天花粉蛋白Lys和Arg残基的化学修饰对其与IgE反应…

用马来酸酐及环己二酮分别对天花粉蛋白上的Ｌｙｓ,Ａｒｇ残基侧链进行修饰,并以竞争性酶免疫测定检查了化学修饰对ＴＣＳ与小鼠抗ＴＣＳ单抗ＴＥ－１（ＩｇＥ）反应活性的影响。两种修饰均使ＴＣＳ与单抗ＴＥ－１反活性下降,推测Ｌｙｓ残基可能直接参了与单抗ＴＥ－１的结合,热变性实验提示ＴＣＳ上单抗ＴＥ－１识别部位需要一定的空间构象。     

天花粉蛋白的定点聚乙二醇修饰

用一种定点修饰天花粉蛋白（ｔｒｉｃｈｏｓａｎｔｈｉｎ,ＴＣＳ）的方法,将聚乙二醇（ＰＥＧ）偶联到预先选定的位点．利用ｎＴＣＳ无半胱氨酸（Ｃｙｓ）残基这一特点,通过定点突变将一个Ｃｙｓ残基引入ＴＣＳ以取代第７位的丝氨酸（Ｓｅｒ）残基．然后,与巯基反应的ＰＥＧ－ｍ ａｌｅｉｍ ｉｄｅ 即可偶联到新引入的Ｃｙｓ 残基上．经纯化得到均一的ＰＥＧ－ＴＣＳ复合物,在ＳＤＳ－ＰＡＧＥ上显示一条区带,表观分子量为３８ ｋＤ．复合物的体外致核糖体失活活性降低了６倍,但其体内引产活性与ｎＴＣＳ相同．定点ＰＥＧ修饰方法为改造ＴＣＳ提供了新途径．     

胸腺肽α_1活性片段和其自旋标记衍生物的合成及免疫活性研究

报道了胸腺肽α_１活性片段Ｔｈｙｍｏｓｉｎα_１［Ｌｙｓ（２３）］（２３－２７）ＯＨ和其自旋标记衍生物的合成及对实验动物免疫功能的影响。其氨基酸序列分别为Ｈ_２Ｎ－Ｌｙｓ－Ｇｌｕ－Ｇｌｕ－Ａｌａ－Ｇｌｕ－ＯＨ,用Ｔｈｙα_１［Ｌｙｓ~（２３）］表示,修饰物为Ｇｌｕ－ＯＨ）,用Ｔｈｙα_１［Ｌｙｓ~（２３）］·Ｒ表示。ＨＰＬＣ测得该肽的纯度在９０％以上,ＥＳＲ谱测定自旋标记衍生物给出氮氧自由基信号,氨基酸组成与预期值相符。实验小鼠腹腔连续注射剂量为０．１、１、１０、１００和１０００μｇ／ｋｇ的该肽１０天后,发现腹腔巨噬细胞吞噬功能、ＥＲＦＣ阳性率及血清溶血素含量明显升高,且最佳剂量在０．１－１０μｇ／ｋｇ范围。实验结果表明人工合成胸腺肽α_１活性片段及其自旋标记衍生物具有显著的免疫促进活性。     

大肠杆菌精氨酰－tRNA合成酶的Lys306为酶活力所必需

将大肠杆菌精氨酰ｔＲＮＡ合成酶（ＡｒｇＲＳ）上Ｌｙｓ３０６用基因点突变的方法分别变为Ａｌａ和Ａｒｇ的密码子;得到变种基因ａｒｇｓ３０６ＫＡ和ａｒｇｓ３０６ＫＲ。变种基因重组在ｐＵＣ１８上,转化到大肠杆菌ＴＧ１中,转化子中ＡｒｇＲＳ及其变种ＡｒｇＲＳ３０６ＫＡ和ＡｒｇＲＳ３０６ＫＲ所表达的蛋白量至少为ＴＧ１表达ＡｒｇＲＳ蛋白量的１００倍。细胞粗抽提液中ＡｒｇＲＳ的比活ＴＧ１、转化子ｐＵＣ１８－ａｒｇｓ、ｐＵＣ１８－ａｒｇｓ３０６ＫＡ和ｐＵＣ１８－ａｒｇｓ３０６ＫＲ分别为１．６５、２１０、１．８和３８单位／毫克。结果表明ＡｒｓＲＳ的Ｌｙｓ３０６为Ａｌａ取代使活力完全丧失;若被Ａｒｇ取代,则活力丧失８０％以上。Ｌｙｓ３０６为ＡｒｇＲＳ活力所必需。     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

大肠杆菌精氨酰－tRNA合成酶的Lys378和381的点突变研究

用点突变的方法将大肠杆菌精氨酰—ｔＲＮＡ合成酶（ＡｒｇＲＳ）的基因ａｒｇｓ上相应于Ｌｙｓ３７８和Ｌｙｓ３８１的密码ＡＡＡ分别变为两氨酸的密码ＧＣＡ和精氨酸的密码子ＣＧＴ,得到了４个ａｒｇｓ的突变体ａｒｇｓ３７８ＫＡ,ａｒｇｓ３７８ＫＲ,ａｒｇｓ３８１ＫＡ和ａｒｇｓ３８１ＫＲ,将它们分别连接到ｐＵＣ１８上,转入大肠杆菌ＴＧ１,在ＴＧ１转化子中,ＡｒｇＲＳ及其变种的表达量约为ＴＧ１中的１２０倍以上。结果表明Ｌｙｓ３７８为Ａｒｇ和Ａｌａ取代分别使活力下降０％和１０％;Ｌｙｓ３８１变为Ａｌａ和Ａｒｇ后,活力分别下降３３％和１０％左右。Ｌｙｓ３７８不为酶活力必需。Ｌｙｓ３８１部位的正电荷对酶活力是重要的。     

［B3－Lys］－胰岛素的研究：受体结合及生物活性

本文用１２５Ｔ－［Ｂ３－Ｌｙｓ］－胰岛素和１２５Ⅰ－胰岛素研究了［Ｂ３－Ｌｙｓ］－胰岛素和胰岛素与人胎盘细胞膜（ＨＰＭ）胰岛素受体结合特性并进行了比较。实验结果表明［Ｂ３－Ｌｙｓ］－胰岛素与ＥＰＭ胰岛素受体结合能力比天然猪胰岛素高。由竞争取代曲线得到的［Ｂ３－Ｌｙｓ］－胰岛素和猪胰岛素的ＩＣ（５０）值分别为０．６５和１．１１ｎｍｏｌ／Ｌ。经Ｓｃａｔｃｈａｒｄ分析得出［Ｂ３－Ｌｙｓ］－胰岛素与ＨＰＭ胰岛素受体中高亲和位点和低亲和位点结合的亲和常数分别为１．７２×１０９和２．２７×１０６Ｌ／ｍｏｌ,而猪胰岛素分别为１．２６×１０９和１．４７×１０６／ｍｏｌ。促脂肪细胞合成脂肪实验结果表明［Ｂ３－Ｌｙｓ］－胰岛素也同样具有比天然猪胰岛素更高的离体生物活力,ＥＣ（５０）分别为０．１７５和０．３０１ｎｍｏｌ／Ｌ。可见［Ｂ３－Ｌｙｓ］－胰岛素的受体结合能力和高体生物活力都为猪胰岛素的１．７倍。     

鸡减蛋综合征病毒（EDSV—76）基因组E1区结构特点分析

ＥＤＳＶ－７６病毒中国株ＡＡ－２经常规方法提取其病毒ＤＮＡ后,建立了限制性内切酶ＰｓｔＩ水解片段的全基因文库。对其中ＰｓｔＩ－Ｇ片段和ＰｓｔＩ－Ａ片段的正反链进行序列测定,获得ＥＤＳＶ Ｅ１区（０－８．８ｍ．ｕ）的核苷酸序列。经分析,ＥＤＳＶ Ｅ１区具有与其他腺病毒Ｅ１区类似的结构。以大于６０个氨基酸残基为标准,ＥＤＳＶ Ｅ１区共有７个开放读码框架（ＯＲＦ）,其中Ｒ１、Ｒ２、ＥｌｂｓＴ和Ｅ１ｂ１Ｔ     

天花粉蛋白Y14F/R22L定点突变及其活性研究

利用多聚酶链式反应（ＰＣＲ）技术,对天然天花粉蛋白（ｎＴＣＳ）基因在Ｔｙｒ１４和Ａｒｇ２２两个保守残基处同时进行定点突变,即Ｔｙｒ１４变成Ｐｈｅ,Ａｒｇ２２变成Ｌｅｕ,然后克隆到ｐＥＴ－８ｃ高效表达载体上,构建成重组质粒ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ．经序列分析,定点突变的结果与预先设计的完全一致,突变后的天花粉蛋白命名为Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ．将ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ转化到Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３,ｐＬｙｓＳ）中,进行表达．经ＣＭ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ柱纯化,ＳＤＳ－ＰＡＧＥ鉴定,纯度可达９０％．ＲＩＰ活性测定显示,Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ的活性比ｎＴＣＳ降低了７．５倍,活性变化不显著,因此,ＴＣＳ的Ｔｒｙ１４和Ａｒｇ２２对维持其活性部位构象并不是必需的．但由于Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ在Ｅ．ｃｏｌｉ中的表达量与ｎＴＣＳ相比明显下降,因此,Ｔｙｒ１４和Ａｒｇ２２可能与ＴＣＳ翻译后的折叠有关．     

缩短的人巨噬细胞集落刺激因子（M—CSF）在大肠杆菌中…

本文用聚合酶链反应（ＰＣＲ）获得了一个缩短的人巨噬细胞集落刺激因子ｃＤＮＡ基因,并克隆在质粒ｐＡＥＴ３ｄ中,在Ｔ７启动子指导下,在大肠杆菌ＢＬ２ＬｙｓＥ中获得了和一个６组氨酸短肽标签的融合表达。重组的融合Ｍ－ＣＳＦ表达量占菌体总蛋白的１２％,表达产物一部分以不溶性包涵体形式存在,另一部分则以可溶性蛋白存在。经过金属螯合新和层析一步纯化,所得的融合（Ｈｉｓ）６－Ｍ－ＣＳＦ在还原型ＳＤＳ－ＰＡＧＥ上基     

Y55 and D78 are crucial amino acid residues of a new IgE epitope on trichosanthin

Trichosanthin (TCS) possesses many biological and pharmaceutical activities, but its strong immunogenicity limits its clinical application. To reduce the immunogenicity of TCS, we modified the reported method for the prediction of antigenic site and identified two crucial amino acid residues (Y55 and D78) for a new epitope. We mutated these two residues into glycine and serine, respectively, and obtained three mutants, Y55G, D78S, and Y55G/D78S. These mutants induced less amount of Ig and IgG antibodies in C57BL/6J mice than wild-type TCS (wTCS) (p<0.01) and almost lost the ability to induce IgE antibody production. The mutants stimulated fewer TCS-specific B cells in C57BL/6J mice than wTCS (p<0.01). Compared with wTCS, Y55G, D78S, and Y55G/D78S lost 26.9%, 17.9%, and 98.7% specific binding ability to anti-TCS monoclonal antibody TCS4E9, respectively. These mutants still retained RNA N-glycosidase activity. In conclusion, Y55 and D78 are two crucial amino acid residues of a new IgE epitope on TCS, and their mutation reduces the immunogenicity of TCS, but still retained the enzymatic activity.     

Identification of the amino acid residues in trichosanthin crucial for IgE response

Trichosanthin (TCS) is the major effective component from Chinese herb Trichosanthes kirilowii. TCS has been approved to be effective in clinical treatment of HIV infection and leukemia, but its allergenicity has limited its clinical usage. To identify amino acid residues in TCS with an important role in IgE induction, TCS-specific IgE mAb (TE1) was used to serve as a probe and TE1 epitope was determined by a random phage-peptide library. Based on phage peptide sequences, TE1 epitope was predicted at amino acid residues 169-174 (QQIGKR) of TCS protein. Based on modeling data, two amino acids (Lys173 and Arg174) on TCS were considered to have a crucial role in binding to TE1. After lysine 173 and arginine 174 were mutated to glycine, the mutant TCS protein specifically lost the binding activity to TE1 mAb and exhibited reduced IgE induction in the immunized mice. The data showed that the IgE epitope of TCS was determined and shown to play a critical role in induction of IgE, and the modification of IgE-epitope may be a useful strategy to reduce the allergenicity of an allergen.     

The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant—free condition

Trichosanthin(TCS) is a potent allergen in mice.It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum.TCS can bring out the IgE responses to ovalbumin(OVA),while OVA itself could not induce IgE responses to it .However,TCS only works when OVA immunization is given one day after TCS immunization.Either time lag in OVA immunization,or immunization of both antigens at the same time,or OVA immunization given first,all has no effect on the induction of IgE responses to OVA.Through analysis of the antibody specificity of hybridoma clones,it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones.The IgE antibldies showed no polyreactivity to different antigens.     

The Antigenic Properties of β-Lactoglobulin Examined with Mouse IgE Antibody

The effects of enzymic or chemical fragmentations and of chemical modifications on the antigenic properties of bovine β-Mactoglobulin were examined using specific mouse IgE antibody. The antigenic reactivity of β-Mactoglobulin derivatives was represented in terms of their ability to neutralize specific IgE antibodies assayed by passive cutaneous anaphylaxis in rat. The tryptic, chymotryptic or peptic hydrolysate free of native β-Mactoglobulin had no antigenic reactivity but the fragments obtained after CNBr cleavage retained the ability to bind the antibody. Modification of the sulfhydryl group, arginine or tryptophan residues and amino groups had no effect on antigenic reactivity but a little decrease in the reactivity was observed on the cleavage of the two disulfide bridges. These results suggest that one sulfhydryl group, two arginine and tryptophan residues and most of the amino groups are out of antigenic sites in β-Mactoglobulin and that the antigenicity depends on the conformation maintained by the disulfide bridges.     

Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2-3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.     

Construction of normal human IgE phage antibody library and the screening of the anti—trichosanthin IgE

Allergen specific IgE response is the major cause of immediate hypersensitivity.However the number of IgEproducing B cells and the amount of IgE,especially the specific IgE,are so low,it greatly impedes the study of the allergic-specifc antibody responses.Here we report the construction of a normal human IgE combinatorial library.The repertoire of IgE VH genes and of κ genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR,and were then constructed to form the phage surface display human Fab(IgEVH) library.A plant protein allergen,trichosanthin(TCS),was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library.Human IgE(Fab) to TCS were detected.     

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.     

Modulation of IgE synthesis by IgE-binding factors released by T cells of asthmatic patients with elevated serum IgE

The culture supernatants of unstimulated T cells (TCS) from asthmatic patients with elevated serum IgE were tested for IgE-binding factors (IgE-BFs) displaying the IgE-potentiating activity. The IgE-BFs were detected by their ability to inhibit the rosetting of RPMI 8866 cells with ox erythrocytes coupled with mouse monoclonal antibody (E-Mab) specific to Fc receptors for IgE (Fc epsilon R). TCS showing the rosette-inhibiting activity significantly enhanced the spontaneous IgE synthesis by B cells of allergic individuals. Interestingly, rosette-inhibiting factors could be removed by absorption with IgE-Sepharose from which they were subsequently eluated with acid buffer, indicating that the rosette inhibition was indeed mediated by IgE-BFs. In addition, such IgE-BFs had affinity for concanavalin A and lost their IgE-potentiating activity after treatment with trypsin and neuraminidase. In contrast, T cells treated with tunicamycin released IgE-suppressing factors capable of inhibiting the IgE-potentiating activity of TCS derived from untreated T cells. On the other hand, the culture supernatants from subpopulations depleted of Fc epsilon R+ T cells but not of Fc gamma R+ T cells contained neither rosette-inhibiting factors nor IgE-potentiating factors, suggesting that IgE-BFs were released by in vivo pre-activated Fc epsilon R+ T cells. With regard to circulating Fc epsilon R+ T cells determined by E-Mab, they were significantly higher in asthmatic patients with elevated serum IgE (0.77 +/- 0.15%) than in normal subjects (0.17 +/- 0.07%) in spite of a very small proportion of T cells bearing Fc epsilon R.     

Demonstration of intracytoplasmic IgE in circulating lymphocytes of allergic individuals

Cells containing intracytoplasmic IgE (IC IgE) were demonstrated in the peripheral blood mononuclear cells of atopic individuals by means of indirect immunofluorescence, employing mouse monoclonal anti-human IgE antibody. IC IgE-positive cells are low-density B cells as shown by centrifugation on discontinuous bovine serum albumin density gradient and by their reactivity with B1 monoclonal antibody specific to B lymphocytes, respectively. Further characterization of these cells by means of a rosette assay employing anti-Ig-coupled bovine erythrocytes indicated that these cells were surface IgE (sIgE) positive and sIgM negative. The data strongly suggest that activated IgE B cells are circulating in the blood of allergic individuals.     

Sites of protein-protein interaction on the mitochondrial F1-ATPase inhibitor protein.

We have investigated the structure of the mitochondrial F1-ATPase inhibitor protein from ox heart by using a differential trace-labelling method. This method has also been used to determine sites on the inhibitor protein involved in binding to both the isolated mitochondrial ATPase (F1) and to a specific anti-inhibitor antibody. Native, free inhibitor was trace-labelled on its lysine and serine residues with [14C]acetic anhydride, and inhibitor protein unfolded in guanidinium chloride or specifically bound to another protein, with [3H]acetic anhydride. Exposure/concealment of residues was deduced from the 14C/3H ratios of the peptides in a proteolytic digest of the inhibitor, after separation by h.p.l.c. None of the lysine or serine residues in the native inhibitor are as exposed as in the unfolded form. There is a gradient of reactivity, with residues 54-58 being most concealed and exposure increasing towards either end of the protein. A slight decrease in reactivity is noted in residues 1-3, suggesting that the N-terminus may be in a fairly restricted environment. These findings are discussed in the light of the predicted structure of the inhibitor protein. All but one of the labelled residues increases in reactivity when inhibitor protein binds to F1. The exception, Lys-24, is only slightly concealed. Hence, F1 binding appears not to involve the lysine or serine residues directly. This finding is consistent with the view that the F1-inhibitor interaction is hydrophobic in nature. Complementary information was provided using an anti-inhibitor antibody that binds to a site on the inhibitor different from that at which F1 binds. Binding of this antibody conceals residues 54, 58, and 65 considerably. This confirms that F1 does not interact with these hydrophilic residues on the inhibitor protein.