Membrane-spanning domain of bovine foamy virus transmembrane protein having cytotoxicity

Foamy viruses (FVs) have broad cellular tropism infecting vertebrates from fish to human being, which indicates that Env protein has a high capability for membrane fusion. Conservative features in all FV transmembrane (TM) proteins include a region of hydrophobic domain called membrane-spanning domain (MSD), which contains several stretches of hydrophobic amino acids. To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli, a series of mutants were constructed and expressed in the E. coli BL21 (DE3) using pET-32a (+) as expressing vector. The results showed that only TM3 without MSD was expressed in E. coli, whereas the other two containing full or part of the MSD (TM1 and TM2) could not be expressed. Furthermore, the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD, especially on the stretches of hydrophobic amino acids. Western blotting analysis showed that TM3 protein was purified with affinity purification. __________ Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2005, 38(2): 49–53 [译自: 南开大学学报 (自然科学版), 2005, 38(2): 49–53]     

Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.     

抗人VEGF受体Ⅱ基因Ⅲ区单链抗体基因的构建和表达

采用RT-PCR技术从分泌抗人血管内皮生长因子受体Ⅱ（kinase insert domaincontaining receptor,KDR）基因Ⅲ区单克隆抗体Ycom1D3的杂交瘤细胞中克隆出VH和VL可变区基因,通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L基因之间引入柔性短肽（Gly₄Ser）₃,体外构建Ycom1D3单链抗体基因Ycom1D3-ScFv）,将其克隆至pAYZ表达载体,在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,Ycom1D3-ScFv在E.coli 16C9中获得表达,重组蛋白的相对分子量为30kD,与预期结果一致。表达产物主要以不溶性包涵体形式存在,经过溶解包涵体,TALON 金属亲合层析基质（TALON metal affinity resin）纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实该单链抗体可与人脐静脉内皮细胞结合,保留了鼠源单抗与KDR抗原的特异性结合活性。抗KDRⅢ单链抗体基因Ycom1D3-ScFv的成功构建和功能性表达为靶向诊断治疗及进一步基因工程改造奠定了基础。     

IL-1018-57-PE40高效表达、纯化及细胞活性之研究

以IL-10的功能短肽(40肽,即IL-10第18号至57号氨基酸)为导向部分与PE40(绿脓杆菌外毒素除去受体结合区后的剩余部分)融合分别构建了IL-101857PE40的胞质和胞周质表达质粒,其中,IL101857PE40在Rosettablue(DE3)中以高效胞质可溶形式表达,在BL21(DE3)pLysS中以胞周质分泌形式表达;表达宿主菌Rosettablue(DE3)超声波破碎后,依次通过硫酸铵盐析、疏水层析、铜离子亲和层析、阴离子交换层析纯化后,得96%重组毒素纯品;细胞活性实验、细胞ELISA和荧光标记实验表明,构建的IL101857PE40符合免疫毒素的作用机理。因此,该实验为PE免疫毒素的规模制备和纯化做了一定的有益的探索。     

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R⁶⁹⁶ (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R⁶⁹⁶, it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R⁶⁹⁶ with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

蜘蛛大壶状腺丝蛋白基因的克隆和原核表达

以悦目金蛛(Argiope amoena)丝腺SMARTRACEcDNA文库为模板进行RT-PCR,克隆了1条大壶状腺丝蛋白(major ampullate spidroin,MaSp)基因cDNA序列。该条cDNA序列编码的氨基酸序列可区分为两部分(1)富含丙氨酸的片段和富含甘氨酸的片段相间排列构成的重复氨基酸序列区,并且富含甘氨酸的片段中有脯氨酸分布;(2)约100个氨基酸残基组成的C末端非重复氨基酸序列区。把MaSp基因cDNA序列亚克隆到质粒pET28b( )中,构建原核表达质粒pET28b( )-MaSp,表达质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE、氨基酸组成测定和N末端氨基酸序列测定的结果表明,表达产物为重组MaSp,表达量约为40mg/L。还对C末端非重复氨基酸序列对重组MaSp在水媒介中溶解性的影响进行了探讨。     

Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene

The nucleotide sequence of the Escherichia coli colicin I receptor gene (cir) has been determined. The predicted mature protein consists of 599 amino acids and has a molecular weight of 67,169. Several previously noted characteristics of other E. coli outer membrane protein sequences were also identified in the sequence of Cir. These include an overall acidic nature, the absence of long hydrophobic stretches of amino acids, and a lack of predicted alpha-helical secondary structure. Because two classes of outer membrane proteins (the TonB-dependent transport proteins and the porins) share some structural features, protein sequences from both of these groups were aligned pairwise and scored for sequence similarity. Statistical evidence suggested that the porins were not related to the proteins in the TonB-dependent group; however, there was a significant relationship between the proteins in the TonB-dependent group. On the basis of the multiple progressive sequence alignment and the similarity scores derived from it, a tree representing evolutionary distance between five TonB-dependent outer membrane transport proteins was generated.     

The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition

Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27-amino acid TM domain. Our mutants included sequential 2-amino acid (Delta2-Delta14) and an 11-amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH(2)-terminal and middle regions, and a deletion of 12 amino acids from the NH(2)-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to Delta12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.     

Expression and sequence analysis of a cDNA encoding the orotidine-5'-monophosphate decarboxylase domain from Ehrlich ascites uridylate synthase

Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.     

Molecular identification of a mechanosensitive channel in archaea

The TM1 domain of the large conductance mechanosensitive (MS) channel of Escherichia coli was used as a genetic probe to search the genomic database of the archaeon Methanoccoccus jannashii for MscL homologs. We report that the hypothetical protein MJ0170 of M. jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL. Moreover, MJ0170 was found to be a conserved homolog of MscS, the second type of E. coli MS channel encoded by the yggB gene. Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly resembled the charged C-terminal amino acid cluster present in Eco-MscL (RKKEE). We cloned and expressed MJ0170 in E. coli, which when reconstituted into liposomes or expressed in the cell membrane of giant E. coli spheroplasts, exhibited similar activity to the bacterial MS channels. Our study suggests that the M. jannashii MS channel and its homologs evolved as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved structural motif among prokaryotic MS channels.     

Nucleotide sequence of the phoM region of Escherichia coli: four open reading frames may constitute an operon.

The phoM gene is one of the positive regulatory genes for the phosphate regulon of Escherichia coli. We analyzed the nucleotide sequence of a 4.7-kilobase chromosomal DNA segment that encompasses the phoM gene and its flanking regions. Four open reading frames (ORFs) were identified in the order ORF1-ORF2-ORF3 (phoM)-ORF4-dye clockwise on the standard E. coli genetic map. Since these ORFs are preceded by a putative promotor sequence upstream of ORF1 and followed by a putative terminator distal to ORF4, they seem to constitute an operon. The 157-amino-acid ORF1 protein contains highly hydrophobic amino acids in the amino-terminal portion, which is a characteristic of a signal peptide. The 229-amino-acid ORF2 protein is highly homologous to the PhoB protein, a positive regulatory protein for the phosphate regulon. The ORF3 (phoM gene) protein contains two stretches of highly hydrophobic residues in the amino-terminal and central regions and, therefore, may be a membrane protein. The 450-amino-acid ORF4 protein contains long hydrophobic regions and is likely to be a membrane protein.     

A chimeric Anabaena/ Escherichia coli KdpD protein (Anacoli KdpD) functionally interacts with E. coli KdpE and activates kdp expression in E. coli

The kdpFABC operon, coding for a high-affinity K(+)-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K(+) starvation or an increase in medium osmolality. Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE. From a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31, a kdpDgene was cloned (GenBank accession no. AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E. coli KdpD. A chimeric kdpD gene was constructed and expressed in E. coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E. coli KdpD were replaced by those from Anabaena KdpD. In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E. coli KdpE, which closely resemble those of the E. coli wild-type KdpD. Cells of E. coli synthesizing Anacoli KdpD expressed kdpFABC in response to K(+) limitation and osmotic upshock. The data demonstrate that Anabaena KdpD can interact with the E. coliKdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.     

浙贝母角鲨烯合酶全长cDNA的克隆及功能分析

鲨烯是甾醇和其他三萜类化合物的关键代谢中间体,其生物合成由鲨烯合酶（squalene synthase,SQS）催化,该酶将2分子法呢基焦磷酸转化为鲨烯。浙贝母异甾体生物碱的生物合成途径与三萜类化合物类似。在本研究中,基于cDNA末端的快速扩增（RACE）技术克隆了浙贝母鲨烯合酶 (FtSQS）基因的全长cDNA,GenBank登录号为KF551097.2。通过生物信息学方法对FtSQS进行详细表征,包括保守区检测、序列同源分析、二级和三级结构预测及系统发育树分析。结果表明,其开放阅读框（ORF）为1 230 bp,编码409个氨基酸,FtSQS氨基酸序列与印度甘松、截形苜蓿、紫衫、马铃薯、柴胡、金铁锁和拟南芥的SQS氨基酸同源性分别达到73.84%、73.23%、72.24%、70.66%、70.66%、69.44%和68.14%。启动子分析表明,FtSQS的5′上游区域具有与生理和环境因素相关的各种潜在因素。为了获得可溶性FtSQS表达,从羧基末端截断24个疏水氨基酸,构建了原核表达载体pGEX-2T-FtSQSΔTM,并在大肠杆菌BL21(DE3)中表达。SDS-PAGE检测到约66 kD的重组FtSQSΔTM蛋白。体外酶促反应证明,FtSQS可以催化FPP转化成鲨烯。qRT-PCR分析FtSQS mRNA在叶中的表达量最高,茎、根次之,而在鳞茎中表达水平最低。这提示,叶子是浙贝母碱生物合成的主要活性器官。FtSQS的鉴定及功能研究为浙贝母次生代谢产物的研究提供了重要依据。     

A proper amino terminus of diphtheria toxin is important for cytotoxicity

A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.     

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full- length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.     

Polarity changes in the transmembrane domain core of HIV-1 Vpu inhibits its anti-tetherin activity

Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs.