methyl Ser. The N-methyl Ser mutant gave rise to a Ki value of 4.70x10^-8 M for furin, and retained over 80% inhibitory activity even after a 3-h incubation with the enzyme. By contrast, the D-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors against furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.Charpter two studies on the two disulfide bonds （Cys14- Cys26 or Cys8- Cys20）of Trichosanthes trypsin inhibitor I. Disulfide bridges between cysteine residues are the key structural and functional elements of protein. Could theπstacking made by two aromatic residues partially compensate for the loss of the disulfide bridge? Two analogues of Trichosanthes trypsin inhibitor I with the Cys14- Cys26 or Cys8- Cys20 disulfide bonds replaced by Tys or Phe and two negative control analogues substitution serine for cysteine at 14 and 26 or 8 and 20 positions were synthesized, respectively. The C14F-C26Y mutant had 1/140 inhibitory activity of native Trichosanthes trypsin inhibitor I to trypsin. But the negative control the C14S-C26S mutant had no activity （ Ki > 0.4 mM） indicating that theπstacking made by C14F and C26Y may partially compensate for the action of the disulfide bond. However, different results obtained from the Cys14- Cys26 mutants that both the C8Y-C20Fand C8S-C20S mutants had no activity（ Ki > 0.4 mM） . So theπstacking made by two aromatic residues may partially compensate for the loss of the disulfide bridge in the some conditions.Charpter three studies on the action of the five amino acid residues （position 1, 2, 3, 4, 5） of carcinoembryonic antigen peptide-1 （CAP-1, YLSGANLNL）. CAP-1 is an HLA-A2 resticted epitope of tumor antigen carcinoembryonic antigen （CEA）. The epitope CAP-1 is an attractive drug target because more than 40% Chinese patients are HLA-A2 positive and more than 90% of gastric cancer, colon cancer and rectum cancer patients are carcinoembryonic antigen expressing positive. Autologous human cultured dendritic cell, loaded with CAP-1 was used for the treatment of the patients with advanced CEA-expressing malignancies in clinic. The analogues with five amino acid residues （position 1, 2, 3, 4, 5） were replaced by other residues were synthesized and tested for the activities of binding to HLA-A2 and generating cytotoxic T Lymphocyte. The analogue with the Gly4 mutation to Pro had a higher activity for binding to HLA-A2 and lower activity for generating cytotoxic T Lymphocyte. Our findings indicate the Gly4 influence the binding of CAP-1 to HLA-A2 and T cell receptor, shedding light on the molecular design of new epitope analogues.Charpter four studies on the relationship of structure and activity of aspartame. An aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester was synthesized with aspartic acid replaced by asparagine and peptide bond replaced by ester bond. The aspartic acid of aspartame could be replaced by asparagine as reported in the literature. In this analogue, the hydrogen of amide group could still form a hydrogen bond with the oxygen of ester bond and the ester bond was isosteric with peptide bond. However, the product was not sweet, showing that the peptide bond could not be replaced by ester bond. The peptide C-N bond behaves as a double bond that is not free to rotate and the C, O, N and H atoms are in the same plane. The replacement of peptide bond by ester bond destroyed the unique conformation of peptide bond, resulting in the loss of sweet taste.更多还原