小鼠富亮氨酸重复结构蛋白Lrig2 基因、蛋白结构及组织定位分析

目的:分析小鼠富亮氨酸重复结构蛋白家族成员Lrig2的基因与蛋白的结构,明确其组织分布和定位,并对其功能进行初步预测。方法:利用生物信息学分析技术对小鼠Lrig2基因的染色体定位、蛋白结构进行分析预测;通过RT-PCR、mRNA原位杂交技术检测Lrig2基因在小鼠不同组织中的表达定位;通过系统进化树分析Lrig2与其他Lrrs蛋白家族成员的同源性。结果:生物信息学分析显示,Lrig2是一种跨膜蛋白受体,是Lrrs蛋白超家族成员之一,胞外区含有15个Lrr模序、3个免疫球蛋白样结构域,存在单一的跨膜结构域;Lrig2在小鼠的多个组织中表达,其中在胸腺、脾脏等组织中表达较强;系统树分析显示,Lrigs蛋白是sLrPs超家族成员,是一种跨膜蛋白受体。结论:Lrig2在免疫组织中表达较强,推测其可能在肿瘤免疫应答进程中发挥重要的效能。     

小鼠富亮氨酸重复结构蛋白Lrig2基因、蛋白结构及组织定位分析

目的：分析小鼠富亮氨酸重复结构蛋白家族成员Lrig2的基因与蛋白的结构,明确其组织分布和定位,并对其功能进行初步预测。方法：利用生物信息学分析技术对小鼠Ln萨基因的染色体定位、蛋白结构进行分析预测;通过RT-PCR、mRNA原位杂交技术检测Lrig2基因在小鼠不同组织中的表达定位;通过系统进化树分析“薛与其他Lrrs蛋白家族成员的同源性。结果：生物信息学分析显示,H薛是一种跨膜蛋白受体,是Lrrs蛋白超家族成员之一,胞外区含有15个m模序、3个免疫球蛋白样结构域,存在单一的跨膜结构域;Lrig2在小鼠的多个组织中表达,其中在胸腺、脾脏等组织中表达较强;系统树分析显示,Lrigs蛋白是sLrPs超家族成员,是一种跨膜蛋白受体。结论：Lrig2在免疫组织中表达较强,推测其可能在肿瘤免疫应答进程中发挥重要的效能。     

聚乙二醇干扰素α-2a对慢性乙型肝炎患者树突状细胞B7-H1表达的影响

目的：观察聚乙二醇干扰素α-2a对慢性乙型肝炎患者外周血树突状细胞功能及B7-H1的影响,探讨慢性乙型肝炎病毒逃逸的的机制。方法：慢性乙型肝炎患者31例,给予聚乙二醇干扰素α-2a180txg抗病毒治疗52周,分别于0、12、26、52周检测肝功能、HBV-DNA;流式细胞术检测外周血mDC表面HLA-DR、CD80、CD86、CD83、CDla、B7一H1水平。根据患者HBV—DNA水平,将患者分为应答组（A组）、非应答组（B组）,10例健康志愿者作正常对照组（C组）。结果：慢性乙肝患者的树突状细胞膜表面分子HLA-DR、CD80、CD86、CD83、CDla的表达均降低。聚乙二醇干扰素α-2a治疗后应答组膜表面分子HLA-DR、CD80、CD86、CD83、cDla的表达高于非应答组65．3±6．2％VS44．2±5．5％,67．2±7．4％VS37.3±7．2％,68．4±3．6％VS42．5±7．3％,65．6±6．8％VS43．2±3．9％,49．4±9．5％VS37．5±7．9％,（P〈0．05）。应答组B7-H1表达水平较治疗前下降,非应答组B7-H1水平无明显变化12．73±3．8％VS25．24±2．92％,（P〈0．05）。结论：慢性乙型肝炎患者树突状细胞功能低下,聚乙二醇干扰素α-2a治疗可以提高树突状细胞功能,降低B7-H1表达,促进HBV-DNA的清除。树突状细胞功能低下及B7-H1高表达是乙型肝炎病毒免疫逃逸的因素之一。     

ADAMs参与哺乳动物精-卵结合与融合

ADAMs家族是含多结构域的跨膜蛋白。睾丸特异的ADAMs,在精子发生与附睾精子转运过程中,经过蛋白水解成为成熟精子的分子形式,与精．卵质膜结合和融合有关。对于精-卵质膜相互作用,ADAMs去整合素域具有关键氨基酸残基和特殊模体。模拟ADAM2和ADAM3去整合素域的短肽能用于鉴别特异性卵子识别蛋白。精子ADAMs去整合素域与卵子膜蛋白整合素β1、α4／α9、α6和CD9相互作用,介导了精卵质膜的结合与融合。     

T细胞免疫球蛋白及黏蛋白域蛋白3在多种疾病中的作用机制

T细胞免疫球蛋白及黏蛋白域蛋白基因编码的蛋白是一类具有共同基序的跨膜糖蛋白,其基本结构包括一个信号肽区、免疫球蛋白样区、黏蛋白样区、跨膜区和有磷酸化位点的胞内区。人类的TIM基因家族包括3个成员(Tim-1,Tim-3,Tim-4),定位于与哮喘、过敏和自身免疫性疾病等有关的染色体5q33.2区域。研究表明,TIM-3与其配体Gal-9相互作用调节T细胞凋亡和免疫耐受,并在过敏性疾病、自身免疫性疾病、肿瘤、感染及母胎界面方面发挥重要作用。虽然,Tim-3在调节机体的天然免疫和特异性免疫应答方面起着重要作用,但是其还有很多作用机制需要进一步研究,为疾病的临床治疗提供理论指导。     

CD36在炎症反应和脂质代谢中的作用

白细胞分化抗原36 (cluster of differentiation 36, CD36)是一种高度糖基化的单链跨膜蛋白,属B型清道夫受体。其功能较为广泛,不仅可以识别"异己"成分,诱发免疫应答,还参与多种生理、病理过程。CD36作为膜受体与其他膜蛋白和胞质蛋白组成不同的信号通路。CD36是TRL4的辅助受体,可识别病原体相关分子模式,级联NF-κB信号通路,在天然免疫过程发挥作用;作为修饰脂蛋白受体,级联MAPK通路,诱发无菌性炎症及脂代谢紊乱,并参与动脉粥样硬化病变;作为外源长链脂肪酸受体,通过AMPK/mTOR信号通路在能量代谢及脂质蓄积过程中发挥调控作用。该文综述了CD36的分子特征及其偶联相关信号通路在天然免疫和脂代谢等方面的作用与机制,展示CD36的多功能特点,为相关生物医学研究提供依据。     

非编码RNA在胞内病原菌免疫逃逸与致病中的作用

非编码RNA(non-coding RNAs,ncRNAs)在细胞增殖、发育、分化、代谢、信号转导以及免疫调控中发挥重要调节作用。越来越多的研究证明,ncRNA在胞内病原菌的致病性和免疫逃逸中发挥重要调控作用。一方面ncRNA是细菌代谢、群体感应和毒力因子表达的调控因子,与胞内病原菌的致病性密切相关;另一方面ncRNA在调节宿主抗胞内病原菌免疫应答中发挥重要作用,深入研究ncRNA如何调节宿主免疫应答将有助于胞内菌免疫逃逸机制的研究。就非编码RNA在胞内病原菌免疫逃逸和致病中的作用作一综述。     

CD47与乳腺癌相关性的研究进展

乳腺癌的发生、发展与许多因素有关,其中免疫系统在其发生与发展过程中发挥重要作用。CD47是高表达于乳腺癌细胞表面的跨膜蛋白,其配体为凝血酶敏感蛋白-1(thrombospondin-1,TSP1)和信号调节蛋白α(signal-regulatory protein alpha,SIRPα),其中CD47/SIRPα信号通路可产生抑制性信号降低巨噬细胞的吞噬作用,产生免疫逃逸。在乳腺癌细胞中CD47表达上调,且高表达的CD47提示预后不良。采用抗CD47抗体可以阻断肿瘤细胞CD47/SIRPα通路介导的抑制吞噬作用,目前抗CD47单克隆抗体的免疫疗法正逐步走向临床试验。本文主要阐述CD47的结构、生理功能及其与肿瘤相关巨噬细胞的关系,并对CD47在乳腺癌中的表达及与预后的关系、在免疫治疗中的作用进行综述。     

天然型调节性T细胞发育分化的研究进展

CD4 CD25 调节性T细胞(Treg)对维持自身免疫耐受及调控免疫应答水平发挥非常重要的作用．Treg的缺失或紊乱导致如多发性硬化症、1型糖尿病等自身免疫性疾病的发生．研究发现,Treg在肿瘤免疫、感染免疫和移植免疫耐受中也发挥关键作用．根据来源不同,可将Treg分为胸腺来源的天然型Treg(natural Treg)和外周诱导型Treg(induced Treg)两群．天然型Treg(nTreg)是由胸腺发育分化成熟的,nTreg存在于胸腺CD4单阳性细胞中,表达CD4、CD25及叉头转录因子Foxp3,主要通过细胞与细胞之间直接接触发挥免疫抑制功能．研究表明,nTreg在胸腺中发育分化受到十分复杂的细胞、分子网络调控．胸腺微环境、T细胞受体、共刺激分子、IL-2等信号都可影响nTreg的发育分化．本文将主要对胸腺nTreg的发育分化过程及分子调控等方面进行综述．     

CD205的结构与功能研究

CD205是一种分子量205KD的C型凝集素,为I型膜蛋白,含有10个糖识别域,系巨噬细胞甘露糖受体家族成员。人CD205基因位于染色体2q24,其CDNA长5166bp,编码产物1722个氨基酸残基。CD205主要表达于树突状细胞和胞腺上皮细胞。在介导胞吞、抗原提呈中起关键作用,还参与稳定胸腺微环境,在胸腺发育及肿瘤免疫中具重要意义。     

Tetraspanins in the humoral immune response

The tetraspanins represent a large superfamily of four-transmembrane proteins that are expressed on all nucleated cells. Tetraspanins play a prominent role in the organization of the plasma membrane by co-ordinating the spatial localization of transmembrane proteins and signalling molecules into 'tetraspanin microdomains'. In immune cells, tetraspanins interact with key leucocyte receptors [including MHC molecules, integrins, CD4/CD8 and the BCR (B-cell receptor) complex] and as such can modulate leucocyte receptor activation and downstream signalling pathways. There is now ample evidence that tetraspanins on B-lymphocytes are important in controlling antibody production. The tetraspanin CD81 interacts with the BCR complex and is critical for CD19 expression and IgG production, whereas the tetraspanin CD37 inhibits IgA production and is important for IgG production. By contrast, the tetraspanins CD9, Tssc6 and CD151 appear dispensable for humoral immune responses. Thus individual tetraspanin family members have specific functions in B-cell biology, which is evidenced by recent studies in tetraspanin-deficient mice and humans. The present review focuses on tetraspanins expressed by B-lymphocytes and discusses novel insights into the function of tetraspanins in the humoral immune response.     

Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.     

中国大鲵研究概况

中国大鲵(Andrias davidianus)为我国特有物种, 也是国家二类保护水生野生动物。野生大鲵多栖息在山区溪流里, 喜穴居, 怕光怕声, 以鱼蟹等水生动物为食, 从数量上看, 以陕西秦岭山区居多。大鲵经济价值极高, 在美食、保健、医药、观赏等方面均具有广泛开发利用的前景, 因而颇受社会各界关注。近年来对大鲵的科学研究涉及多个方面, 包括疾病方面, 如病毒性疾病、细菌性疾病、真菌性疾病、寄生虫性疾病等; 生理与发育方面, 如生长、发育、应激反应等; 遗传与进化方面; 保护与开发方面, 如开发药品、食品、保健品等。综上, 围绕大鲵从不同角度所展开的研究非常丰富。大鲵基因水平的研究仍是未来研究的热点和趋势, 其成果将为日后更深层的探究奠定基础。     

Genetic diversity of Chinese giant salamander (Andrias davidianus) based on the novel microsatellite markers

Chinese giant salamander (Andrias davidianus) is a rare amphibian species in the world. Microsatellite markers are a promising tool for accurate estimation population structure and genetic diversity. In this paper, we isolated novel microsatellite marker for Chinese giant salamander using fast isolation by AFLP of sequences containing repeats (FIASCO) method. More than 50% sequences in 132 clones had repeat number over ten times with di or trinucleeotide repeat except of (GA)₁₂. Seventy pairs of primers were designed and eleven polymorphic microsatellite loci were characterized for wild and cultivated Chinese giant salamander populations. The allele number was from 3 to 9 in different loci. Polymorphism information content was from 0.544 to 0.702 in cultivated population. The results implied more alleles and PIC were in the wild population than cultivated population. The observed heterozygosities in two populations were higher than 0.553. The data analysis suggested that the cultivated population has lower genetic diversity than wild population, which it??s perhaps owing to inbreeding in artificial breeding. To our knowledge, it??s the first time to isolated microsatellite markers for Chinese giant salamander. The result indicated that the markers were suitable for the population genetic analysis of Chinese giant salamander.     

Domain analysis of the tetraspanins: studies of CD9/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding

CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface. CD9 molecules localize on the cell surface, while those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.     

Multiple levels of interactions within the tetraspanin web

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.     

Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology

Here we demonstrate that multiple tetraspanin (transmembrane 4 superfamily) proteins are palmitoylated, in either the Golgi or a post-Golgi compartment. Using CD151 as a model tetraspanin, we identified and mutated intracellular N-terminal and C-terminal cysteine palmitoylation sites. Simultaneous mutations of C11, C15, C242, and C243 (each to serine) eliminated >90% of CD151 palmitoylation. Notably, palmitoylation had minimal influence on the density of tetraspanin protein complexes, did not promote tetraspanin localization into detergent-resistant microdomains, and was not required for CD151-alpha 3 beta 1 integrin association. However, the CD151 tetra mutant showed markedly diminished associations with other cell surface proteins, including other transmembrane 4 superfamily proteins (CD9, CD63). Thus, palmitoylation may be critical for assembly of the large network of cell surface tetraspanin-protein interactions, sometimes called the "tetraspanin web." Also, compared with wild-type CD151, the tetra mutant was much more diffusely distributed and showed markedly diminished stability during biosynthesis. Finally, expression of the tetra-CD151 mutant profoundly altered alpha 3 integrin-deficient kidney epithelial cells, such that they converted from a dispersed, elongated morphology to an epithelium-like cobblestone clustering. These results point to novel biochemical and biological functions for tetraspanin palmitoylation.