Three-Dimensional Analysis of CD6 Site-Directed Mutagenesis and Monoclonal Antibody Binding Studies Using the X-ray Structure of Mac-2 Binding Protein and a Molecular Model of the CD6 Ligand Binding Domain

The extracellular region of CD6 consists of three scavenger receptor cysteine-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues important for the CD6-ALCAM interaction have previously been identified by mutagenesis. A total of 22 CD6 residues were classified according to their importance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The three-dimensional structure of the SRCR domain of Mac-2 binding protein has recently been determined, providing a structural prototype for the SRCR protein superfamily. This has made a thorough three-dimensional analysis of CD6 mutagenesis and mAb binding experiments possible. Mutation of buried residues compromised both mAb and ALCAM binding, consistent with the presence of structural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were found to map to the surface in the same region of the domain. This suggests that the CD6 ligand binding site and epitopes of tested mAbs overlap and provides an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the ALCAM binding site.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050263Abbreviations ALCAM activated leukocyte cell adhesion molecule - CD6D3 third (membrane-proxi-mal) extracellular domain of CD6 - IgSF immunoglobulin superfamily - mAb monoclonal antibody - M2BP Mac-2 binding protein - SRCR scavenger receptor cysteine-rich domain - SRCRSF scavenger receptor cysteine-rich protein superfamily     

Effect of γ-radiation on the ratio of [18F]2-fluoro-2-deoxy-D-glucose to glucose utilization in human glioblastoma cellsin vitro

The glucose consumption in tumoursin vivo as reflected by uptake of [¹⁸F]2-fluoro-2-deoxy-D-glucose (¹⁸FDG) using positron emission tomography (PET) is currently under investigation as a measure of tumour response to radiotherapy. The calculation of cerebral metabolic rate of glucose from¹⁸FDG-PET data requires a proportionality factor referred to as the lumped constant. In the presentin vitro study, the utilizations of¹⁸FDG and glucose have been measured in a human glioblastoma cell line (86HG-39) as a function of γ-radiation dose with various post-irradiation times and of different fractionation modes. The ratio of utilization of¹⁸FDG to that of glucose (R_F/G), assumed to correspond to the lumped constant, was observed to increase 12 and 24 h after single fraction γ-exposure by factors ranging from 1.2 to 1.5 compared with the non-irradiated controls. It decreased after multiple fraction γ-exposure (4 × 2 Gy) by a factor of 0.7 compared with the single fraction schedule (1 × 8 Gy). The results suggest that the affinities of glucose transporters or hexokiriase enzyme or both for¹⁸FDG and glucose could be influenced by γ-irradiation in this tumour cell linein vitro. Apparent changes of the glucose consumption determined with PET in human tumours following radiotherapy may, therefore, not be solely due to changes in cellular metabolism or cell number but may also be due to changes in R _F/G .     

恒温和变温下中国对虾生长和能量收支的比较

对比研究了模拟自然昼夜温度变化节律的4个变温(22±2)、(25±2)、(28±2)和(31±2)℃与相应的恒温22、25、28和31℃下中国对虾(F ennerop enaeus ch inensis O sbeck)生长和能量收支的差异。结果表明,对虾在(22±2)℃、(25±2)℃和(28±2)℃变温条件下的生长率显著高于相应的恒温,但(31±2)℃与恒温31℃相比没有显著差异。与相应的恒温相比,(25±2)℃、(28±2)℃和(31±2)℃变温下对虾的摄食量显著增大,(22±2)℃、(25±2)℃和(28±2)℃变温下对虾的饵料转化率则显著提高。但变温下对虾对食物的消化率与相应的恒温相比没有显著差异。能量收支研究结果则发现,(22±2)℃、(25±2)℃和(28±2)℃变温下对虾摄食能中,用于生长的能量比例显著增加,而(31±2)℃与31℃相比则未见显著差异。从而表明,变温促长的主要机制可归因于变温下摄食量的增大、饵料转化率的提高及其摄食能中用于生长能比例的增加。     

基于氨基酸组分和位点保守信息识别蛋白质HEME结合残基

血红素是一种重要的、常用的配体,在电子传递、催化、信号转导和基因表达等方面发挥着重要作用,准确预测蛋白质与血红素相互作用的结合残基是结构生物信息学的主要挑战之一。本文下载整理了Biolip数据库中HEME配体与蛋白质结合的信息,统计分析了结合残基和非结合残基的氨基酸组分和位点保守性信息并将其作为预测特征参数,用Fisher-PSSM判别法识别HEME结合残基,计算结果表明优化特征参数的Fisher-PSSM判别法得到了较好的预测结果。     

Asia Ⅰ型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位.本研究设计和合成了由Asia Ⅰ型FMDV VP1蛋白136～160aa和198～211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因.利用BamH I、EcoR I和Xho I位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG.100μg FshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200 ID_(50)剂量FMDV攻击时得到了完全保护.由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防.     

尿激酶型纤溶酶原激活剂单克隆抗体的生产及其在抗原纯化中的应用

从ＣＨＯ工程细胞培养上清初步纯化的ｕＰＡ免疫ＢＡＬＢ／ｃ小鼠,通过杂交瘤技术制备单克隆抗体细胞株,取其中一株３８－１－７株作高密度大量培养,细胞密度达１３．２×１０６／ｍＬ时,抗体滴度为１∶６１．４４×１０４。用自制的ｕＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱纯化抗体。抗体滴度提高２４３倍。纯化后的抗体与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ珠交联,制成ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析抗体柱,亲和力常数：１．２８×１０９（ｍｏｌ／Ｌ）－１,交联率：８３．５％。直接从培养上清纯化ｕＰＡ,纯度为９６．３％,回收率：８１．６％±１９,纯化倍数：５０倍左右,比活１．１１±０．２９×１０５。试验结果表明该法效果好,方法简单、操作方便、值得进一步研究和应用。     

PURE ribosome display and its application in antibody technology

Ribosome display utilizes formation of the mRNA–ribosome–polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA–ribosome–polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Interaction Between Tetanus Toxin and Rabbit Kidney: A Comparison with Rat Brain Preparations

125I-Tetanus toxin is bound by basolateral membranes from rabbit kidneys. Fixation is specific, as it is minimally inhibited by the nonbinding (fragment B) moiety of tetanus toxin, whereas the binding moiety (fragment C) is equivalent to the native toxin in inhibiting fixation. Competition is also pronounced with mildly toxoided toxin. Association and dissociation of 125I-toxin are delayed in kidney when compared to brain membranes. The binding sites in kidney membranes are partially sensitive to neuraminidase and resist heating to 56 degrees C, in contrast to those in brain membranes which are very sensitive to both treatments. The binding sites of the two preparations can be discriminated further by variation of the ionic environment. Sodium dodecyl sulfate-disc gel electrophoresis followed by transfer to nitrocellulose, and TLC with consecutive overlay indicate that tetanus toxin exclusively binds to long-chain gangliosides from rat brain. Binding sites in kidney membranes from rabbits and rats can be made visible by the overlay technique. They are apparently heterogeneous and more hydrophobic. We conclude that rabbit kidney contains binding sites for tetanus toxin which resemble gangliosides but differ from the major gangliosides in brain both chemically and with respect to their interaction with tetanus toxin.     

Kunitz 型丝氨酸蛋白酶抑制剂结构与功能研究

蛋白酶抑制剂在酶学及蛋白质的结构与功能关系研究中有重要意义,Kunitz型丝氨酸蛋白酶抑制剂是其中最重要的,也是研究最广泛的蛋白酶抑制剂之一．该类蛋白酶抑制剂三维结构高度保守：由一个明显的疏水核心、三对高度保守的二硫键桥、三链β-折叠和一个N端3 10螺旋及一个C端α-螺旋组成．3对二硫键对分子空间结构的稳定起着非常重要的作用．这一类型抑制剂有5个主要的活性位点：P1、P1’、P3、P3’、P4,它们都位于一个溶剂暴露的环上．P1位点是抑制作用的关键活性位点,抑制剂的专一性由P1位点氨基酸残基的性质决定;P1’位点氨基酸残基的侧链大小对抑制剂．酶的结合常数有很大影响,用大的侧链残基取代会导致结合常数降低;P4位点残基被取代经常产生负效应,会导致活性区域环的构象发生很大改变,从而影响酶与抑制剂的结合．