Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D.

The B-cell antigen receptor of the IgM and IgD class is a multimeric complex consisting of the membrane-bound form of the immunoglobulin molecule and two other proteins, Ig-alpha and Ig-beta. The Ig-alpha and Ig-beta proteins form a disulphide-linked alpha/beta heterodimer and are encoded by the mb-1 (ref 9, 10) and B29 genes, respectively. Surface expression of the membrane-bound IgM molecule requires assembly with the alpha/beta heterodimer. The IgD molecule, however, can be expressed on the cell surface in an alpha/beta-dependent and -independent form. We show here that in the alpha/beta-independent form the IgD molecule is anchored in the plasma membrane through a glycosyl-phosphatidylinositol linker. In the presence of the alpha/beta heterodimer, most of the otherwise glycosyl-phosphatidylinositol-linked IgD molecule is expressed on the cell surface as transmembrane proteins.     

Synthesis in E. coli of alpha 1-antitrypsin variants of therapeutic potential for emphysema and thrombosis

The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components. The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal. Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking. Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein. To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA. We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli. The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation. The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor. The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder.     

Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfecta type II

Although the perinatal lethal form of osteogenesis imperfecta (OI type II) occasionally results from large rearrangements within the genes encoding type I collagen, most mutations are far more subtle. The complexity of the human collagen genes precludes cloning and sequencing each gene from every patient, and we have therefore developed an approach to localizing mutations at the protein level. We report here that cells cultured from 15 infants with OI type II synthesized both normal type I procollagen and a form that was unstable, poorly secreted and excessively modified. Abnormal procollagen from different strains was overmodified to different extents. The patterns of overmodification we observed are best explained by mutations that disrupt the Gly-X-Y sequence of pro alpha chains, and thus alter the rate of propagation of triple helix from COOH-terminus to NH2-terminus. As a consequence, a given mutation allows overmodification of all three chains in a molecule NH2-terminal to its position in the triple helix.     

DNA restriction fragments associated with alpha 1-antitrypsin indicate a single origin for deficiency allele PI Z

The alpha 1-protease inhibitor, or alpha-antitrypsin (AAT), a major plasma inhibitor of leukocyte elastase and bacterial proteases, is encoded at the PI locus on chromosome 14 (14q24.3-q32.1). A deficiency of AAT in individuals homozygous for the PI Z allele occurs in about 1 in 2,000-8,000 caucasians and is associated with an increased risk of early adult onset emphysema and liver disease in childhood. We have now used DNA polymorphisms associated with the AAT gene to investigate the origin of the PI Z allele. Using two genomic probes extending into the 5' and 3' flanking regions, respectively, we have identified eight polymorphic restriction sites. Extensive linkage disequilibrium occurs throughout the probed region with the PI Z allele, but not with normal PI M alleles. The Z allele occurs mainly with one haplotype, indicating a single, relatively recent, origin in caucasians.     

Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis

An old puzzle in protein biochemistry concerns the ready conversion of ovalbumin, by proteolysis, to the much more stable derivative, plakalbumin. Ovalbumin is now known to belong to the serpin superfamily, most of which are serine proteinase inhibitors. We report here studies of two such members of the family, the human plasma proteins alpha 1-antitrypsin and antithrombin, and show that they undergo a similar change in stability on selective proteolysis. This change, which is accompanied by a loss of inhibitory activity, can best be considered as an irreversible molecular transition from a native stressed (S) conformation, to a more ordered relaxed (R) form. The maintenance of the native S conformation, and hence the maintenance of inhibitory activity, is critically dependent on the integrity of an exposed loop of polypeptide. We propose that the susceptibility of this peptide loop to proteolytic cleavage gives it an incidental role as a physiological switch which allows the inactivation of individual inhibitors by specific proteolysis. The vulnerability of this exposed loop in each inhibitor also explains the pathological action of a number of venoms and toxins. In particular, the demonstration here of the cleavage of antithrombin, by leukocyte elastase, explains an observed change in blood coagulation that accompanies severe inflammation and which can result in fatal thrombosis.     

Was the loss of the D helix in alpha globin a functionally neutral mutation?

Proteins in the globin family are found in a variety of species from bacteria to man. From the many globin sequences known, evolutionary trees have been constructed showing that alpha and beta globins diverged from a common ancestor between 425 and 500 million years ago, after vertebrate species had appeared and roughly when sharks and bony vertebrates diverged. The alpha and beta globins assemble to form tetrameric haemoglobin, alpha 2 beta 2, which can switch between quaternary states having high and low oxygen affinity. This allows the protein to bind oxygen cooperatively and therefore efficiently transport oxygen from the lungs to respiring tissues. The alpha and beta globins have closely related tertiary structures, being alpha-helical proteins with similar haem-binding sites. Most globins consist of eight helices, designated A to H from the N terminus, connected by short nonhelical segments, but all known vertebrate alpha globins lack a D helix. Because the loss of this helix by alpha globin occurred shortly before tetrameric haemoglobin appeared, it might be a functionally important mutation required for a tetramer assembly or allostery. We have now tested this idea by engineering human haemoglobins containing beta subunits without a D helix and alpha subunits with a D helix. Both of these mutations have little effect on the oxygen-binding properties of the molecule. Thus it is possible that deletion of the D helix in the alpha subunit was caused by a neutral mutation.     

Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization

Repeating intermolecular protein association by means of beta-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer's, Huntington's and Parkinson's and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered beta-sheet linkages leading to intracellular accretion and disease. These 'serpinopathies' include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in alpha(1)-antitrypsin (alpha(1)AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the beta-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel beta-strands inserting in the centre of the principal beta-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible beta-sheet expansion.     

Plasma protease inhibitors in mouse and man: divergence within the reactive centre regions

The plasma protease inhibitors control a wide variety of physiological functions including blood coagulation, complement activation and aspects of the inflammatory response. The inhibitors function by forming a 1:1 complex with a specific protease within the reactive centre region of the inhibitor. Little is known about the evolutionary relationships of these inhibitors. We report here the sequences of cDNAs which represent the C-terminal halves of the two major murine plasma protease inhibitors. One of these, murine alpha 1-antitrypsin, more appropriately called alpha 1-proteinase inhibitor (alpha 1-PI), has diverged from its human counterpart at a vital position in the reactive centre but this has not led to a physiologically significant change in function. Also, we have determined the partial sequence of a recently characterized protein termed contrapsin, which inhibits trypsin-like proteases. We show, surprisingly, that contrapsin is highly homologous to human alpha 1-antichymotrypsin, an inhibitor of chymotrypsin-like proteases. The reactive centre regions of these two inhibitors have diverged considerably, which may account for the differences in specificity. We propose that the genes for contrapsin and human alpha 1-antichymotrypsin are the descendents of a single gene that have evolved since rodent and primate divergence to encode proteins with different functions.     

甘露(聚)糖结合凝集素外显子Ⅰ 54位密码子突变的检测方法

对人源甘露糖结合凝集素基因54位密码子点突变的情况进行测定,建立了相关实验室检测方法.采用PCR扩增目的片段,再对产物采用PCR-RFLP测定点突变.检测的哈萨克40例血样54位密码子的点突变情况:野生型为20例,占50%;突变杂合型为20,占50%;突变纯合子为0例,占0%.PCR-RFLP适合实验室检测MBL外显子I54密码子突变情况,可以作为分析依据.     

Extreme value distributions of mixing two sequences with the same MDA

Suppose [(i), i>or=1] and [Y(i), i>or=1] are two independent sequences with distribution functions F(X)(x) and F(Y)(x), respectively. Z(i,n) is the combination of X(i) and Y(i) with a probability p(n) for each i with 1相似文献   

Spectrin assembly in avian erythroid development is determined by competing reactions of subunit homo- and hetero-oligomerization

Erythroid differentiation entails the biogenesis of a membrane skeleton, a network of proteins underlying and interacting with the plasma membrane, whose major constituent is the heterodimeric protein spectrin, composed of two structurally similar but distinct subunits, alpha (relative molecular mass (Mr) 240,000) and beta (Mr 220,000), which interact side-on with each other to form a long rod-like molecule. Interaction of this network with the membrane is mediated by the binding of the beta subunit to ankyrin, which in turn binds to the cytoplasmic domain of the transmembrane anion transporter (also referred to as band 3). Purified alpha and beta subunits of spectrin from the membrane of mature red blood cells will spontaneously heterodimerize, suggesting that assembly of the spectrin-actin skeleton is a simple self-assembly process, but in vivo studies with developing chicken embryo erythroid cells have indicated that assembly in vivo is more complex. We now present evidence that newly synthesized spectrin subunits in vivo or in vitro rapidly adopt one of two competing conformations, a heterodimer or a homo-oligomer. These competing reactions seem to determine the overall extent of spectrin assembled during erythroid development by determining which conformation will assemble onto the membrane-skeleton (the heterodimer) and which conformations are targeted for degradation (the homo-oligomers).     

Identification of a factor that links apoptotic cells to phagocytes

Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif--it bound particularly strongly to cells expressing alpha(v)beta(3) integrin. The NIH3T3 cell transformants that expressed a high level of alpha(v)beta(3) integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.     

一种蛋白A的Z结构域衍生物的构建及对IgG亲和力的研究

蛋白A是金葡菌中一种重要的致病因子,有较强结合多种哺乳动物的IgG的能力,因此降低机体产生特异性SPA抗体的水平,使金葡菌具免疫逃逸功能.研究一种与IgG亲和力较弱的蛋白A衍生物,对金葡菌疫苗的开发具重要意义.研究表明,蛋白A的Z结构域中位于第13、14位的Phe,Tyr是两个与IgG结合的关键位点.本文首次通过PCR突变技术,将这两个关键氨基酸均改造为Gly,构建突变体ZFY.Discovery Studio3.5受体-配体相互作用力模块分析显示,突变体ZFY与IgG结合力降低到-61.68kcal/mol,仅为原相互作用力的10.69%;构建得到ZFY蛋白,经ITC测定,ZFY与兔IgG的亲和常数降低到1.2×104 M-1,仅为原亲和常数的0.39%,证明了ZFY是一种与IgG亲和力较弱的蛋白A衍生物,为实现新型蛋白A疫苗治疗金葡菌感染奠定基础.     

Molecular mechanisms of serpin deficiencies

The study of serpin deficiency is currently one of the most active areas in basic medical research. Recently, three hypotheses concerning serpin deficiency have been proposed, which are referred to as the conformational disturbance hypothesis (CDH) , loop-sheet polymerisation hypothesis (LSPH) and multiple binding site hypothesis (MB-SH) . CDH was put forward to explicit serpin deficiency due to conformational change of reactive loop of serpins as a result of mutations occurring away from the reactive site residues and LSPH was to explain deficient serpins due to the formation of polymers. MBSH was proposed to explain the mechanism of the formation of stable enzyme-serpin complex via more than one binding site and blockage or mutation in any of the sites resulting in serpin deficiency. A combination of these mechanisms may be critical in understanding the roles of the many documented mutations and autoimmunities which result in qualitative and quantitative serpin deficiency.     

A mutation that prevents GTP-dependent activation of the alpha chain of Gs

Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.     

高吸水性树脂的制备及其表征

采用反相悬浮聚合法制备丙烯酸钾-丙烯酸(PKAA)共聚物,研究了中和度、单体浓度、引发剂用量等因素对聚合过程及产物吸水性的影响。实验表明,在单体的质量分数为50%、丙烯酸中和度为80%、引发剂质量为单体质量的0.071%的条件下,所得树脂的吸水率最高。利用显微摄影、红外光谱、热失重分析等对树脂的表面形态、分子结构和热稳定性进行了表征,树脂的红外光谱分析显示树脂中存在大量亲水性基团,由热分析得知,其热分解温度约为380℃,热稳定性良好。     

聚吡咯结构与导电性能的研究

利用化学氧化法制备导电聚吡咯,通过改变制备温度得到了不同的样品.用固体~(13)C NMR谱、FTIR光谱、Raman光谱、XRD和XPS等手段进行分析.结果表明聚吡咯的电导率随着制备温度的升高而逐渐降低,说明不同温度下制备的聚吡咯内部结构存在差异.当制备温度较低时,生成的聚吡咯主要结构是以a-a方式连接的线性分子链,这种连接方式使得高分子同时趋向于形成平面化的构型和构象,保证了高分子具有较高的规整度、共轭度和电导率.随着制备温度的升高,分子链中α-α连接方式的比例逐渐降低,分子的结构遭破坏,有序度降低,趋向于生成构象相对扭曲、缠结的空间稳定态,最终导致了分子共轭链长变短,材料的导电性能降低.     

半乳糖-聚乙二醇-聚-L-赖氨酸共聚物的制备与分析

 将相对分子质量为4 000的聚乙二醇中活性较弱的羟基转化为氨基,利用乳糖分子中含有半缩醛结构,可与双端氨基聚乙二醇（AT-PEG）分子中的伯氨基形成Schiff 碱,然后在氰基硼氢化钠作用下选择性还原为稳定的C-N 单键,使半乳糖连接在PEG的一个氨基上,合成半乳糖-单端氨基聚乙二醇（Gal-PEG-NH₂）,以Gal-PEG-NH₂为大分子引发剂,引发N^ε-苄氧羰基-L- 赖氨酸-N-羧酸酐［Lys(Z) NCA］开环聚合制备一系列半乳-糖-聚乙二醇-聚-L-赖氨酸（PLL-PEG-Gal）嵌段共聚物,通过调节Lys(Z) NCA和 Gal-PEG-NH₂的摩尔比例（［A］/［I］）可控制聚合物的相对分子质量。利用IR、¹³C-NMR、UV等方法进行共聚物结构分析,并对其接糖率进行了分析。结果表明,乳糖与双端氨基聚乙二醇反应生成的带有一侧氨基的聚乙二醇能引发Lys(Z) NCA 聚合生成Gal-PEG-PLL,其中半乳糖的接入率为8.3%。随着［A］/［I］的增加,共聚物的相对分子质量变大。     

中国人HLA-G1的cDNA克隆、序列分析、表达和蛋白质空间结构预测

从中国人外周血单个核细胞、胎盘组织和肝癌组织等样品中克隆了包含完整HLA-G1读框的cDNA;与国外同行获得的该基因及其蛋白质序列比较分析表明,该基因虽然有着细微的种族特异性,但高度保守;并获得了它的截断型重组蛋白,根据蛋白一级结构和同源比较方法,模建了它及其与特异性受体KIR2DL4形成复合体的空间结构模拟,预测了它们之间相互作用的特征.     

Immunohistochemical localization of endogenous nerve growth factor

Nerve growth factor (NGF) has been proposed as a trophic molecule essential for the development of sympathetic and primary sensory neurones. In newborn mice and rats, administration of nerve growth factor results in an increase in the number of surviving neurones, whereas administration of antiserum to NGF decreases neuronal survival. Thus it has been proposed that the factor is produced and secreted by the relevant target tissues to provide trophic support for the ingrowing nerves. The site of synthesis of nerve growth factor is still unknown, and it has been emphasized that a precise physiological role for the molecule cannot be ascribed until the cell types that produce it are known. I report here the use of immunohistochemistry to localize endogenous NGF in the rat iris, a tissue in which there is sound biochemical evidence for the production of NGF activity. Surprisingly, the results reveal that NGF can be detected readily in Schwann cells, but not in smooth muscle cells of the iris when it is sympathetically denervated or cultured.