杜氏盐藻盐适应蛋白GPI基因的鉴定及其对食管鳞癌细胞侵袭的影响
|
摘要
杜氏盐藻(Dunaliella salina, D. salina)是一种高度耐盐的单细胞真核绿藻,可在氯化钠浓度为0.05 M～5M的环境中生存,是迄今为止所发现的最耐盐的生物之一。围绕杜氏盐藻进行耐盐机制的研究已经引起广泛关注,并且一些盐适应相关蛋白也已经被陆续分离、鉴定。尽管盐藻的基因组信息非常有限,但蛋白质组学方法却提供了一种分离盐藻盐适应相关蛋白非常有效的技术手段。
许多参与糖代谢的酶都已经被证实参与了植物的盐胁迫应答过程,同时这些糖代谢相关的酶在莱茵衣藻(Chlamydomonas reinhardti)和盐藻以及果蝇(Drosophila)等模式生物中也被证实跟它们的运动、感觉器官-鞭毛/纤毛具有相关性。鞭毛和纤毛是细胞表面的特化细胞器,形态和结构相似,在不同的物种中具有高度保守性。除了具有典型的运动功能,纤毛/鞭毛在生物的感官和生长发育过程中也发挥至关重要的作用,在多种组织细胞中其结构和功能相关基因的缺失或突变可能导致多种疾病的发生。最近的一些研究表明纤毛可能在肿瘤细胞中起重要作用,一些重要的信号通路如：Hedgehog (Hh)、Smoothened和Wnt似乎都跟鞭毛成分相关。
葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase, GPI)广泛存在于真核生物、原核生物和一些古细菌中,催化糖酵解和糖异生过程中葡萄糖-6-磷酸和果糖-6-磷酸的相互转化,而且是“进化学说”的证据之一。在哺乳动物细胞中,除了作为糖代谢的关键酶它还具有细胞因子的功能,能够通过和它的受体结合来促进细胞的运动。相关的研究证实杜氏盐藻GPI在盐藻的鞭毛功能中可能发挥作用,但它是否具有细胞因子功能尚不清楚。
本研究中我们通过不同盐浓度培养杜氏盐藻,利用蛋白质组学方法获得了盐藻的差异表达图谱,分离出了21种在高盐诱导条件下蛋白表达增加达到3倍的蛋白并通过质谱分析鉴定出其中的12种蛋白,包括糖代谢的关键酶GPI(DsGPI);为了进一步验证其在分子水平也参与了盐胁迫的过程,我们通过快速扩增cDNA末端(Rapid amplification of cDNA end, RACE)技术得到了DsGPI基因cDNA全长,利用原核表达验证该cDNA的正确性,同时通过实时荧光定量分析该基因在高盐和光照诱导条件下的变化情况；由于杜氏盐藻GPI具有和哺乳动物GPI相似的结构和一些相同的关键酶活位点,我们将杜氏盐藻GPI转染MDCK细胞验证其是否具有和人GPI相似的细胞定位,转染食管鳞癌细胞验证其是否具有和人GPI相似的促进肿瘤细胞运动的能力。结果显示,DsGPI不但在分子水平而且蛋白水平都参与了盐胁迫的过程,在高盐诱导条件下二者的表达量均明显升高。杜氏盐藻GPI转染MDCK细胞后和人GPI(AMF)具有相似的细胞定位：主要集中的细胞核区域；同时Western blot结果也证实杜氏盐藻GPI能够在食管鳞癌细胞中稳定过表达,而且能够增强食管鳞癌细胞的侵袭能力,提示其可能也具有细胞因子功能。为进一步探讨DsGPI作为细胞因子影响癌细胞增殖、凋亡以及其鞭毛/纤毛中的定位提供了实验基础。
方法
1第一部分杜氏盐藻盐适应蛋白的蛋白质组学研究
1.1杜氏盐藻藻株的鉴定
取适量藻细胞混合液轻涂于半固体培养基培养板上,培养10～14天至板上长出单藻落,随机挑选60个单藻落转接于试管中培养。7-10天后在显微镜下观察藻体形态,要求大小均一。分别选取体积较大和体积较小的藻细胞扩大培养,生长至对数生长期后提取DNA并设计引物扩增内部间隔序列(internal transcribed space,ITS),测序后根据比对结果确定纯化的杜氏盐藻藻株。
1.2双向电泳分离不同盐浓度培养下的杜氏盐藻总蛋白
三氯醋酸-丙酮沉淀法提取1.5 M和3.5 M NaCl培养的杜氏盐藻总蛋白,Brandford法进行蛋白定量。分别取400μg和800μg在等电点pI范围为3～10的24cm胶条上进行第一向等电聚焦,然后通过SDS-PAGE进行第二向电泳,并分别用硝酸银和考马斯亮蓝R250染色。
1.3双向电泳图谱分析和质谱鉴定高盐诱导条件下增强表达的蛋白
二维凝胶分别经硝酸银和考马斯亮蓝R250染色并显色后,用Image-Scanner进行图像扫描获得凝胶图像,经ImageMaster 2D Platinum software凝胶图像分析软件进行图像调整、蛋白点检测、蛋白点匹配和数据分析后结合肉眼观察,确定差异表达的蛋白质点；在匹配的蛋白质点中选取在高盐条件下表达量增加达到3倍的蛋白点,从凝胶上挖下,胰酶消化后,经基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS)获得肽质量指纹图谱(PMF),通过软件搜索蛋白质数据库并结合生物信息学方法鉴定蛋白质点。
2第二部分杜氏盐藻GPI基因cDNA的克隆、表达和功能分析
2.1杜氏盐藻GPI基因cDNA全长的克隆
根据质谱鉴定获得的DsGPI的肽段信息,结合其它物种中GPI氨基酸的保守性分析,设计兼并引物,RT-PCR获得DsGPI基因cDNA片段；根据片段序列信息,利用3'RACE和5'RACE的方法分别向3’下游和5’上游扩增杜氏盐藻GPI基因的3’和5'cDNA末端片段；将上述片段拼接得到DsGPI基因cDNA全长,然后设计引物PCR扩增全长,测序后进行核苷酸和氨基酸的生物信息学分析。
2.2杜氏盐藻GPI基因cDNA全长的原核表达
将在两端添加适当酶切位点的杜氏盐藻GPI cDNA编码序列定向克隆于原核表达载体pET-28a(+)中构建重组质粒,筛选并测序鉴定重组质粒。转化E-coliBL21,并用IPTG诱导DsGPI融合蛋白在BL21中表达。SDS-PAGE后,对DsGPI融合蛋白在大肠杆菌BL21中的表达进行分析。
2.3杜氏盐藻GPI基因的功能分析
将对数生长期的盐藻置于暗环境中培养24小时后进行光照培养,以继续在暗环境培养的盐藻为对照,分别在0,1,2,4,8,12,16,20和24小时取样并提取RNA,实时荧光定量PCR分析DsGPI基因在光诱导条件下的变化趋势；取对数生长期的盐藻分别接入新鲜1.5 M、2.5 M和3.5 M NaCl培养基中,分别在0,1,2,4,6,8,10和12小时取样并提取RNA,实时荧光定量分析DsGPI基因在盐诱导条件下的变化趋势。
3第三部分杜氏盐藻GPI基因对食管鳞癌细胞侵袭能力的影响
3.1杜氏盐藻GPI多克隆抗体的制备
利用Ni-NTA组氨酸亲和柱纯化原核表达的DsGPI融合蛋白,免疫新西兰白兔制备DsGPI多克隆抗体,并利用间接ELISA进行效价评测和Western blots进行抗体特异性分析。
3.2杜氏盐藻GPI在MDCK细胞中的定位
构建EGFP-C1-DsGPI真核表达载体并利用脂质体转染细胞,同时以EGFP-C1-GPI、EGFP-C1空载体转染的细胞及未处理的细胞为对照,荧光显微镜观察杜氏盐藻GPI在MDCK细胞中的定位。
3.3杜氏盐藻GPI对食管鳞癌细胞EC9706侵袭能力的影响
以pcDNA3.1(+)质粒为基础,分别构建pcDNA3.1(+)-DsGPI和pcDNA3.1(+)-GPI质粒并转染EC9706细胞,同时以pcDNA3.1(+)空质粒转染的细胞和未经处理的细胞为对照,Western blots鉴定重组质粒在EC9706细胞中的蛋白表达情况,然后利用Transwell小室试验检测EC9706细胞侵袭能力变化。
4统计学处理
Western blots结果采用TotalLab2.0软件分析,同一实验均重复三次以上。用SPSS13.0统计软件进行统计学分析,统计学数据用均数±标准差(x±s)表示,行单因素方差分析(one-way ANOVA), P<0.05具差异性。
结果
1第一部分杜氏盐藻盐适应蛋白的蛋白质组学研究
1．1杜氏盐藻藻株的鉴定
从半固体培养基上挑取的单个藻细胞经在试管中培养后,形态均一,无互相混合；以体积较大和体积较小的藻DNA为模板,用ITS引物分别扩增出长度～600bp和～1100 bp的片段；经测序并比对后证实体积较大的藻为杜氏盐藻(Dunaliella salina UTEX 1644),体积较小的藻为(Dunaliella viridis);后续的实验以体积较大的纯净的杜氏盐藻藻株为研究对象。
1.2双向电泳分离不同盐浓度培养下的杜氏盐藻总蛋白
TCA-丙酮沉淀法提取的杜氏盐藻总蛋白能够有效的去除盐藻中的色素等成分,并且易溶解于上样缓冲液；等电聚焦过程中电压上稳定上升并且均达到了预期的8000 V,说明样品处理符合条件；二维凝胶染色后Image-Scanner扫描获得的凝胶图像清晰,蛋白质点均匀,重复性好；
1.3双向电泳图谱分析和质谱鉴定高盐诱导条件下增强表达的蛋白
经ImageMaster 2D Platinum software凝胶图像分析软件后共检测到803个蛋白点,共匹配739对,匹配率为92.1%；在这些匹配的蛋白质点中有21个蛋白点在3.5 M NaCl的条件下增强表达达到3倍以上,这些蛋白点并被选择进行下一步的质谱鉴定；所选择的21个蛋白经质谱分析和数据库比对后,有9个未能找到理想的匹配对象,剩余的12个均被鉴定,包括：葡萄糖-6-磷酸异构酶,铁-超氧化物歧化酶,ATP合成酶,热休克蛋白70B,30S核糖体蛋白S4,反转录转座子蛋白(retrotransposon protein),动力蛋白1-α重链,G蛋白,钙依赖性蛋白激酶,乙醇脱氢酶和2个暂未命名的蛋白。
2第二部分杜氏盐藻GPI基因cDNA的克隆、表达和功能分析
2.1杜氏盐藻GPI基因cDNA全长的克隆
利用兼并引物扩增得到771 bp长的DsGPI基因cDNA片段,3'RACE和5'RACE分别得到了687 bp和1206 bp的片段,去除重叠区域后得到了全长为2338 bp的序列,此序列中包含一个1980 bp长的开放阅读框；此阅读框对应的659个氨基酸长度序列经BLAST分析后显示其与其它物种的GPI具有很高的同源性,说明克隆的序列就是杜氏盐藻的GPI基因cDNA序列。
2.2杜氏盐藻GPI基因cDNA全长的原核表达
杜氏盐藻的GPI cDNA编码序列定向克隆于原核表达载体pET-28a(+)后转染大肠杆菌株BL21,经IPTG诱导后成功表达DsGPI融合蛋白。SDS-PAGE结果表明：在～78 kD出现一条特异性条带,而对照组无此特异条带,说明所克隆得到的杜氏盐藻GPI cDNA序列正确。
2.3杜氏盐藻GPI基因的功能分析
实时荧光定量PCR分析显示,DsGPI基因在光诱导的前2个小时其表达量会迅速下降,而后又恢复到正常水平并维持相对稳定；而在高盐诱导条件下,DsGPI基因的表达在开始的前2个小时有所下降而后迅速升高并且分别在3.5 MNaCl诱导16小时后到达峰值,此时其表达量增至14倍；在2.5 M NaCl诱导20小时后到达峰值,此时其表达量增至8倍。
3第三部分杜氏盐藻GPI基因对食管鳞癌细胞侵袭能力的影响
3.1杜氏盐藻GPI多克隆抗体的制备
经Ni-NTA组氨酸亲和柱纯化的DsGPI融合蛋白其纯度高达96.3%；间接ELISA检测到该多抗的效价高达1：256 K～1：512 K,同时Western blots条带单一,说明该抗体具有特异性。
3.2杜氏盐藻GPI在MDCK细胞中的定位
荧光显微镜观察显示转染EGFP-C1-DsGPI和EGFP-C1-GPI质粒的MDCK细胞在细胞核区域富集有绿色荧光蛋白,而EGFP-C1空载体转染的细胞在胞质中均匀分布绿色荧光蛋白,未经处理的细胞无绿色荧光显现,说明杜氏盐藻GPI具有和人GPI (AMF)相同的细胞定位。
3.3杜氏盐藻GPI对食管鳞癌细胞侵袭能力的影响
Western blots结果显示转染pcDNA3.1(+)-DsGPI的EC9706细胞其GPI的表达分别是用pcDNA3.1(+)空质粒转染细胞和未经处理的细胞中GPI的表达量的2.2±0.06倍(P<0.05)和1.9±0.13倍(P<0.05),转染pcDNA3.1(+)-GPI质粒的EC9706细胞其AMF的表达量分别是用pcDNA3.1(+)空质粒转染细胞和未经处理的细胞中AMF的表达量的3.3±0.11倍(P<0.05)和3．0±0.19倍(P<0.05)；560 nm的OD值数据分析显示转染pcDNA3.1 (+)或未进行转染的细胞其OD值分别为2.64±0.06和2.47±0.10,而转染pcDNA3.1-DsGPI和pcDNA3.1-AMF的细胞其OD值分别提高到3.86±0.14(P<0.05)和4.18±0.11(P<0.05),提示DsGPI具有和AMF促进肿瘤细胞侵袭的相似功能。
结论
1.本实验分离、纯化并鉴定了杜氏盐藻藻株,获得了形态均一的杜氏盐藻细胞,为以杜氏盐藻为材料进行研究奠定了可靠的基础；获得了杜氏盐藻在高盐诱导条件下的差异蛋白表达图谱,在蛋白质组水平对盐藻盐适应蛋白进行了初步分离；利用质谱分析了21个在高盐诱导条件增强表达达到3倍的蛋白点并鉴定出了其中的12种蛋白,获得这些蛋白的部分肽段序列,为进一步详细研究这些蛋白和基因功能提供了相关信息。
2.克隆得到杜氏盐藻GPI的cDNA序列,全长2338 bp,包含一个1980bp的开放阅读框并通过原核表达进行了验证；通过实时荧光定量PCR证实该基因的表达受光和高盐条件所诱导,而且盐浓度越高诱导效果越强,在分子水平证实DsGPI参与了高盐诱导,进一步验证了蛋白质组学的研究结果；利用纯化的原核表达蛋白成功制备了效价高、特异性强的盐藻GPI多克隆抗体。
3.构建杜氏盐藻GPI的真核表达载体,转染MDCK细胞后证实：杜氏盐藻GPI和人GPI (AMF)一样,都定位在细胞核；盐藻GPI也能在EC9706细胞中表达,Transwell小室证实,盐藻GPI具有跟人GPI相似的细胞因子功能,能够提高EC9706细胞的侵袭能力；杜氏盐藻GPI促进肿瘤细胞侵袭运动的功能,为进一步探讨盐藻GPI在体外对肿瘤细胞增殖、凋亡以及对体内肿瘤发生、发展的影响以及其在盐藻鞭毛/纤毛中的定位奠定了基础。
The unicellular eukaryotic green alga Dunaliella salina(D.salina) grows over a wide range of salt concentrations from 0.05 to 5.0 M and is one of the most halotolerant organisms. D.salina has received considerable attention in studies of halotolerance mechanisms and some salt-induced proteins have been isolated and identified. Although the genome of D. salina is not completely available, proteome analysis is an efficient technique to detect and characterize proteins from D. salina.
Many glycometabolic enzymes have been identified being involved in responding to salt stress and meanwhile, they have been proved to be related to the flagella/cilia of D.salina, the model organism Chlamydomonas reinhardti and Drosophila et al. Flagella/cilia are specialized and widespread cell organelles. More and more evidences suggest that flagella/cilia serve diverse roles in motility, sensory signaling and development of many eukaryots and implicate cilia in polycystic kidney disease (PKD) and developmental disorders, even in cancer.
Being one of the evidences for evolution theory, glucose-6-phosphate isomerase (GPI) is universally distributed among eukaryotes, bacteria and some archaea and catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate, which is an essential reaction involved in both catabolic glycolysis and anabolic gluconeogenesis. GPIs also act as an atuocrine motility factor (AMF) that can promote tumor cells metastasis and invasion by binding to their receptors in animals. Although GPI is related to flagella/cilia in D.salina, whether it has the cytokine function is still unknown.
In this study, we analyzed changes of proteins between algal cells cultured at 1.5 or 3.5 M NaCl using proteomic method and obtained the differential expression maps. Twenty one proteins increased by more than 3-fold in 3.5 M NaCl than that in 1.5 M NaCl medium were isolated and analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS), making twelve proteins were identified including GPI. In order to verify GPI is involved in responding to salt stress at gene level, the full-length cDNA of GPI was obtained using rapid amplification of cDNA end technique and heterologous expressed to. Real-time quantitative RT-PCR was used to detect its expression changes in response to light and salinity. Bioinformatics analysis revealed that the GPI from D.salina(DsGPI) have the similar structure and key active sites with that from mammalian cells and hence, it was used to transfect esophageal squamous cell carcinoma cells to check whether it has the potential ability like human GPI to promote invasion of tumor cells. The results showed that DsGPI is involved in responding to salt stress not only at the gene level but also at the protein level and that DsGPI has the same cellular localization with human GPI that mainly localized in the nucleus and also distributed in cytoplasm in esophageal squamous cell carcinoma cell (ESCC) line EC9706 cells. In addition, western blots confirmed that DsGPI was stable expressed in EC9706 and able to enhance the invasion ability of EC9706, suggesting that DsGPI may has the cytokine functions like human GPI.
Methods
1 Proteomics analysis of salt-induced proteins from D.salina
1.1 Identification of D. salina strain
Contaminated algal cells containing not only one species were spread on semisolid medium and cultured for 10 to 14 days and then, single cells were picked out and continue cultured in test tube, respectively. Seven to 10 days later, algal cells were observed under microscope and samples with the larger or the smaller uniform size were cultured to logarithmic phase and harvested for DNA extraction. A pair of primers was designed to amplify the internal transcribed space and the PCR results were blasted to determine the pure D.salina species.
1.2 Two-dimensional gel electrophoresis (2-DE)
Proteins of algal cells cultured in 1.5M and 3.5M NaCl medium were precipitated by Trichloroacetic acid-actone and dissolved in a buffer, respectively. Protein concentrations were determined using the Bradford method. A total of 400μg soluble proteins were applied to isoelectric focusing (IEF) on IPGphor using 24 cm ImmobilineTM DryStrips (pH 3-10, linear; GE Healthcare) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
1.3 Analysis of the 2-DE gels and identification of salt-induced proteins by MS analysis
The 2-DE gels stained with Coomassie Brilliant Blue (CBB) R-250 or AgNO3 were scanned using Image-Scanner (GE Healthcare). Protein spots from the 3.5 M NaCl medium were compared to those from the 1.5 M NaCl medium (control) using ImageMaster 2D Platinum software After images were adjusted, protein spots were detected and matched, and protein intensities were measured, the differential expressed proteins were determined combined with eye observation. Each sample was replicated at least three times, and protein spots for comparative analyses were detected on all of the gels.
Among the matched proteins, proteins up-regulated in by more than three times from the 2-DE gels were selected and digested by trypsin, the resulting peptides were extracted and analyzed using MALDI-TOF/TOF MS (Applied Biosystems). MS data were collected and protein identification was searched against the database of plants in NCBI using the MASCOT search engine. An identified protein was matched with the known protein(s) in the database only when its score is greater than 65 (P<0.05)
2 Cloning, heterologous expression and functional analysis of the DsGPI gene
2.1 Cloning of full-length cDNA of DsGPI gene
A pair of degenerated PCR primers "GARTTYGGNATHGAYCC" (sense) and "NACNCCCCAYTGRTCRAA"(antisense) corresponding to the highly conserved amino acid residues "EFGIDP" and "FDQWGV" was used to amplify a cDNA fragment of the DsGPI, and then 5'-and 3'-RACEs were performed. The putative 5'-and 3'-RACE cDNAs and the partial sequences cloned using the degenarate primers were organized to form a cDNA contigue that was applied to bioinformatics analysis.
2.2 Heterologous expression of DsGPI
The DsGPI cDNA was subcloned into the prokaryotic protein expression vector pET28a(+) (Novagen) with NdeI andoRI sites and recombinant plasmid was screened and identified by sequencing. Recombinant plasmids were expressed in E. coli BL21 and IPTG was added in to produce recombinant fusion DsGPI with a 6×His-tag that was subjected to SDS-PAGE.
2.3 Analysis of DsGPI gene expression
To confirm whether DsGPI responds to salt stress, algal cells were collected at 0,1,2,4,8,12,16,20 and 24 h after being transferred from 1.5 to 3.5 M NaCl medium, and thecells transferred to fresh 1.5 M NaCl medium were served as controls. Besides, to investigate whether DsGPI is lightrelated, algal cells having been cultured in the dark for 48 h were exposed to light and then collected at 1,2,4,8, 10 and 12 h, and cells without exposure to light were used as controls. Real time quantitative RT-PCR was performed for the expression change of DsGPI.
3 Effects of DsGPI gene on the invasion ability of EC9706 cells
3.1 Preparation of DsGPI polyclonal antibody
The DsGPI fusion proteins expressed in E. coli cells were purified using Ni-NTA His Bind column and subjected to immunize albino rabbit for the preparation of polyclonal antibody. The antibody titer and specificity were determined by ELISIA and Western blots, respectively.
3.2 Localization of DsGPI in MDCK cells
The complete cDNA sequences of DsGPI was cloned into the plasmid pEGFP-C1 to create a recombinant plasmid pEGFP-C1-DsGPI by using BamH I and EcoR I sites and identified. The same method was used to construct recombinant plasmid pEGFP-C1-GPI with complete cDNA sequences of human GPI.Both recombinant plasmids transfected in MDCK cells with blank plasmid pEGFP-C1 and cells without transfection were used as control. Localization the EGFP fusion proteins were examined by fluorescence microscopy.
3.3 Invasion ability Changes of EC9706 cells
Recombinant plasmids pcDNA3.1(+)-DsGPI and pcDNA3.1(+)-GPI were constructed and transfected in EC9706 cells, respectively. Cells transfected with blank plasmid pcDNA3.1(+) and cells without transfection were used as control. Protein expressions were examined by Western blots and cell invasion assay was performed using transwell chambers.
4 Statistical analysis
Results of Western blotting were analyzed by TotalLab2.0 software and all experiments were replicated at least three times. The data was performed by one-way analysis of variance using SPSS version13.0 (SPSS, Chicago, USA). Summary statistics were expressed as the means±the standard deviations, except as otherwise stated. In all statistical analyses, a P value of< 0.05 was considered statistically significant, and all P values were two-sided.
Results
1 Proteomics analysis of salt-induced proteins from D.salina
1.1 Identification of D.salina
The contaminated algal cells were purified as two groups, one with the larger and the other with the smaller cell volume. The internal transcribed spaces (ITS) cloned by PCR and sequenced of the two species showed that the larger one resulted a 680 bp while the smaller one resulted a 1042 bp fragment. Analysis of the ITS sequences indicated that one (the larger) was Dunaliella salina UTEX 1644 and the other (the smaller) was Dunaliella viridis.
1.2 Two-dimensional gel electrophoresis (2-DE)
Pigments such as chlorophyll were removed and the protein precipitation was easy to dissolve in the IEF buffer using the TCA-actone method for the extraction of total proteins; the voltage gradually increased up to the final 8000 v during the process of IEF suggesting that the sample was properly treated.
1.3 Analysis of the 2-DE gels and identification of salt-induced proteins by MS analysis
Eight hundreds and three protein spots were reproducibly detected in each CBB-stained gel and 739 spots were matched with a matching rate of 92.1%. Intensity quantification of protein spots revealed that intensities of a total of 21 protein spots were increased by over threefold at 3.5 M NaCl as compared to control gels and these protein spots were selected for MS analysis.
The selected 21 protein spots digested by trypsin were applied to MALDI-TOF/TOF MS. According to the search results by MASCOT, there was not a good match (score<65) in nine spots while 12 spots had a good match (score>65). Of the 12 spots, one protein spot was identified as DsGPI, two spots were unknown proteins, and the nine proteins have been identified as follows:Fe superoxide dismutase, ATP synthase, heat shock protein 70B,30S ribosomal protein S4, retrotransposon protein, dynein 1-alpha heavy chain (flagellar inner arm), G-protein, calcium-dependent protein kinase, and alcohol dehydrogenase.
2 Cloning, heterologous expression and functional analysis of the DsGPI gene
2.1 Cloning of full-length cDNA of DsGPI gene
PCR amplification with degenerated primers resulted in a specific PCR product of 771 bp long, A 1,206 bp and a 687 bp of products were obtained by 5'and 3' RACE inner PCR, respectively. Based on fact that 123 bp of the 1206 bp and 137 bp of the 687 bp were overlapped with the 771 bp of PCR product, respectively, a full-length sequence of 2338 bp (GenBank accession number FJ210719), in which an ORF of 1,980 bp was included, was deduced. Bioinformatics analysis showed that DsGPI amino acids shared high identities with the known GPIs from other species and potential active site residues that have been proved to be extremely conserved in the GPIs from other organisms.
2.2 Heterologous expression of DsGPI
After competent cells of E. coli BL21 transformed with the pET28a(+)-DsGPI were induced by IPTG, the predicted. abundant fusion proteins with a molecular weight of-75 kDa were identified by SDS-PAGE, while null or trace of the protein band appeared in control E. coli BL21 cells that were untransformed or transformed but without induction.
2.3 Analysis of DsGPI gene expression
Real time quantitative RT-PCR demonstrated that when the algal cells were transferred to higher NaCl (2.5 and 3.5 M) from lower (1.5 M) concentrations, DsGPI expressions were abruptly reduced at the first 2 h but then rapidly increased by up to eightfold or 14-fold in 2.5 or 3.5 M NaCl, respectively, suggesting that DsGPI is highly induced by salt and may play an important role in adaptation to salt stress. Furthermore, after algal cells that had been cultured in the dark for 48 h were exposed to light, expression of the DsGPI gene was reduced quickly in the first 2 h and then maintained at a relatively stable level.
3 Effects of DsGPI gene on the invasion ability of EC9706 cells
3.1 Preparation of DsGPI polyclonal antibody
The fusion proteins purified using Ni-NTA spin column according to the affinity of His-tag to Ni2+ resulted in a purity reaching to 96.3%;The titer of anti-serum was determined as 1:256K-1:2512K by ELISA and Western blotting showed that the anti-serum from the immunized albino rabbit could specially bind with DsGPI protein in the total soluble proteins of BL21.
3.2 Localization of DsGPI in MDCK cells
As shown by fluorescence microscopy, EGFP fusion proteins were mainly localized in the nucleus area in MDCK cells transfected with plamids of pEGFP-Cl-DsGPI or pEGFP-Cl-GPI while uniform distributed with blank plasmid of EGFP-C1 and without fluorescence in untreated cells, suggesting that DsGPI has the similar cellular localization with that of human GPI (AMF).
3.3 Invasion ability Changes of EC9706 cells
Results of Western blots revealed that expression of DsGPI was incresed by 2.2±0.06 fold (P< 0.05) and 1.9±0.13 fold (P< 0.05) in EC9706 cells transfected with pcDNA3.1(+)-DsGPI compared with the expression of AMF in EC9706 cells that transfected with blank pcDNA3.1(+) or without transfection; and that expression of AMF was incresed by 3.3±0.11 fold (P< 0.05) and 3.0±0.19 fold (P< 0.05)in EC9706 cells transfected with pcDNA3.1(+)-AMF compared with that with blank pcDNA3.1(+) or without transfection. In cell invasion assay, the values of OD of cells transfected with and without blank pcDNA3.1 (+) were 2.64±0.06 and 2.47±0.10, but increased to 3.86±0.14(P< 0.05) and 4.18±0.11 (P< 0.05) in the cells transfected with pcDNA3.1-DsGPI and pcDNA3.1-AMF (P< 0.05), respectively, suggesting that DsGPI can acts as the similar roles in promoting cell invasion.
Conclusions
1. D. salina was successfully isolated, purified and identified based on nuclear ITS rDNA sequences; two-dimensional gel electrophoresis maps of proteins from D.salina cells cultured in 1.5 M or 3.5 M NaCl medium were constructed and salt-induced proteins were isolated; twenty-one protein spots whose intensities were elevated 3-to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by MALDI-TOF MS and 12 proteins had a good match with databases providing useful peptides information for further studies on functions of these proteins and corresponding genes. The identified proteins are as follows: glucose-6-phosphate isomerase, Fe superoxide dismutase, ATP synthase, heat shock protein 70B,30S ribosomal protein S4, kinetochore protein, dynein 1-alpha heavy chain (flagellar inner arm), G-protein, calcium-dependent protein kinase, alcohol dehydrogenase and two unknown proteins.
2. A full-length cDNA of GPI gene of 2338 bp in length has firstly been obtained from D. salina containing a complete open reading frame 1980 bp that has been verified by bioinformatics analysis and heterologous expression; expression of DsGPI gene is related to light induction and involves in responding to salt stress; polyclonal antibody of DsGPI has been prepared with specificity and a titer of 1:250K.
3. Eukaryotic expression plasmids pEGFP-Cl-DsGPI and pcDNA 3.1-DsGPI containing full-length cDNA of DsGPI the are successfully constructed; DsGPI mainly localizes in the nucleus in MDCK cells as that of human GPI(AMF); DsGPI can enhance the invasion ability of EC9706 cells function as an cytokine. The cytokine function of DsGPI that promotes invasion of tumor cells and its correlation to flagella/cilia provides the possibility to study the functions of DsGPI in flagella/cilia and the relationship between flagella/cilia and tumorigenesis.
许多参与糖代谢的酶都已经被证实参与了植物的盐胁迫应答过程,同时这些糖代谢相关的酶在莱茵衣藻(Chlamydomonas reinhardti)和盐藻以及果蝇(Drosophila)等模式生物中也被证实跟它们的运动、感觉器官-鞭毛/纤毛具有相关性。鞭毛和纤毛是细胞表面的特化细胞器,形态和结构相似,在不同的物种中具有高度保守性。除了具有典型的运动功能,纤毛/鞭毛在生物的感官和生长发育过程中也发挥至关重要的作用,在多种组织细胞中其结构和功能相关基因的缺失或突变可能导致多种疾病的发生。最近的一些研究表明纤毛可能在肿瘤细胞中起重要作用,一些重要的信号通路如：Hedgehog (Hh)、Smoothened和Wnt似乎都跟鞭毛成分相关。
葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase, GPI)广泛存在于真核生物、原核生物和一些古细菌中,催化糖酵解和糖异生过程中葡萄糖-6-磷酸和果糖-6-磷酸的相互转化,而且是“进化学说”的证据之一。在哺乳动物细胞中,除了作为糖代谢的关键酶它还具有细胞因子的功能,能够通过和它的受体结合来促进细胞的运动。相关的研究证实杜氏盐藻GPI在盐藻的鞭毛功能中可能发挥作用,但它是否具有细胞因子功能尚不清楚。
本研究中我们通过不同盐浓度培养杜氏盐藻,利用蛋白质组学方法获得了盐藻的差异表达图谱,分离出了21种在高盐诱导条件下蛋白表达增加达到3倍的蛋白并通过质谱分析鉴定出其中的12种蛋白,包括糖代谢的关键酶GPI(DsGPI);为了进一步验证其在分子水平也参与了盐胁迫的过程,我们通过快速扩增cDNA末端(Rapid amplification of cDNA end, RACE)技术得到了DsGPI基因cDNA全长,利用原核表达验证该cDNA的正确性,同时通过实时荧光定量分析该基因在高盐和光照诱导条件下的变化情况；由于杜氏盐藻GPI具有和哺乳动物GPI相似的结构和一些相同的关键酶活位点,我们将杜氏盐藻GPI转染MDCK细胞验证其是否具有和人GPI相似的细胞定位,转染食管鳞癌细胞验证其是否具有和人GPI相似的促进肿瘤细胞运动的能力。结果显示,DsGPI不但在分子水平而且蛋白水平都参与了盐胁迫的过程,在高盐诱导条件下二者的表达量均明显升高。杜氏盐藻GPI转染MDCK细胞后和人GPI(AMF)具有相似的细胞定位：主要集中的细胞核区域；同时Western blot结果也证实杜氏盐藻GPI能够在食管鳞癌细胞中稳定过表达,而且能够增强食管鳞癌细胞的侵袭能力,提示其可能也具有细胞因子功能。为进一步探讨DsGPI作为细胞因子影响癌细胞增殖、凋亡以及其鞭毛/纤毛中的定位提供了实验基础。
方法
1第一部分杜氏盐藻盐适应蛋白的蛋白质组学研究
1.1杜氏盐藻藻株的鉴定
取适量藻细胞混合液轻涂于半固体培养基培养板上,培养10～14天至板上长出单藻落,随机挑选60个单藻落转接于试管中培养。7-10天后在显微镜下观察藻体形态,要求大小均一。分别选取体积较大和体积较小的藻细胞扩大培养,生长至对数生长期后提取DNA并设计引物扩增内部间隔序列(internal transcribed space,ITS),测序后根据比对结果确定纯化的杜氏盐藻藻株。
1.2双向电泳分离不同盐浓度培养下的杜氏盐藻总蛋白
三氯醋酸-丙酮沉淀法提取1.5 M和3.5 M NaCl培养的杜氏盐藻总蛋白,Brandford法进行蛋白定量。分别取400μg和800μg在等电点pI范围为3～10的24cm胶条上进行第一向等电聚焦,然后通过SDS-PAGE进行第二向电泳,并分别用硝酸银和考马斯亮蓝R250染色。
1.3双向电泳图谱分析和质谱鉴定高盐诱导条件下增强表达的蛋白
二维凝胶分别经硝酸银和考马斯亮蓝R250染色并显色后,用Image-Scanner进行图像扫描获得凝胶图像,经ImageMaster 2D Platinum software凝胶图像分析软件进行图像调整、蛋白点检测、蛋白点匹配和数据分析后结合肉眼观察,确定差异表达的蛋白质点；在匹配的蛋白质点中选取在高盐条件下表达量增加达到3倍的蛋白点,从凝胶上挖下,胰酶消化后,经基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS)获得肽质量指纹图谱(PMF),通过软件搜索蛋白质数据库并结合生物信息学方法鉴定蛋白质点。
2第二部分杜氏盐藻GPI基因cDNA的克隆、表达和功能分析
2.1杜氏盐藻GPI基因cDNA全长的克隆
根据质谱鉴定获得的DsGPI的肽段信息,结合其它物种中GPI氨基酸的保守性分析,设计兼并引物,RT-PCR获得DsGPI基因cDNA片段；根据片段序列信息,利用3'RACE和5'RACE的方法分别向3’下游和5’上游扩增杜氏盐藻GPI基因的3’和5'cDNA末端片段；将上述片段拼接得到DsGPI基因cDNA全长,然后设计引物PCR扩增全长,测序后进行核苷酸和氨基酸的生物信息学分析。
2.2杜氏盐藻GPI基因cDNA全长的原核表达
将在两端添加适当酶切位点的杜氏盐藻GPI cDNA编码序列定向克隆于原核表达载体pET-28a(+)中构建重组质粒,筛选并测序鉴定重组质粒。转化E-coliBL21,并用IPTG诱导DsGPI融合蛋白在BL21中表达。SDS-PAGE后,对DsGPI融合蛋白在大肠杆菌BL21中的表达进行分析。
2.3杜氏盐藻GPI基因的功能分析
将对数生长期的盐藻置于暗环境中培养24小时后进行光照培养,以继续在暗环境培养的盐藻为对照,分别在0,1,2,4,8,12,16,20和24小时取样并提取RNA,实时荧光定量PCR分析DsGPI基因在光诱导条件下的变化趋势；取对数生长期的盐藻分别接入新鲜1.5 M、2.5 M和3.5 M NaCl培养基中,分别在0,1,2,4,6,8,10和12小时取样并提取RNA,实时荧光定量分析DsGPI基因在盐诱导条件下的变化趋势。
3第三部分杜氏盐藻GPI基因对食管鳞癌细胞侵袭能力的影响
3.1杜氏盐藻GPI多克隆抗体的制备
利用Ni-NTA组氨酸亲和柱纯化原核表达的DsGPI融合蛋白,免疫新西兰白兔制备DsGPI多克隆抗体,并利用间接ELISA进行效价评测和Western blots进行抗体特异性分析。
3.2杜氏盐藻GPI在MDCK细胞中的定位
构建EGFP-C1-DsGPI真核表达载体并利用脂质体转染细胞,同时以EGFP-C1-GPI、EGFP-C1空载体转染的细胞及未处理的细胞为对照,荧光显微镜观察杜氏盐藻GPI在MDCK细胞中的定位。
3.3杜氏盐藻GPI对食管鳞癌细胞EC9706侵袭能力的影响
以pcDNA3.1(+)质粒为基础,分别构建pcDNA3.1(+)-DsGPI和pcDNA3.1(+)-GPI质粒并转染EC9706细胞,同时以pcDNA3.1(+)空质粒转染的细胞和未经处理的细胞为对照,Western blots鉴定重组质粒在EC9706细胞中的蛋白表达情况,然后利用Transwell小室试验检测EC9706细胞侵袭能力变化。
4统计学处理
Western blots结果采用TotalLab2.0软件分析,同一实验均重复三次以上。用SPSS13.0统计软件进行统计学分析,统计学数据用均数±标准差(x±s)表示,行单因素方差分析(one-way ANOVA), P<0.05具差异性。
结果
1第一部分杜氏盐藻盐适应蛋白的蛋白质组学研究
1．1杜氏盐藻藻株的鉴定
从半固体培养基上挑取的单个藻细胞经在试管中培养后,形态均一,无互相混合；以体积较大和体积较小的藻DNA为模板,用ITS引物分别扩增出长度～600bp和～1100 bp的片段；经测序并比对后证实体积较大的藻为杜氏盐藻(Dunaliella salina UTEX 1644),体积较小的藻为(Dunaliella viridis);后续的实验以体积较大的纯净的杜氏盐藻藻株为研究对象。
1.2双向电泳分离不同盐浓度培养下的杜氏盐藻总蛋白
TCA-丙酮沉淀法提取的杜氏盐藻总蛋白能够有效的去除盐藻中的色素等成分,并且易溶解于上样缓冲液；等电聚焦过程中电压上稳定上升并且均达到了预期的8000 V,说明样品处理符合条件；二维凝胶染色后Image-Scanner扫描获得的凝胶图像清晰,蛋白质点均匀,重复性好；
1.3双向电泳图谱分析和质谱鉴定高盐诱导条件下增强表达的蛋白
经ImageMaster 2D Platinum software凝胶图像分析软件后共检测到803个蛋白点,共匹配739对,匹配率为92.1%；在这些匹配的蛋白质点中有21个蛋白点在3.5 M NaCl的条件下增强表达达到3倍以上,这些蛋白点并被选择进行下一步的质谱鉴定；所选择的21个蛋白经质谱分析和数据库比对后,有9个未能找到理想的匹配对象,剩余的12个均被鉴定,包括：葡萄糖-6-磷酸异构酶,铁-超氧化物歧化酶,ATP合成酶,热休克蛋白70B,30S核糖体蛋白S4,反转录转座子蛋白(retrotransposon protein),动力蛋白1-α重链,G蛋白,钙依赖性蛋白激酶,乙醇脱氢酶和2个暂未命名的蛋白。
2第二部分杜氏盐藻GPI基因cDNA的克隆、表达和功能分析
2.1杜氏盐藻GPI基因cDNA全长的克隆
利用兼并引物扩增得到771 bp长的DsGPI基因cDNA片段,3'RACE和5'RACE分别得到了687 bp和1206 bp的片段,去除重叠区域后得到了全长为2338 bp的序列,此序列中包含一个1980 bp长的开放阅读框；此阅读框对应的659个氨基酸长度序列经BLAST分析后显示其与其它物种的GPI具有很高的同源性,说明克隆的序列就是杜氏盐藻的GPI基因cDNA序列。
2.2杜氏盐藻GPI基因cDNA全长的原核表达
杜氏盐藻的GPI cDNA编码序列定向克隆于原核表达载体pET-28a(+)后转染大肠杆菌株BL21,经IPTG诱导后成功表达DsGPI融合蛋白。SDS-PAGE结果表明：在～78 kD出现一条特异性条带,而对照组无此特异条带,说明所克隆得到的杜氏盐藻GPI cDNA序列正确。
2.3杜氏盐藻GPI基因的功能分析
实时荧光定量PCR分析显示,DsGPI基因在光诱导的前2个小时其表达量会迅速下降,而后又恢复到正常水平并维持相对稳定；而在高盐诱导条件下,DsGPI基因的表达在开始的前2个小时有所下降而后迅速升高并且分别在3.5 MNaCl诱导16小时后到达峰值,此时其表达量增至14倍；在2.5 M NaCl诱导20小时后到达峰值,此时其表达量增至8倍。
3第三部分杜氏盐藻GPI基因对食管鳞癌细胞侵袭能力的影响
3.1杜氏盐藻GPI多克隆抗体的制备
经Ni-NTA组氨酸亲和柱纯化的DsGPI融合蛋白其纯度高达96.3%；间接ELISA检测到该多抗的效价高达1：256 K～1：512 K,同时Western blots条带单一,说明该抗体具有特异性。
3.2杜氏盐藻GPI在MDCK细胞中的定位
荧光显微镜观察显示转染EGFP-C1-DsGPI和EGFP-C1-GPI质粒的MDCK细胞在细胞核区域富集有绿色荧光蛋白,而EGFP-C1空载体转染的细胞在胞质中均匀分布绿色荧光蛋白,未经处理的细胞无绿色荧光显现,说明杜氏盐藻GPI具有和人GPI (AMF)相同的细胞定位。
3.3杜氏盐藻GPI对食管鳞癌细胞侵袭能力的影响
Western blots结果显示转染pcDNA3.1(+)-DsGPI的EC9706细胞其GPI的表达分别是用pcDNA3.1(+)空质粒转染细胞和未经处理的细胞中GPI的表达量的2.2±0.06倍(P<0.05)和1.9±0.13倍(P<0.05),转染pcDNA3.1(+)-GPI质粒的EC9706细胞其AMF的表达量分别是用pcDNA3.1(+)空质粒转染细胞和未经处理的细胞中AMF的表达量的3.3±0.11倍(P<0.05)和3．0±0.19倍(P<0.05)；560 nm的OD值数据分析显示转染pcDNA3.1 (+)或未进行转染的细胞其OD值分别为2.64±0.06和2.47±0.10,而转染pcDNA3.1-DsGPI和pcDNA3.1-AMF的细胞其OD值分别提高到3.86±0.14(P<0.05)和4.18±0.11(P<0.05),提示DsGPI具有和AMF促进肿瘤细胞侵袭的相似功能。
结论
1.本实验分离、纯化并鉴定了杜氏盐藻藻株,获得了形态均一的杜氏盐藻细胞,为以杜氏盐藻为材料进行研究奠定了可靠的基础；获得了杜氏盐藻在高盐诱导条件下的差异蛋白表达图谱,在蛋白质组水平对盐藻盐适应蛋白进行了初步分离；利用质谱分析了21个在高盐诱导条件增强表达达到3倍的蛋白点并鉴定出了其中的12种蛋白,获得这些蛋白的部分肽段序列,为进一步详细研究这些蛋白和基因功能提供了相关信息。
2.克隆得到杜氏盐藻GPI的cDNA序列,全长2338 bp,包含一个1980bp的开放阅读框并通过原核表达进行了验证；通过实时荧光定量PCR证实该基因的表达受光和高盐条件所诱导,而且盐浓度越高诱导效果越强,在分子水平证实DsGPI参与了高盐诱导,进一步验证了蛋白质组学的研究结果；利用纯化的原核表达蛋白成功制备了效价高、特异性强的盐藻GPI多克隆抗体。
3.构建杜氏盐藻GPI的真核表达载体,转染MDCK细胞后证实：杜氏盐藻GPI和人GPI (AMF)一样,都定位在细胞核；盐藻GPI也能在EC9706细胞中表达,Transwell小室证实,盐藻GPI具有跟人GPI相似的细胞因子功能,能够提高EC9706细胞的侵袭能力；杜氏盐藻GPI促进肿瘤细胞侵袭运动的功能,为进一步探讨盐藻GPI在体外对肿瘤细胞增殖、凋亡以及对体内肿瘤发生、发展的影响以及其在盐藻鞭毛/纤毛中的定位奠定了基础。
The unicellular eukaryotic green alga Dunaliella salina(D.salina) grows over a wide range of salt concentrations from 0.05 to 5.0 M and is one of the most halotolerant organisms. D.salina has received considerable attention in studies of halotolerance mechanisms and some salt-induced proteins have been isolated and identified. Although the genome of D. salina is not completely available, proteome analysis is an efficient technique to detect and characterize proteins from D. salina.
Many glycometabolic enzymes have been identified being involved in responding to salt stress and meanwhile, they have been proved to be related to the flagella/cilia of D.salina, the model organism Chlamydomonas reinhardti and Drosophila et al. Flagella/cilia are specialized and widespread cell organelles. More and more evidences suggest that flagella/cilia serve diverse roles in motility, sensory signaling and development of many eukaryots and implicate cilia in polycystic kidney disease (PKD) and developmental disorders, even in cancer.
Being one of the evidences for evolution theory, glucose-6-phosphate isomerase (GPI) is universally distributed among eukaryotes, bacteria and some archaea and catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate, which is an essential reaction involved in both catabolic glycolysis and anabolic gluconeogenesis. GPIs also act as an atuocrine motility factor (AMF) that can promote tumor cells metastasis and invasion by binding to their receptors in animals. Although GPI is related to flagella/cilia in D.salina, whether it has the cytokine function is still unknown.
In this study, we analyzed changes of proteins between algal cells cultured at 1.5 or 3.5 M NaCl using proteomic method and obtained the differential expression maps. Twenty one proteins increased by more than 3-fold in 3.5 M NaCl than that in 1.5 M NaCl medium were isolated and analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS), making twelve proteins were identified including GPI. In order to verify GPI is involved in responding to salt stress at gene level, the full-length cDNA of GPI was obtained using rapid amplification of cDNA end technique and heterologous expressed to. Real-time quantitative RT-PCR was used to detect its expression changes in response to light and salinity. Bioinformatics analysis revealed that the GPI from D.salina(DsGPI) have the similar structure and key active sites with that from mammalian cells and hence, it was used to transfect esophageal squamous cell carcinoma cells to check whether it has the potential ability like human GPI to promote invasion of tumor cells. The results showed that DsGPI is involved in responding to salt stress not only at the gene level but also at the protein level and that DsGPI has the same cellular localization with human GPI that mainly localized in the nucleus and also distributed in cytoplasm in esophageal squamous cell carcinoma cell (ESCC) line EC9706 cells. In addition, western blots confirmed that DsGPI was stable expressed in EC9706 and able to enhance the invasion ability of EC9706, suggesting that DsGPI may has the cytokine functions like human GPI.
Methods
1 Proteomics analysis of salt-induced proteins from D.salina
1.1 Identification of D. salina strain
Contaminated algal cells containing not only one species were spread on semisolid medium and cultured for 10 to 14 days and then, single cells were picked out and continue cultured in test tube, respectively. Seven to 10 days later, algal cells were observed under microscope and samples with the larger or the smaller uniform size were cultured to logarithmic phase and harvested for DNA extraction. A pair of primers was designed to amplify the internal transcribed space and the PCR results were blasted to determine the pure D.salina species.
1.2 Two-dimensional gel electrophoresis (2-DE)
Proteins of algal cells cultured in 1.5M and 3.5M NaCl medium were precipitated by Trichloroacetic acid-actone and dissolved in a buffer, respectively. Protein concentrations were determined using the Bradford method. A total of 400μg soluble proteins were applied to isoelectric focusing (IEF) on IPGphor using 24 cm ImmobilineTM DryStrips (pH 3-10, linear; GE Healthcare) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
1.3 Analysis of the 2-DE gels and identification of salt-induced proteins by MS analysis
The 2-DE gels stained with Coomassie Brilliant Blue (CBB) R-250 or AgNO3 were scanned using Image-Scanner (GE Healthcare). Protein spots from the 3.5 M NaCl medium were compared to those from the 1.5 M NaCl medium (control) using ImageMaster 2D Platinum software After images were adjusted, protein spots were detected and matched, and protein intensities were measured, the differential expressed proteins were determined combined with eye observation. Each sample was replicated at least three times, and protein spots for comparative analyses were detected on all of the gels.
Among the matched proteins, proteins up-regulated in by more than three times from the 2-DE gels were selected and digested by trypsin, the resulting peptides were extracted and analyzed using MALDI-TOF/TOF MS (Applied Biosystems). MS data were collected and protein identification was searched against the database of plants in NCBI using the MASCOT search engine. An identified protein was matched with the known protein(s) in the database only when its score is greater than 65 (P<0.05)
2 Cloning, heterologous expression and functional analysis of the DsGPI gene
2.1 Cloning of full-length cDNA of DsGPI gene
A pair of degenerated PCR primers "GARTTYGGNATHGAYCC" (sense) and "NACNCCCCAYTGRTCRAA"(antisense) corresponding to the highly conserved amino acid residues "EFGIDP" and "FDQWGV" was used to amplify a cDNA fragment of the DsGPI, and then 5'-and 3'-RACEs were performed. The putative 5'-and 3'-RACE cDNAs and the partial sequences cloned using the degenarate primers were organized to form a cDNA contigue that was applied to bioinformatics analysis.
2.2 Heterologous expression of DsGPI
The DsGPI cDNA was subcloned into the prokaryotic protein expression vector pET28a(+) (Novagen) with NdeI andoRI sites and recombinant plasmid was screened and identified by sequencing. Recombinant plasmids were expressed in E. coli BL21 and IPTG was added in to produce recombinant fusion DsGPI with a 6×His-tag that was subjected to SDS-PAGE.
2.3 Analysis of DsGPI gene expression
To confirm whether DsGPI responds to salt stress, algal cells were collected at 0,1,2,4,8,12,16,20 and 24 h after being transferred from 1.5 to 3.5 M NaCl medium, and thecells transferred to fresh 1.5 M NaCl medium were served as controls. Besides, to investigate whether DsGPI is lightrelated, algal cells having been cultured in the dark for 48 h were exposed to light and then collected at 1,2,4,8, 10 and 12 h, and cells without exposure to light were used as controls. Real time quantitative RT-PCR was performed for the expression change of DsGPI.
3 Effects of DsGPI gene on the invasion ability of EC9706 cells
3.1 Preparation of DsGPI polyclonal antibody
The DsGPI fusion proteins expressed in E. coli cells were purified using Ni-NTA His Bind column and subjected to immunize albino rabbit for the preparation of polyclonal antibody. The antibody titer and specificity were determined by ELISIA and Western blots, respectively.
3.2 Localization of DsGPI in MDCK cells
The complete cDNA sequences of DsGPI was cloned into the plasmid pEGFP-C1 to create a recombinant plasmid pEGFP-C1-DsGPI by using BamH I and EcoR I sites and identified. The same method was used to construct recombinant plasmid pEGFP-C1-GPI with complete cDNA sequences of human GPI.Both recombinant plasmids transfected in MDCK cells with blank plasmid pEGFP-C1 and cells without transfection were used as control. Localization the EGFP fusion proteins were examined by fluorescence microscopy.
3.3 Invasion ability Changes of EC9706 cells
Recombinant plasmids pcDNA3.1(+)-DsGPI and pcDNA3.1(+)-GPI were constructed and transfected in EC9706 cells, respectively. Cells transfected with blank plasmid pcDNA3.1(+) and cells without transfection were used as control. Protein expressions were examined by Western blots and cell invasion assay was performed using transwell chambers.
4 Statistical analysis
Results of Western blotting were analyzed by TotalLab2.0 software and all experiments were replicated at least three times. The data was performed by one-way analysis of variance using SPSS version13.0 (SPSS, Chicago, USA). Summary statistics were expressed as the means±the standard deviations, except as otherwise stated. In all statistical analyses, a P value of< 0.05 was considered statistically significant, and all P values were two-sided.
Results
1 Proteomics analysis of salt-induced proteins from D.salina
1.1 Identification of D.salina
The contaminated algal cells were purified as two groups, one with the larger and the other with the smaller cell volume. The internal transcribed spaces (ITS) cloned by PCR and sequenced of the two species showed that the larger one resulted a 680 bp while the smaller one resulted a 1042 bp fragment. Analysis of the ITS sequences indicated that one (the larger) was Dunaliella salina UTEX 1644 and the other (the smaller) was Dunaliella viridis.
1.2 Two-dimensional gel electrophoresis (2-DE)
Pigments such as chlorophyll were removed and the protein precipitation was easy to dissolve in the IEF buffer using the TCA-actone method for the extraction of total proteins; the voltage gradually increased up to the final 8000 v during the process of IEF suggesting that the sample was properly treated.
1.3 Analysis of the 2-DE gels and identification of salt-induced proteins by MS analysis
Eight hundreds and three protein spots were reproducibly detected in each CBB-stained gel and 739 spots were matched with a matching rate of 92.1%. Intensity quantification of protein spots revealed that intensities of a total of 21 protein spots were increased by over threefold at 3.5 M NaCl as compared to control gels and these protein spots were selected for MS analysis.
The selected 21 protein spots digested by trypsin were applied to MALDI-TOF/TOF MS. According to the search results by MASCOT, there was not a good match (score<65) in nine spots while 12 spots had a good match (score>65). Of the 12 spots, one protein spot was identified as DsGPI, two spots were unknown proteins, and the nine proteins have been identified as follows:Fe superoxide dismutase, ATP synthase, heat shock protein 70B,30S ribosomal protein S4, retrotransposon protein, dynein 1-alpha heavy chain (flagellar inner arm), G-protein, calcium-dependent protein kinase, and alcohol dehydrogenase.
2 Cloning, heterologous expression and functional analysis of the DsGPI gene
2.1 Cloning of full-length cDNA of DsGPI gene
PCR amplification with degenerated primers resulted in a specific PCR product of 771 bp long, A 1,206 bp and a 687 bp of products were obtained by 5'and 3' RACE inner PCR, respectively. Based on fact that 123 bp of the 1206 bp and 137 bp of the 687 bp were overlapped with the 771 bp of PCR product, respectively, a full-length sequence of 2338 bp (GenBank accession number FJ210719), in which an ORF of 1,980 bp was included, was deduced. Bioinformatics analysis showed that DsGPI amino acids shared high identities with the known GPIs from other species and potential active site residues that have been proved to be extremely conserved in the GPIs from other organisms.
2.2 Heterologous expression of DsGPI
After competent cells of E. coli BL21 transformed with the pET28a(+)-DsGPI were induced by IPTG, the predicted. abundant fusion proteins with a molecular weight of-75 kDa were identified by SDS-PAGE, while null or trace of the protein band appeared in control E. coli BL21 cells that were untransformed or transformed but without induction.
2.3 Analysis of DsGPI gene expression
Real time quantitative RT-PCR demonstrated that when the algal cells were transferred to higher NaCl (2.5 and 3.5 M) from lower (1.5 M) concentrations, DsGPI expressions were abruptly reduced at the first 2 h but then rapidly increased by up to eightfold or 14-fold in 2.5 or 3.5 M NaCl, respectively, suggesting that DsGPI is highly induced by salt and may play an important role in adaptation to salt stress. Furthermore, after algal cells that had been cultured in the dark for 48 h were exposed to light, expression of the DsGPI gene was reduced quickly in the first 2 h and then maintained at a relatively stable level.
3 Effects of DsGPI gene on the invasion ability of EC9706 cells
3.1 Preparation of DsGPI polyclonal antibody
The fusion proteins purified using Ni-NTA spin column according to the affinity of His-tag to Ni2+ resulted in a purity reaching to 96.3%;The titer of anti-serum was determined as 1:256K-1:2512K by ELISA and Western blotting showed that the anti-serum from the immunized albino rabbit could specially bind with DsGPI protein in the total soluble proteins of BL21.
3.2 Localization of DsGPI in MDCK cells
As shown by fluorescence microscopy, EGFP fusion proteins were mainly localized in the nucleus area in MDCK cells transfected with plamids of pEGFP-Cl-DsGPI or pEGFP-Cl-GPI while uniform distributed with blank plasmid of EGFP-C1 and without fluorescence in untreated cells, suggesting that DsGPI has the similar cellular localization with that of human GPI (AMF).
3.3 Invasion ability Changes of EC9706 cells
Results of Western blots revealed that expression of DsGPI was incresed by 2.2±0.06 fold (P< 0.05) and 1.9±0.13 fold (P< 0.05) in EC9706 cells transfected with pcDNA3.1(+)-DsGPI compared with the expression of AMF in EC9706 cells that transfected with blank pcDNA3.1(+) or without transfection; and that expression of AMF was incresed by 3.3±0.11 fold (P< 0.05) and 3.0±0.19 fold (P< 0.05)in EC9706 cells transfected with pcDNA3.1(+)-AMF compared with that with blank pcDNA3.1(+) or without transfection. In cell invasion assay, the values of OD of cells transfected with and without blank pcDNA3.1 (+) were 2.64±0.06 and 2.47±0.10, but increased to 3.86±0.14(P< 0.05) and 4.18±0.11 (P< 0.05) in the cells transfected with pcDNA3.1-DsGPI and pcDNA3.1-AMF (P< 0.05), respectively, suggesting that DsGPI can acts as the similar roles in promoting cell invasion.
Conclusions
1. D. salina was successfully isolated, purified and identified based on nuclear ITS rDNA sequences; two-dimensional gel electrophoresis maps of proteins from D.salina cells cultured in 1.5 M or 3.5 M NaCl medium were constructed and salt-induced proteins were isolated; twenty-one protein spots whose intensities were elevated 3-to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by MALDI-TOF MS and 12 proteins had a good match with databases providing useful peptides information for further studies on functions of these proteins and corresponding genes. The identified proteins are as follows: glucose-6-phosphate isomerase, Fe superoxide dismutase, ATP synthase, heat shock protein 70B,30S ribosomal protein S4, kinetochore protein, dynein 1-alpha heavy chain (flagellar inner arm), G-protein, calcium-dependent protein kinase, alcohol dehydrogenase and two unknown proteins.
2. A full-length cDNA of GPI gene of 2338 bp in length has firstly been obtained from D. salina containing a complete open reading frame 1980 bp that has been verified by bioinformatics analysis and heterologous expression; expression of DsGPI gene is related to light induction and involves in responding to salt stress; polyclonal antibody of DsGPI has been prepared with specificity and a titer of 1:250K.
3. Eukaryotic expression plasmids pEGFP-Cl-DsGPI and pcDNA 3.1-DsGPI containing full-length cDNA of DsGPI the are successfully constructed; DsGPI mainly localizes in the nucleus in MDCK cells as that of human GPI(AMF); DsGPI can enhance the invasion ability of EC9706 cells function as an cytokine. The cytokine function of DsGPI that promotes invasion of tumor cells and its correlation to flagella/cilia provides the possibility to study the functions of DsGPI in flagella/cilia and the relationship between flagella/cilia and tumorigenesis.
引文
[1]Parida AK, Das AB. Salt tolerance and salinity effects on plants:a review. Ecotoxicol Environ Saf,2005,60:324-349
[2]Klahn S, Marquardt DM, Rollwitz I, et al. Expression of the ggpPS gene for glucosylglycerol biosynthesis from Azotobacter vinelandii improves the salt tolerance of Arabidopsis thaliana. J Exp Bot,2009,60:1679-1689
[3]Yang Q, Chen ZZ, Zhou XF, et al. Over expression of SOS (Salt Overly Sensitive) genes increases salt tolerance in transgenic Arabidopsis. Mol Plant,2009,2:22-31
[4]Tuteja N. Mechanisms of high salinity tolerance in plants. Methods,2007, Enzymol 428:419-438
[5]Munns R. Genes and salt tolerance:bring them together. New Phytol,2005,167:645-663
[6]Mishra A, Mandoli A, Jha B. Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan. J Ind Microbiol Biotechnol,2008, 35:1093-1101
[7]Liska AJ, Shevchenko A, Pick U, et al. Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics. Plant Physiol,2004,136:2806-2817
[8]Wagner V, Boesger J, Mittag M. Sub-proteome analysis in the green flagellate alga Chlamydomonas reinhardtii. J Basic Microbiol,2009,49(1):32-41
[9]Jia Y, Xue L, Li J, et al. Isolation and proteomic analysis of the halotolerant alga Dunaliella salina flagella using shotgun strategy. Mol Biol Rep,2010,37(2):711-716.
[10]Stauber EJ, Hippler M. Chlamydomonas reinhardtii proteomics. Plant Physiol Biochem, 2004,42(12):989-1001
[11]Mans DA, Voest EE, Giles RH. All along the watchtower:is the cilium a tumor suppressor organelle? Biochim Biophys Acta,2008,1786(2):114-125
[12]Pan J, Wang Q, Snell WJ. Cilium-generated signaling and cilia-related disorders. Laboratory Investigation,2005,85:452-463
[13]Kozminski KG, Johnson KA, Forscher P, et al. A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci USA,1993,90:5519-5523
[14]Watanabe H, Takehana K, Date M, et al. Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res,1996,56(13):2960-2963
[15]Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci U S A,1986,83(10):3302-3306
[16]Yakirevich E, Naot Y. Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol Reprod,2000,62(4):1016-1023
[17]Schulz LC, Bahr JM. Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret. Proc Natl Acad Sci U S A,2003,100(14):8561-8566
[18]Cao MJ, Osatomi K, Matsuda R, et al. Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus). Biochem Biophys Res Commun,2000,272(2):485-489
[19]Yanagawa T, Watanabe H, Takeuchi T, et al. Overexpression of autocrine motility factor in metastatic tumor cells:possible association with augmented expression of KIF3A and GDI-beta. Lab Invest,2004,84(4):513-522
[20]Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motility factor signaling enhances pancreatic cancer metastasis. Clin Cancer Res,2004,10(22):7775-7784
[21]Dobashi Y, Watanabe H, Sato Y, et al. Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas. J Pathol,2006,210(4):431-440
[22]Popova OV, Yang O, Dietz KJ, et al. Differential transcript regulation in Arabidopsis thaliana and the halotolerant Lobularia maritima indicates genes with potential function in plant salt adaptation. Gene,2008,423:142-148
[23]Katz A, Waridel P, Shevchenko A, et al. Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis. Mol Cell Proteomics,2007,6:1459-1472
[24]Goyal A. Osmoregulation in Dunaliella, Part II:Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta. Plant Physiol Biochem, 2007,45:705-710
[25]Jorrin-Novo JV, Maldonado AM, Echevarria-Zomeno S, et al. Plant proteomics update (2007-2008):Second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge. J Proteomics,2009,72(3):285-314
[26]Gonza lez Mariela A, Coleman Annette W, G 6 mez'Patricia I, et al. Phylogenetic relationship among various strains of Dunaliella (Chlorophyceae) based on nuclear ITS rDNA sequences. J Phycol,2001,37:604-611
[27]王伟,许政暟,宋任涛.利用ITS序列对两个盐藻株的分子鉴定.上海大学学报(自然科学版),2006,12(1)：84-88
[28]Ben-Amotz A, Avron M. The biotechnology of cultivating the halotolerant alga Dunaliella. Trends Biotech,1990,8:121-126
[29]张弛,胡鸿钧,李中奎,等.衣藻属的系统发育分析-基于形态形状和nrDNA ITS序列.武汉植物学研究,2000,18(3)：189-194
[30]Gomez Patricia I, Gonzalea Mariela A. Genetic variation among seven strains of Dunaliella salina (Chlorophyta) with industrial potential, based on RAPD banding patterns and on nuclear ITS rDNA sequences. Aquaculture,2004,233:149-161
[31]Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics,2004,4(12):3665-3685
[32]李义良,朱华清,张红锋.双向电泳技术.生物学教学,2004,29(4)：64
[33]Rabilloud T. Solubilization of proteins for electrophoretic analyses. Electrophoresis,1996, 17(5):813-829
[34]Chambers G, Lawrie L, Cash P, et al. Proteomics:a new approach to the study of disease. J Pathol,2000,192(3):280-288
[35]Zhou M, Veenstra TD. Proteomic analysis of protein complexes. Proteomics,2007, 7(16):2688-2697
[36]Lopez JL. Two-dimensional electrophoresis in proteome expression analysis. J Chromatogr B Analyt Technol Biomed Life Sci,2007,849(1-2):190-202
[37]Bodzon-Kulakowska A, Bierczynska-Krzysik A, Dylag T, et al. Methods for samples preparation in proteomic research. J Chromatogr B Analyt Technol Biomed Life Sci,2007, 49(1-2):1-31
[38]Berkelman T. Quantitation of protein in samples prepared for 2-D electrophoresis. Methods Mol Biol,2008,424:43-49
[39]Marengo E, Robotti E, Bobba M.2D-PAGE maps analysis. Methods Mol Biol,2008, 428:291-325
[40]Pan C, Xu S, Zhou H, et al. Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS. Anal Bioanal Chem,2007,387(1):193-204
[41]Bonk T, Humeny A. MALDI-TOF-MS analysis of protein and DNA. Neuroscientist,2001, 7(1):6-12
[42]Grauvogel C, Brinkmann H, Petersen J. Evolution of the glucose-6-phosphate isomerase:the plasticity of primary metabolism in photosynthetic eukaryotes. Mol Biol Evol,2007, 24:1611-1621
[43]Katz LA. Transkingdom transfer of the phosphoglucose isomerase gene. J Mol Evol,1996 43:453-459
[44]Goyal A, Brown AD, Gimmler H. Regulation of salt-induced starch degradation in Dunaliella tertiolecta. J Plant Physiol,1987,127:77-96
[45]Chen H, Jiang JG. Osmotic responses of Dunaliella to the changes of salinity. J Cell Physiol, 2009,219:251-258
[46]Jithesh MN, Prashanth SR, Sivaprakash KR, et al. Antioxidative response mechanisms in halophytes:their role in stress defence. J Genet,2006,85:237-254
[47]Ray S, Agarwal P, Arora R, et al. Expression analysis of calcium-dependent protein kinase gene family during reproductive development and abiotic stress conditions in rice (Oryza sativa L. ssp. indica). Mol Genet Genomics,2007,278:493-505
[48]Stepien P, Johnson GN. Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella:role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol,2009,149:1154-1165
[49]Wang X, Yang P, Gao Q, et al. Proteomic analysis of the response to high-salinity stress in Physcomitrella patens. Planta,2008,228:167-177
[50]范吉星,邓用川,黄惜等.红海榄根部总蛋白质提取方法的改进和双向电泳体系的建立.湖南农业大学学报(自然科学版),2008,34(5)：549-553
[51]Phillips TL, Porter DW, Gracy RW. Nature of the multiple forms of sweet-potato glucose phosphate isomerase. Biochem J.1975,147(2):381-384
[52]Faik P, Walker JI, Redmill AA, et al. Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences. Nature.1988,332(6163):455-457
[53]Chaput M, Claes V, Portetelle D, et al. The neurotrophic factor neuroleukin is 90% homologous with phosphohexose isomerase. Nature.1988,332(6163):454-455
[54]Wang R, Okamoto M, Xing X,et al. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol.2003, 132(2):556-567
[55]Munjral N, Gupta AK, Kaur N. Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules. Indian J Biochem Biophys.2007,44(3):186-189
[56]Cordenunsi BR, Oliveira do Nascimento JR, Vieira da Mota R, et al. Phosphoglucose isomerase from bananas:partial characterization and relation to main changes in carbohydrate composition during ripening. Biosci Biotechnol Biochem,2001, 65(10):2174-2180
[57]Yu TS, Lue WL, Wang SM, et al. Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation. Plant Physiol,2000,123(1):319-326
[58]Funasaka T, Raz A. The role of autocrine motility factor in tumor and tumor microenvironment. Cancer Metastasis Rev,2007,26(3-4):725-735
[59]Yanagawa T, Funasaka T, Tsutsumi S, et al. Novel roles of the autocrine motility factor/phosphoglucose isomerase in tumor malignancy. Endocr Relat Cancer,2004, 11(4):749-59
[60]Chiu CG, St-Pierre P, Nabi IR, et al. Autocrine motility factor receptor:a clinical review. Expert Rev Anticancer Ther,2008,8(2):207-217
[61]Pazour GJ, Agrin N, Leszyk J et al. Proteomic analysis of a eukaryotic cilium. J Cell Biol, 2005,170:103-113
[62]卢圣栋.现代分子生物学实验技术.中国协和医科大学出版社,1999年12月出版.第二版
[63]J 萨姆布鲁克,D W 拉塞尔.分子克隆实验指南.科学出版社,2002年8月第三版
[64]Cordeiro AT, Michels PA, Delboni LF, et al. The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features. Eur J Biochem,2004, 271(13):2765-2772
[65]Sun YJ, Chou CC, Chen WS, et al. The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin. Proc Natl Acad Sci U S A. 1999,96(10):5412-5417
[66]Yamamoto H, Miwa H, Kunishima N. Crystal structure of glucose-6-phosphate isomerase from Thermus thermophilus HB8 showing a snapshot of active dimeric state. J Mol Biol, 2008,382(3):747-762
[67]Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods,2001,25:402-408
[68]Oren A. A hundred years of Dunaliella research:1905-2005. Salina systems,2005,1:2
[69]Borowitzka LJ, Brown AD. The salt relations of marine and halophilic species of the unicellular green alga, Dunaliella. The role of glycerol as a compatible solute. Arch Microbiol,1974,96:37-52
[70]Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci USA,1986,83(10):3302-3306
[71]Shimizu K, Tani M, Watanabe H, et al. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett,1999,456(2):295-300
[72]Haga A, Tanaka N, Funasaka T, et al. The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF. J Mol Biol,2006,358(3):741-753
[73]Funasaka T, Hogan V, Raz A. Phosphoglucose isomerase/autocrine motility factor mediates epithelial and mesenchymal phenotype conversions in breast cancer. Cancer Res,2009, 69(13):5349-5356
[74]Shih WL, Liao MH, Yu FL, et al. AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration. Cancer Lett,2008,270(2):202-217
[75]Luo Y, Luo Y, Long JM, et al. A link between maze learning and hippocampal expression of neuroleukin and its receptor gp78. J Neurochem,2002,80(2):354-361
[76]Merchant SS, Prochnik SE, Vallon O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science,2007,318(5848):245-250
[77]Olsen BR. When primary cilia fail. Matrix Biol,2009,8(8):443-444
[78]Nigg EA, Raff JW. Centrioles, centrosomes, and cilia in health and disease. Cell,2009, 139(4):663-678
[79]Baker K, Beales PL. Making sense of cilia in disease:the human ciliopathies. Am J Med Genet C Semin Med Genet,2009,151:281-295
[80]Hurd TW, Margolis B. Cystin, cilia, and cysts:unraveling trafficking determinants. J Am Soc Nephrol,2009,20(12):2485-2486
[81]Doherty D. Joubert syndrome:insights into brain development, cilium biology, and complex disease. Semin Pediatr Neurol,2009,16(3):143-154
[82]Roy S. The motile cilium in development and disease:emerging new insights. Bioessays, 2009,31(7):694-699
[83]Lancaster MA, Gleeson JG. The primary cilium as a cellular signaling center:lessons from disease. Curr Opin Genet Dev,2009,19(3):220-229
[84]Veland IR, Awan A, Pedersen LB, et al. Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol,2009,111(3):39-53
[85]Lee YL, Li TT. Crystallization and preliminary crystallographic study of the phosphoglucose isomerase from Bacillus subtilis. Acta Crystallogr Sect F Struct Biol Cryst Commun.2008, 64:1181-1183
[86]Arsenieva D, Appavu BL, Mazock GH, et al. Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution. Proteins, 2009,74(1):72-80
[87]Lee JH, Jeffery CJ. The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate. Protein Sci,2005,14(3):727-734
[88]Andzhaparidze OG, Bektemirov TA, Burgasova MP. Influence of a complex of poly(I):poly(C) and poly-l-lysine on the course of vaccinia in monkeys. Vopr Virusol,1977, (3):339-343
[89]Anthony LS, Stevenson MM, Skamene E. Enhancement of resistance to Listeria monocytogenes infection in mice by pyrimidine analogs. Clin Invest Med,1984, 7(4):343-348
[90]Stiehm ER, Keller MA, Vyas GN. Preparation and use of therapeutic antibodies primarily of human origin. Biologicals,2008,36(6):363-374
[91]Khatri I, Forstner G, Forstner J. Preparation of polyclonal antibodies to native and modified mucin antigens. Methods Mol Biol,1993,14:225-235
[92]Mayne R. Preparation and applications of monoclonal antibodies to different collagen types. Clin Biochem,1988,21(2):111-115
[93]Xu W, Chiao JW. Biochemical characteristics of a human myeloid leukemia cell differentiation factor. Prep Biochem Biotechnol,1996,26(1):21-30
[94]Gurney ME, Heinrich SP, Lee MR, et al. Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons. Science,1986,234(4776):566-574
[95]Yakirevich E, Naot Y. Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol Reprod,2000,62(4):1016-1023
[96]Schulz LC, Bahr JM. Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret. Proc Natl Acad Sci U S A,2003,100(14):8561-8566
[97]Cao MJ, Osatomi K, Matsuda R, et al. Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus). Biochem Biophys Res Commun,2000,272(2):485-489
[98]Repiso A, Andres R, Climent F, et al. Expression patterns in mouse embryos of neuroleukin/glucose-6-phosphate isomerase and autocrine motility factor receptor. Anat Histol Embryol,2008,37(5):380-382
[99]Fairbank M, St-Pierre P, Nabi IR. The complex biology of autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and its receptor, the gp78/AMFR E3 ubiquitin ligase. Mol Biosyst,2009,5(8):793-801
[100]Tsutsumi S, Hogan V, Nabi IR, et al. Overexpression of the autocrine motility factor/phosphoglucose isomerase induces transformation and survival of NIH-3T3 fibroblasts. Cancer Res.2003,63(1):242-249
[101]Yanagawa T, Watanabe H, Takeuchi T, et al. Overexpression of autocrine motility factor in metastatic tumor cells:possible association with augmented expression of KIF3A and GDI-beta. Lab Invest.2004,84(4):513-522
[102]Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motility factor signaling enhances pancreatic cancer metastasis. Clin Cancer Res.2004,10(22):7775-7784
[103]Silletti S, Paku S, Raz A. Autocrine motility factor and the extracellular matrix. II. Degradation or remodeling of substratum components directs the motile response of tumor cells. Int J Cancer.1998,76(1):129-135
[104]Yu FL, Liao MH, Lee JW, et al. Induction of hepatoma cells migration by phosphoglucose isomerase/autocrine motility factor through the upregulation of matrix metalloproteinase-3. Biochem Biophys Res Commun.2004,314(1):76-82
[105]Haga A, Funasaka T, Deyashiki Y, et al. Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway. FEBS Lett.2008,582(13):1877-1882
[106]Tsutsumi S, Gupta SK, Hogan V, et al. Activation of small GTPase Rho is required for autocrine motility factor signaling. Cancer Res.2002,62(15):4484-4490
[107]Talukder AH, Bagheri-Yarmand R, et al. Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors. Clin Cancer Res.2002,8(10):3285-3289
[108]Dobashi Y, Watanabe H, Sato Y, et al. Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas. J Pathol.2006,210(4):431-440
[1]Chiu CG, St-Pierre P, Nabi IR, et al. Autocrine motility factor receptor:a clinical review. Expert Rev Anticancer Ther.2008,8(2):207-217
[2]Fairbank M, St-Pierre P, Nabi IR. The complex biology of autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and its receptor, the gp78/AMFR E3 ubiquitin ligase. Mol Biosyst.2009,5(8):793-801
[3]Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci U S A.1986,83(10):3302-3306
[4]Watanabe H, Takehana K, Date M, et al. Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res.1996,56(13):2960-2963
[5]Kim JW, Dang CV. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci.2005, 30(3):142-150
[6]Amraei M, Nabi IR. Species specificity of the cytokine function of phosphoglucose isomerase. FEBS Lett.2002,525(1-3):151-155
[7]Arsenieva D, Jeffery CJ. Conformational changes in phosphoglucose isomerase induced by ligand binding. J Mol Biol.2002,323(1):77-84
[8]Tsutsumi S, Gupta SK, Hogan V, et al. The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function. FEBS Lett.2003,534(1-3):49-53
[9]Xu W, Chiao JW. Biochemical characteristics of a human myeloid leukemia cell differentiation factor. Prep Biochem Biotechnol.1996,26(1):21-30
[10]Gurney ME, Heinrich SP, Lee MR, et al. Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons. Science.1986,234(4776):566-574
[11]Yakirevich E, Naot Y. Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol Reprod.2000,62(4):1016-1023
[12]Schulz LC, Bahr JM. Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret. Proc Natl Acad Sci U S A.2003,100(14):8561-8566
[13]Cao MJ, Osatomi K, Matsuda R, et al. Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus). Biochem Biophys Res Commun.2000,272(2):485-489
[14]Repiso A, Andres R, Climent F, et al. Expression patterns in mouse embryos of neuroleukin/glucose-6-phosphate isomerase and autocrine motility factor receptor. Anat Histol Embryol.2008,37(5):380-382
[15]Baumann M, Kappl A, Lang T, et al. The diagnostic validity of the serum tumor marker phosphohexose isomerase (PHI) in patients with gastrointestinal, kidney, and breast cancer. Cancer Invest.1990,8(34):351-356
[16]Niinaka Y, Paku S, Haga A, et al. Expression and secretion of neuroleukin/phosphohexose isomerase/maturation factor as autocrine motility factor by tumor cells. Cancer Res.1998, 58(12):2667-2674
[17]Rubartelli A, Cozzolino F, Talio M, et al. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J.1990,9(5):1503-1510
[18]Lagana A, Duchaine T, Raz A, et al. Expression of autocrine motility factor/phosphohexose isomerase in Cos7 cells. Biochem Biophys Res Commun.2000,273(l):213-278
[19]Yanagawa T, Funasaka T, Tsutsumi S, et al. Differential regulation of phosphoglucose isomerase/autocrine motility factor activities by protein kinase CK2 phosphorylation. J Biol Chem.2005,280(11):10419-10426
[20]Yanagawa T, Funasaka T, Tsutsumi S, et al. Regulation of phosphoglucose isomerase/autocrine motility factor activities by the poly(ADP-ribose) polymerase family-14. Cancer Res.2007,67(18):8682-8689
[21]Nabi IR, Raz A. Cell shape modulation alters glycosylation of a metastatic melanoma cell-surface antigen. Int J Cancer.1987,40(3):396-402
[22]Nabi IR, Raz A. Loss of metastatic responsiveness to cell shape modulation in a newly characterized B16 melanoma adhesive cell variant. Cancer Res.1988,48(5):1258-1264
[23]Nabi IR, Watanabe H, Raz A. Identification of B16-F1 melanoma autocrine motility-like factor receptor. Cancer Res.1990,50(2):409-414
[24]Registre M, Goetz JG, St Pierre P, et al. The gene product of the gp78/AMFR ubiquitin E3 ligase cDNA is selectively recognized by the 3F3A antibody within a subdomain of the endoplasmic reticulum. Biochem Biophys Res Commun.2004,320(4):1316-1322
[25]Goetz JG, Genty H, St-Pierre P, et al. Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca2+ levels. J Cell Sci.2007,120(Pt 20):3553-3564
[26]Watanabe H, Nabi IR, Raz A. The relationship between motility factor receptor internalization and the lung colonization capacity of murine melanoma cells. Cancer Res. 1991,51(10):2699-2705
[27]Haga A, Komazaki S, Funasaka T, et al. AMF/G6PI induces differentiation of leukemic cells via an unknown receptor that differs from gp78. Leuk Lymphoma.2006,47(10):2234-2243
[28]Shimizu K, Tani M, Watanabe H, et al. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett.1999,456(2):295-300
[29]Mishra S, Raz A, Murphy LJ. Insulin-like growth factor binding protein-3 interacts with autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and inhibits the AMF/PGI function. Cancer Res.2004,64(7):2516-2522
[30]Lagana A, Goetz JG, Y N, et al. pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate. J Cell Sci.2005, 118(Pt 18):4175-4185
[31]Watanabe H, Carmi P, Hogan V, et al. Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor. J Biol Chem.1991,266(20):13442-13448
[32]Ponting CP. Proteins of the endoplasmic-reticulum-associated degradation pathway:domain detection and function prediction. Biochem J.2000,351(Pt 2):527-535
[33]Biederer T, Volkwein C, Sommer T. Role of Cuelp in ubiquitination and degradation at the ER surface. Science.1997,278(5344):1806-1809
[34]Chen B, Mariano J, Tsai YC, et al. The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad Sci U S A.2006,103(2):341-346
[35]Li W, Tu D, Brunger AT, et al. A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate. Nature.2007,446(7133):333-337
[36]Neer EJ. Heterotrimeric G proteins:organizers of transmembrane signals. Cell.1995, 80(2):249-257
[37]Milligan G, Mitchell FM, Mullaney I, et al. The role and specificity of guanine nucleotide binding proteins in receptor-effector coupling. Symp Soc Exp Biol.1990,44:157-172
[38]Silletti S, Raz A. Autocrine motility factor is a growth factor. Biochem Biophys Res Commun.1993,194(1):446-457
[39]Silletti S, Paku S, Raz A. Autocrine motility factor and the extracellular matrix. I. Coordinate regulation of melanoma cell adhesion, spreading and migration involves focal contact reorganization. Int J Cancer.1998,76(1):120-128
[40]Kristiansen K. Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors:molecular modeling and mutagenesis approaches to receptor structure and function. Pharmacol Ther.2004,103(1):21-80
[41]Salahpour A, Angers S, Mercier JF, et al. Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting. J Biol Chem.2004,279(32):33390-33397
[42]Alber T. Structure of the leucine zipper. Curr Opin Genet Dev.1992,2(2):205-10
[43]Franco R, Casado V, Cortes A, et al. Basic concepts in G-protein-coupled receptor homo-and heterodimerization. ScientificWorldJournal.2007,7:48-57
[44]Benlimame N, Simard D, Nabi IR. Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle. J Cell Biol.1995,129(2):459-471
[45]Benlimame N, Le PU, Nabi IR. Localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum. Mol Biol Cell.1998,9(7):1773-1786
[46]Wang HJ, Benlimame N, Nabi I. The AMF-R tubule is a smooth ilimaquinone-sensitive subdomain of the endoplasmic reticulum. J Cell Sci.1997,110 (Pt 24):3043-3053
[47]Wang HJ, Guay G, Pogan L, et al. Calcium regulates the association between mitochondria and a smooth subdomain of the endoplasmic reticulum. J Cell Biol.2000,150(6):1489-1498
[48]Meusser B, Hirsch C, Jarosch E, et al. ERAD:the long road to destruction. Nat Cell Biol. 2005,7(8):766-772
[49]Zhong X, Shen Y, Ballar P, et al. AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation. J Biol Chem. 2004,279(44):45676-45684
[50]Tsai YC, Mendoza A, Mariano JM, et al. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nat Med.2007,13(12):1504-1509
[51]Song BL, Sever N, DeBose-Boyd RA. Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase. Mol Cell.2005,19(6):829-840
[52]Morito D, Hirao K, Oda Y, et al. Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508. Mol Biol Cell.2008,19(4):1328-1336
[53]Liang JS, Kim T, Fang S, et al. Overexpression of the tumor autocrine motility factor receptor Gp78, a ubiquitin protein ligase, results in increased ubiquitinylation and decreased secretion of apolipoprotein B100 in HepG2 cells. J Biol Chem.2003,278(26):23984-23988
[54]Lee JN, Song B, DeBose-Boyd RA, et al. Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78. J Biol Chem.2006,281(51):39308-39315
[55]Cao J, Wang J, Qi W, et al. Ufdl is a cofactor of gp78 and plays a key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase. Cell Metab.2007, 6(2):115-128
[56]Ballar P, Shen Y, Yang H, et al. The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation. J Biol Chem.2006, 281(46):35359-35368
[57]Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem.1998,67:425-479
[58]Sun F, Zhang R, Gong X, et al. Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants. J Biol Chem.2006,281(48):36856-36863
[59]Carlson EJ, Pitonzo D, Skach WR. p97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation. EMBO J.2006, 25(19):4557-4566
[60]Wang B, Heath-Engel H, Zhang D, et al. BAP31 interacts with Sec61 translocons and promotes retrotranslocation of CFTRDeltaF508 via the derlin-1 complex. Cell.2008, 133(6):1080-1092
[61]Ye Y, Shibata Y, Kikkert M, et al. Inaugural Article:Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane. Proc Natl Acad Sci U S A.2005,102(40):14132-14138
[62]Ye Y, Shibata Y, Yun C, et al. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature.2004,429(6994):841-847
[63]Li G, Zhao G, Zhou X, et al. The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor. Proc Natl Acad Sci U S A.2006,103(22):8348-8353
[64]Ballar P, Zhong Y, Nagahama M, et al. Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation. J Biol Chem.2007,282(47):33908-33914.
[65]Ye Y, Meyer HH, Rapoport TA. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature.2001,414(6864):652-656
[66]Bays NW, Hampton RY. Cdc48-Ufdl-Npl4:stuck in the middle with Ub. Curr Biol.2002, 12(10):R366-R371
[67]Ye Y, Meyer HH, Rapoport TA. Function of the p97-Ufdl-Npl4 complex in retrotranslocation from the ER to the cytosol:dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J Cell Biol.2003,162(1):71-84
[68]Meyer HH, Wang Y, Warren G Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufdl-Npl4. EMBO J.2002,21(21):5645-5652
[69]DeLaBarre B, Brunger AT. Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains. Nat Struct Biol.2003,10(10):856-863
[70]Pye VE, Beuron F, Keetch CA, et al. Structural insights into the p97-Ufdl-Npl4 complex. Proc Natl Acad Sci U S A.2007,104(2):467-472
[71]Park S, Isaacson R, Kim HT, et al. Ufdl exhibits the AAA-ATPase fold with two distinct ubiquitin interaction sites. Structure.2005,13(7):995-1005
[72]Protzel C, Kakies C, Kleist B, et al. Down-regulation of the metastasis suppressor protein KAI1/CD82 correlates with occurrence of metastasis, prognosis and presence of HPV DNA in human penile squamous cell carcinoma. Virchows Arch.2008,452(4):369-375
[73]Abe M, Sugiura T, Takahashi M, et al. A novel function of CD82/KAI-1 on E-cadherin-mediated homophilic cellular adhesion of cancer cells. Cancer Lett.2008, 266(2):163-170
[74]Briese J, Schulte HM, Sajin M, et al. Correlations between reduced expression of the metastasis suppressor gene KAI-1 and accumulation of p53 in uterine carcinomas and sarcomas. Virchows Arch.2008,453(1):89-96
[75]Xu JH, Guo XZ, Ren LN, et al. KAI1 is a potential target for anti-metastasis in pancreatic cancer cells. World J Gastroenterol.2008,14(7):1126-1132
[76]Yang JM, Peng ZH, Si SH, et al. KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97-H cells in vitro and in animal models. Liver Int.2008, 28(1):132-139
[77]Voeltz GK, Prinz WA, Shibata Y, et al. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell.2006,124(3):573-586
[78]Rizzuto R, Brini M, Murgia M, et al. Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science.1993,262(5134):744-747.
[79]Szabadkai G, Bianchi K, Varnai P, et al. Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J Cell Biol.2006,175(6):901-911
[80]Ross CA, Meldolesi J, Milner TA, et al. Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature.1989,339(6224):468-470
[81]Pearce MM, Wang Y, Kelley GG, et al. SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors and other substrates in mammalian cells. J Biol Chem.2007,282(28):20104-20115
[82]Le PU, Guay G, Altschuler Y, et al. Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J Biol Chem.2002,277(5):3371-3379
[83]Tsutsumi S, Hogan V, Nabi IR, et al. Overexpression of the autocrine motility factor/phosphoglucose isomerase induces transformation and survival of NIH-3T3 fibroblasts. Cancer Res.2003,63(1):242-249
[84]Kojic LD, Wiseman SM, Ghaidi F, et al. Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase:a potential drug delivery route for tumor cells. PLoS One. 2008,3(10):e3597
[85]Haga A, Funasaka T, Niinaka Y, et al. Autocrine motility factor signaling induces tumor apoptotic resistance by regulations Apaf-1 and Caspase-9 apoptosome expression. Int J Cancer.2003,107(5):707-714
[86]Yanagawa T, Watanabe H, Takeuchi T, et al. Overexpression of autocrine motility factor in metastatic tumor cells:possible association with augmented expression of KIF3A and GDI-beta. Lab Invest.2004,84(4):513-522
[87]Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motility factor signaling enhances pancreatic cancer metastasis. Clin Cancer Res.2004,10(22):7775-7784
[88]Silletti S, Paku S, Raz A. Autocrine motility factor and the extracellular matrix. II. Degradation or remodeling of substratum components directs the motile response of tumor cells. Int J Cancer.1998,76(1):129-135
[89]Yu FL, Liao MH, Lee JW, et al. Induction of hepatoma cells migration by phosphoglucose isomerase/autocrine motility factor through the upregulation of matrix metalloproteinase-3. Biochem Biophys Res Commun.2004,314(1):76-82
[90]Haga A, Funasaka T, Deyashiki Y, et al. Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway. FEBS Lett.2008,582(13):1877-1882
[91]Tsutsumi S, Gupta SK, Hogan V, et al. Activation of small GTPase Rho is required for autocrine motility factor signaling. Cancer Res.2002,62(15):4484-4490
[92]Wang W, Yang LY, Yang ZL, et al. Elevated expression of autocrine motility factor receptor correlates with overexpression of RhoC and indicates poor prognosis in hepatocellular carcinoma. Dig Dis Sci.2007,52(3):770-775
[93]Talukder AH, Bagheri-Yarmand R, et al. Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors. Clin Cancer Res.2002,8(10):3285-3289
[94]Dobashi Y, Watanabe H, Sato Y, et al. Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas. J Pathol.2006,210(4):431-440
[95]Onishi Y, Tsukada K, Yokota J, et al. Overexpression of autocrine motility factor receptor (AMFR) in NIH3T3 fibroblasts induces cell transformation. Clin Exp Metastasis.2003, 20(1):51-58
[96]Otto T, Birchmeier W, Schmidt U, et al. Inverse relation of E-cadherin and autocrine motility factor receptor expression as a prognostic factor in patients with bladder carcinomas. Cancer Res.1994,54(12):3120-3123
[97]Simard D, Nabi IR. Inverse relation of autocrine motility factor receptor and E-cadherin expression following MDCK epithelial cell transformation. Biochem Biophys Res Commun. 1996,219(1):122-127
[98]Nabi IR, Watanabe H, Raz A. Autocrine motility factor and its receptor:role in cell locomotion and metastasis. Cancer Metastasis Rev.1992,11(1):5-20
[99]Silletti S, Raz A. Regulation of autocrine motility factor receptor expression in tumor cell locomotion and metastasis. Curr Top Microbiol Immunol.1996,213 (Pt 2):137-169
[100]Torimura T, Ueno T, Kin M, et al. Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of betal integrins. Hepatology. 2001,34(1):62-71
[101]Funasaka T, Hu H, Yanagawa T, et al. Down-regulation of phosphoglucose isomerase/autocrine motility factor results in mesenchymal-to-epithelial transition of human lung fibrosarcoma cells. Cancer Res.2007,67(9):4236-4243
[102]Funasaka T, Haga A, Raz A, et al. Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility. Biochem Biophys Res Commun.2001, 285(1):118-128
[103]Funasaka T, Haga A, Raz A, et al. Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid. Biochem Biophys Res Commun.2002,293(1):192-200
[104]Gong W, Jiang Y, Wang L, et al. Expression of autocrine motility factor correlates with the angiogenic phenotype of and poor prognosis for human gastric cancer. Clin Cancer Res. 2005, 11(16):5778-5783
[2]Klahn S, Marquardt DM, Rollwitz I, et al. Expression of the ggpPS gene for glucosylglycerol biosynthesis from Azotobacter vinelandii improves the salt tolerance of Arabidopsis thaliana. J Exp Bot,2009,60:1679-1689
[3]Yang Q, Chen ZZ, Zhou XF, et al. Over expression of SOS (Salt Overly Sensitive) genes increases salt tolerance in transgenic Arabidopsis. Mol Plant,2009,2:22-31
[4]Tuteja N. Mechanisms of high salinity tolerance in plants. Methods,2007, Enzymol 428:419-438
[5]Munns R. Genes and salt tolerance:bring them together. New Phytol,2005,167:645-663
[6]Mishra A, Mandoli A, Jha B. Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan. J Ind Microbiol Biotechnol,2008, 35:1093-1101
[7]Liska AJ, Shevchenko A, Pick U, et al. Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics. Plant Physiol,2004,136:2806-2817
[8]Wagner V, Boesger J, Mittag M. Sub-proteome analysis in the green flagellate alga Chlamydomonas reinhardtii. J Basic Microbiol,2009,49(1):32-41
[9]Jia Y, Xue L, Li J, et al. Isolation and proteomic analysis of the halotolerant alga Dunaliella salina flagella using shotgun strategy. Mol Biol Rep,2010,37(2):711-716.
[10]Stauber EJ, Hippler M. Chlamydomonas reinhardtii proteomics. Plant Physiol Biochem, 2004,42(12):989-1001
[11]Mans DA, Voest EE, Giles RH. All along the watchtower:is the cilium a tumor suppressor organelle? Biochim Biophys Acta,2008,1786(2):114-125
[12]Pan J, Wang Q, Snell WJ. Cilium-generated signaling and cilia-related disorders. Laboratory Investigation,2005,85:452-463
[13]Kozminski KG, Johnson KA, Forscher P, et al. A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci USA,1993,90:5519-5523
[14]Watanabe H, Takehana K, Date M, et al. Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res,1996,56(13):2960-2963
[15]Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci U S A,1986,83(10):3302-3306
[16]Yakirevich E, Naot Y. Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol Reprod,2000,62(4):1016-1023
[17]Schulz LC, Bahr JM. Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret. Proc Natl Acad Sci U S A,2003,100(14):8561-8566
[18]Cao MJ, Osatomi K, Matsuda R, et al. Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus). Biochem Biophys Res Commun,2000,272(2):485-489
[19]Yanagawa T, Watanabe H, Takeuchi T, et al. Overexpression of autocrine motility factor in metastatic tumor cells:possible association with augmented expression of KIF3A and GDI-beta. Lab Invest,2004,84(4):513-522
[20]Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motility factor signaling enhances pancreatic cancer metastasis. Clin Cancer Res,2004,10(22):7775-7784
[21]Dobashi Y, Watanabe H, Sato Y, et al. Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas. J Pathol,2006,210(4):431-440
[22]Popova OV, Yang O, Dietz KJ, et al. Differential transcript regulation in Arabidopsis thaliana and the halotolerant Lobularia maritima indicates genes with potential function in plant salt adaptation. Gene,2008,423:142-148
[23]Katz A, Waridel P, Shevchenko A, et al. Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis. Mol Cell Proteomics,2007,6:1459-1472
[24]Goyal A. Osmoregulation in Dunaliella, Part II:Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta. Plant Physiol Biochem, 2007,45:705-710
[25]Jorrin-Novo JV, Maldonado AM, Echevarria-Zomeno S, et al. Plant proteomics update (2007-2008):Second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge. J Proteomics,2009,72(3):285-314
[26]Gonza lez Mariela A, Coleman Annette W, G 6 mez'Patricia I, et al. Phylogenetic relationship among various strains of Dunaliella (Chlorophyceae) based on nuclear ITS rDNA sequences. J Phycol,2001,37:604-611
[27]王伟,许政暟,宋任涛.利用ITS序列对两个盐藻株的分子鉴定.上海大学学报(自然科学版),2006,12(1)：84-88
[28]Ben-Amotz A, Avron M. The biotechnology of cultivating the halotolerant alga Dunaliella. Trends Biotech,1990,8:121-126
[29]张弛,胡鸿钧,李中奎,等.衣藻属的系统发育分析-基于形态形状和nrDNA ITS序列.武汉植物学研究,2000,18(3)：189-194
[30]Gomez Patricia I, Gonzalea Mariela A. Genetic variation among seven strains of Dunaliella salina (Chlorophyta) with industrial potential, based on RAPD banding patterns and on nuclear ITS rDNA sequences. Aquaculture,2004,233:149-161
[31]Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics,2004,4(12):3665-3685
[32]李义良,朱华清,张红锋.双向电泳技术.生物学教学,2004,29(4)：64
[33]Rabilloud T. Solubilization of proteins for electrophoretic analyses. Electrophoresis,1996, 17(5):813-829
[34]Chambers G, Lawrie L, Cash P, et al. Proteomics:a new approach to the study of disease. J Pathol,2000,192(3):280-288
[35]Zhou M, Veenstra TD. Proteomic analysis of protein complexes. Proteomics,2007, 7(16):2688-2697
[36]Lopez JL. Two-dimensional electrophoresis in proteome expression analysis. J Chromatogr B Analyt Technol Biomed Life Sci,2007,849(1-2):190-202
[37]Bodzon-Kulakowska A, Bierczynska-Krzysik A, Dylag T, et al. Methods for samples preparation in proteomic research. J Chromatogr B Analyt Technol Biomed Life Sci,2007, 49(1-2):1-31
[38]Berkelman T. Quantitation of protein in samples prepared for 2-D electrophoresis. Methods Mol Biol,2008,424:43-49
[39]Marengo E, Robotti E, Bobba M.2D-PAGE maps analysis. Methods Mol Biol,2008, 428:291-325
[40]Pan C, Xu S, Zhou H, et al. Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS. Anal Bioanal Chem,2007,387(1):193-204
[41]Bonk T, Humeny A. MALDI-TOF-MS analysis of protein and DNA. Neuroscientist,2001, 7(1):6-12
[42]Grauvogel C, Brinkmann H, Petersen J. Evolution of the glucose-6-phosphate isomerase:the plasticity of primary metabolism in photosynthetic eukaryotes. Mol Biol Evol,2007, 24:1611-1621
[43]Katz LA. Transkingdom transfer of the phosphoglucose isomerase gene. J Mol Evol,1996 43:453-459
[44]Goyal A, Brown AD, Gimmler H. Regulation of salt-induced starch degradation in Dunaliella tertiolecta. J Plant Physiol,1987,127:77-96
[45]Chen H, Jiang JG. Osmotic responses of Dunaliella to the changes of salinity. J Cell Physiol, 2009,219:251-258
[46]Jithesh MN, Prashanth SR, Sivaprakash KR, et al. Antioxidative response mechanisms in halophytes:their role in stress defence. J Genet,2006,85:237-254
[47]Ray S, Agarwal P, Arora R, et al. Expression analysis of calcium-dependent protein kinase gene family during reproductive development and abiotic stress conditions in rice (Oryza sativa L. ssp. indica). Mol Genet Genomics,2007,278:493-505
[48]Stepien P, Johnson GN. Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella:role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol,2009,149:1154-1165
[49]Wang X, Yang P, Gao Q, et al. Proteomic analysis of the response to high-salinity stress in Physcomitrella patens. Planta,2008,228:167-177
[50]范吉星,邓用川,黄惜等.红海榄根部总蛋白质提取方法的改进和双向电泳体系的建立.湖南农业大学学报(自然科学版),2008,34(5)：549-553
[51]Phillips TL, Porter DW, Gracy RW. Nature of the multiple forms of sweet-potato glucose phosphate isomerase. Biochem J.1975,147(2):381-384
[52]Faik P, Walker JI, Redmill AA, et al. Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences. Nature.1988,332(6163):455-457
[53]Chaput M, Claes V, Portetelle D, et al. The neurotrophic factor neuroleukin is 90% homologous with phosphohexose isomerase. Nature.1988,332(6163):454-455
[54]Wang R, Okamoto M, Xing X,et al. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol.2003, 132(2):556-567
[55]Munjral N, Gupta AK, Kaur N. Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules. Indian J Biochem Biophys.2007,44(3):186-189
[56]Cordenunsi BR, Oliveira do Nascimento JR, Vieira da Mota R, et al. Phosphoglucose isomerase from bananas:partial characterization and relation to main changes in carbohydrate composition during ripening. Biosci Biotechnol Biochem,2001, 65(10):2174-2180
[57]Yu TS, Lue WL, Wang SM, et al. Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation. Plant Physiol,2000,123(1):319-326
[58]Funasaka T, Raz A. The role of autocrine motility factor in tumor and tumor microenvironment. Cancer Metastasis Rev,2007,26(3-4):725-735
[59]Yanagawa T, Funasaka T, Tsutsumi S, et al. Novel roles of the autocrine motility factor/phosphoglucose isomerase in tumor malignancy. Endocr Relat Cancer,2004, 11(4):749-59
[60]Chiu CG, St-Pierre P, Nabi IR, et al. Autocrine motility factor receptor:a clinical review. Expert Rev Anticancer Ther,2008,8(2):207-217
[61]Pazour GJ, Agrin N, Leszyk J et al. Proteomic analysis of a eukaryotic cilium. J Cell Biol, 2005,170:103-113
[62]卢圣栋.现代分子生物学实验技术.中国协和医科大学出版社,1999年12月出版.第二版
[63]J 萨姆布鲁克,D W 拉塞尔.分子克隆实验指南.科学出版社,2002年8月第三版
[64]Cordeiro AT, Michels PA, Delboni LF, et al. The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features. Eur J Biochem,2004, 271(13):2765-2772
[65]Sun YJ, Chou CC, Chen WS, et al. The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin. Proc Natl Acad Sci U S A. 1999,96(10):5412-5417
[66]Yamamoto H, Miwa H, Kunishima N. Crystal structure of glucose-6-phosphate isomerase from Thermus thermophilus HB8 showing a snapshot of active dimeric state. J Mol Biol, 2008,382(3):747-762
[67]Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods,2001,25:402-408
[68]Oren A. A hundred years of Dunaliella research:1905-2005. Salina systems,2005,1:2
[69]Borowitzka LJ, Brown AD. The salt relations of marine and halophilic species of the unicellular green alga, Dunaliella. The role of glycerol as a compatible solute. Arch Microbiol,1974,96:37-52
[70]Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci USA,1986,83(10):3302-3306
[71]Shimizu K, Tani M, Watanabe H, et al. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett,1999,456(2):295-300
[72]Haga A, Tanaka N, Funasaka T, et al. The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF. J Mol Biol,2006,358(3):741-753
[73]Funasaka T, Hogan V, Raz A. Phosphoglucose isomerase/autocrine motility factor mediates epithelial and mesenchymal phenotype conversions in breast cancer. Cancer Res,2009, 69(13):5349-5356
[74]Shih WL, Liao MH, Yu FL, et al. AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration. Cancer Lett,2008,270(2):202-217
[75]Luo Y, Luo Y, Long JM, et al. A link between maze learning and hippocampal expression of neuroleukin and its receptor gp78. J Neurochem,2002,80(2):354-361
[76]Merchant SS, Prochnik SE, Vallon O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science,2007,318(5848):245-250
[77]Olsen BR. When primary cilia fail. Matrix Biol,2009,8(8):443-444
[78]Nigg EA, Raff JW. Centrioles, centrosomes, and cilia in health and disease. Cell,2009, 139(4):663-678
[79]Baker K, Beales PL. Making sense of cilia in disease:the human ciliopathies. Am J Med Genet C Semin Med Genet,2009,151:281-295
[80]Hurd TW, Margolis B. Cystin, cilia, and cysts:unraveling trafficking determinants. J Am Soc Nephrol,2009,20(12):2485-2486
[81]Doherty D. Joubert syndrome:insights into brain development, cilium biology, and complex disease. Semin Pediatr Neurol,2009,16(3):143-154
[82]Roy S. The motile cilium in development and disease:emerging new insights. Bioessays, 2009,31(7):694-699
[83]Lancaster MA, Gleeson JG. The primary cilium as a cellular signaling center:lessons from disease. Curr Opin Genet Dev,2009,19(3):220-229
[84]Veland IR, Awan A, Pedersen LB, et al. Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol,2009,111(3):39-53
[85]Lee YL, Li TT. Crystallization and preliminary crystallographic study of the phosphoglucose isomerase from Bacillus subtilis. Acta Crystallogr Sect F Struct Biol Cryst Commun.2008, 64:1181-1183
[86]Arsenieva D, Appavu BL, Mazock GH, et al. Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution. Proteins, 2009,74(1):72-80
[87]Lee JH, Jeffery CJ. The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate. Protein Sci,2005,14(3):727-734
[88]Andzhaparidze OG, Bektemirov TA, Burgasova MP. Influence of a complex of poly(I):poly(C) and poly-l-lysine on the course of vaccinia in monkeys. Vopr Virusol,1977, (3):339-343
[89]Anthony LS, Stevenson MM, Skamene E. Enhancement of resistance to Listeria monocytogenes infection in mice by pyrimidine analogs. Clin Invest Med,1984, 7(4):343-348
[90]Stiehm ER, Keller MA, Vyas GN. Preparation and use of therapeutic antibodies primarily of human origin. Biologicals,2008,36(6):363-374
[91]Khatri I, Forstner G, Forstner J. Preparation of polyclonal antibodies to native and modified mucin antigens. Methods Mol Biol,1993,14:225-235
[92]Mayne R. Preparation and applications of monoclonal antibodies to different collagen types. Clin Biochem,1988,21(2):111-115
[93]Xu W, Chiao JW. Biochemical characteristics of a human myeloid leukemia cell differentiation factor. Prep Biochem Biotechnol,1996,26(1):21-30
[94]Gurney ME, Heinrich SP, Lee MR, et al. Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons. Science,1986,234(4776):566-574
[95]Yakirevich E, Naot Y. Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol Reprod,2000,62(4):1016-1023
[96]Schulz LC, Bahr JM. Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret. Proc Natl Acad Sci U S A,2003,100(14):8561-8566
[97]Cao MJ, Osatomi K, Matsuda R, et al. Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus). Biochem Biophys Res Commun,2000,272(2):485-489
[98]Repiso A, Andres R, Climent F, et al. Expression patterns in mouse embryos of neuroleukin/glucose-6-phosphate isomerase and autocrine motility factor receptor. Anat Histol Embryol,2008,37(5):380-382
[99]Fairbank M, St-Pierre P, Nabi IR. The complex biology of autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and its receptor, the gp78/AMFR E3 ubiquitin ligase. Mol Biosyst,2009,5(8):793-801
[100]Tsutsumi S, Hogan V, Nabi IR, et al. Overexpression of the autocrine motility factor/phosphoglucose isomerase induces transformation and survival of NIH-3T3 fibroblasts. Cancer Res.2003,63(1):242-249
[101]Yanagawa T, Watanabe H, Takeuchi T, et al. Overexpression of autocrine motility factor in metastatic tumor cells:possible association with augmented expression of KIF3A and GDI-beta. Lab Invest.2004,84(4):513-522
[102]Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motility factor signaling enhances pancreatic cancer metastasis. Clin Cancer Res.2004,10(22):7775-7784
[103]Silletti S, Paku S, Raz A. Autocrine motility factor and the extracellular matrix. II. Degradation or remodeling of substratum components directs the motile response of tumor cells. Int J Cancer.1998,76(1):129-135
[104]Yu FL, Liao MH, Lee JW, et al. Induction of hepatoma cells migration by phosphoglucose isomerase/autocrine motility factor through the upregulation of matrix metalloproteinase-3. Biochem Biophys Res Commun.2004,314(1):76-82
[105]Haga A, Funasaka T, Deyashiki Y, et al. Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway. FEBS Lett.2008,582(13):1877-1882
[106]Tsutsumi S, Gupta SK, Hogan V, et al. Activation of small GTPase Rho is required for autocrine motility factor signaling. Cancer Res.2002,62(15):4484-4490
[107]Talukder AH, Bagheri-Yarmand R, et al. Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors. Clin Cancer Res.2002,8(10):3285-3289
[108]Dobashi Y, Watanabe H, Sato Y, et al. Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas. J Pathol.2006,210(4):431-440
[1]Chiu CG, St-Pierre P, Nabi IR, et al. Autocrine motility factor receptor:a clinical review. Expert Rev Anticancer Ther.2008,8(2):207-217
[2]Fairbank M, St-Pierre P, Nabi IR. The complex biology of autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and its receptor, the gp78/AMFR E3 ubiquitin ligase. Mol Biosyst.2009,5(8):793-801
[3]Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci U S A.1986,83(10):3302-3306
[4]Watanabe H, Takehana K, Date M, et al. Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res.1996,56(13):2960-2963
[5]Kim JW, Dang CV. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci.2005, 30(3):142-150
[6]Amraei M, Nabi IR. Species specificity of the cytokine function of phosphoglucose isomerase. FEBS Lett.2002,525(1-3):151-155
[7]Arsenieva D, Jeffery CJ. Conformational changes in phosphoglucose isomerase induced by ligand binding. J Mol Biol.2002,323(1):77-84
[8]Tsutsumi S, Gupta SK, Hogan V, et al. The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function. FEBS Lett.2003,534(1-3):49-53
[9]Xu W, Chiao JW. Biochemical characteristics of a human myeloid leukemia cell differentiation factor. Prep Biochem Biotechnol.1996,26(1):21-30
[10]Gurney ME, Heinrich SP, Lee MR, et al. Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons. Science.1986,234(4776):566-574
[11]Yakirevich E, Naot Y. Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol Reprod.2000,62(4):1016-1023
[12]Schulz LC, Bahr JM. Glucose-6-phosphate isomerase is necessary for embryo implantation in the domestic ferret. Proc Natl Acad Sci U S A.2003,100(14):8561-8566
[13]Cao MJ, Osatomi K, Matsuda R, et al. Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus). Biochem Biophys Res Commun.2000,272(2):485-489
[14]Repiso A, Andres R, Climent F, et al. Expression patterns in mouse embryos of neuroleukin/glucose-6-phosphate isomerase and autocrine motility factor receptor. Anat Histol Embryol.2008,37(5):380-382
[15]Baumann M, Kappl A, Lang T, et al. The diagnostic validity of the serum tumor marker phosphohexose isomerase (PHI) in patients with gastrointestinal, kidney, and breast cancer. Cancer Invest.1990,8(34):351-356
[16]Niinaka Y, Paku S, Haga A, et al. Expression and secretion of neuroleukin/phosphohexose isomerase/maturation factor as autocrine motility factor by tumor cells. Cancer Res.1998, 58(12):2667-2674
[17]Rubartelli A, Cozzolino F, Talio M, et al. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J.1990,9(5):1503-1510
[18]Lagana A, Duchaine T, Raz A, et al. Expression of autocrine motility factor/phosphohexose isomerase in Cos7 cells. Biochem Biophys Res Commun.2000,273(l):213-278
[19]Yanagawa T, Funasaka T, Tsutsumi S, et al. Differential regulation of phosphoglucose isomerase/autocrine motility factor activities by protein kinase CK2 phosphorylation. J Biol Chem.2005,280(11):10419-10426
[20]Yanagawa T, Funasaka T, Tsutsumi S, et al. Regulation of phosphoglucose isomerase/autocrine motility factor activities by the poly(ADP-ribose) polymerase family-14. Cancer Res.2007,67(18):8682-8689
[21]Nabi IR, Raz A. Cell shape modulation alters glycosylation of a metastatic melanoma cell-surface antigen. Int J Cancer.1987,40(3):396-402
[22]Nabi IR, Raz A. Loss of metastatic responsiveness to cell shape modulation in a newly characterized B16 melanoma adhesive cell variant. Cancer Res.1988,48(5):1258-1264
[23]Nabi IR, Watanabe H, Raz A. Identification of B16-F1 melanoma autocrine motility-like factor receptor. Cancer Res.1990,50(2):409-414
[24]Registre M, Goetz JG, St Pierre P, et al. The gene product of the gp78/AMFR ubiquitin E3 ligase cDNA is selectively recognized by the 3F3A antibody within a subdomain of the endoplasmic reticulum. Biochem Biophys Res Commun.2004,320(4):1316-1322
[25]Goetz JG, Genty H, St-Pierre P, et al. Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca2+ levels. J Cell Sci.2007,120(Pt 20):3553-3564
[26]Watanabe H, Nabi IR, Raz A. The relationship between motility factor receptor internalization and the lung colonization capacity of murine melanoma cells. Cancer Res. 1991,51(10):2699-2705
[27]Haga A, Komazaki S, Funasaka T, et al. AMF/G6PI induces differentiation of leukemic cells via an unknown receptor that differs from gp78. Leuk Lymphoma.2006,47(10):2234-2243
[28]Shimizu K, Tani M, Watanabe H, et al. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett.1999,456(2):295-300
[29]Mishra S, Raz A, Murphy LJ. Insulin-like growth factor binding protein-3 interacts with autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and inhibits the AMF/PGI function. Cancer Res.2004,64(7):2516-2522
[30]Lagana A, Goetz JG, Y N, et al. pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate. J Cell Sci.2005, 118(Pt 18):4175-4185
[31]Watanabe H, Carmi P, Hogan V, et al. Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor. J Biol Chem.1991,266(20):13442-13448
[32]Ponting CP. Proteins of the endoplasmic-reticulum-associated degradation pathway:domain detection and function prediction. Biochem J.2000,351(Pt 2):527-535
[33]Biederer T, Volkwein C, Sommer T. Role of Cuelp in ubiquitination and degradation at the ER surface. Science.1997,278(5344):1806-1809
[34]Chen B, Mariano J, Tsai YC, et al. The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad Sci U S A.2006,103(2):341-346
[35]Li W, Tu D, Brunger AT, et al. A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate. Nature.2007,446(7133):333-337
[36]Neer EJ. Heterotrimeric G proteins:organizers of transmembrane signals. Cell.1995, 80(2):249-257
[37]Milligan G, Mitchell FM, Mullaney I, et al. The role and specificity of guanine nucleotide binding proteins in receptor-effector coupling. Symp Soc Exp Biol.1990,44:157-172
[38]Silletti S, Raz A. Autocrine motility factor is a growth factor. Biochem Biophys Res Commun.1993,194(1):446-457
[39]Silletti S, Paku S, Raz A. Autocrine motility factor and the extracellular matrix. I. Coordinate regulation of melanoma cell adhesion, spreading and migration involves focal contact reorganization. Int J Cancer.1998,76(1):120-128
[40]Kristiansen K. Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors:molecular modeling and mutagenesis approaches to receptor structure and function. Pharmacol Ther.2004,103(1):21-80
[41]Salahpour A, Angers S, Mercier JF, et al. Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting. J Biol Chem.2004,279(32):33390-33397
[42]Alber T. Structure of the leucine zipper. Curr Opin Genet Dev.1992,2(2):205-10
[43]Franco R, Casado V, Cortes A, et al. Basic concepts in G-protein-coupled receptor homo-and heterodimerization. ScientificWorldJournal.2007,7:48-57
[44]Benlimame N, Simard D, Nabi IR. Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle. J Cell Biol.1995,129(2):459-471
[45]Benlimame N, Le PU, Nabi IR. Localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum. Mol Biol Cell.1998,9(7):1773-1786
[46]Wang HJ, Benlimame N, Nabi I. The AMF-R tubule is a smooth ilimaquinone-sensitive subdomain of the endoplasmic reticulum. J Cell Sci.1997,110 (Pt 24):3043-3053
[47]Wang HJ, Guay G, Pogan L, et al. Calcium regulates the association between mitochondria and a smooth subdomain of the endoplasmic reticulum. J Cell Biol.2000,150(6):1489-1498
[48]Meusser B, Hirsch C, Jarosch E, et al. ERAD:the long road to destruction. Nat Cell Biol. 2005,7(8):766-772
[49]Zhong X, Shen Y, Ballar P, et al. AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation. J Biol Chem. 2004,279(44):45676-45684
[50]Tsai YC, Mendoza A, Mariano JM, et al. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nat Med.2007,13(12):1504-1509
[51]Song BL, Sever N, DeBose-Boyd RA. Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase. Mol Cell.2005,19(6):829-840
[52]Morito D, Hirao K, Oda Y, et al. Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508. Mol Biol Cell.2008,19(4):1328-1336
[53]Liang JS, Kim T, Fang S, et al. Overexpression of the tumor autocrine motility factor receptor Gp78, a ubiquitin protein ligase, results in increased ubiquitinylation and decreased secretion of apolipoprotein B100 in HepG2 cells. J Biol Chem.2003,278(26):23984-23988
[54]Lee JN, Song B, DeBose-Boyd RA, et al. Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78. J Biol Chem.2006,281(51):39308-39315
[55]Cao J, Wang J, Qi W, et al. Ufdl is a cofactor of gp78 and plays a key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase. Cell Metab.2007, 6(2):115-128
[56]Ballar P, Shen Y, Yang H, et al. The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation. J Biol Chem.2006, 281(46):35359-35368
[57]Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem.1998,67:425-479
[58]Sun F, Zhang R, Gong X, et al. Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants. J Biol Chem.2006,281(48):36856-36863
[59]Carlson EJ, Pitonzo D, Skach WR. p97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation. EMBO J.2006, 25(19):4557-4566
[60]Wang B, Heath-Engel H, Zhang D, et al. BAP31 interacts with Sec61 translocons and promotes retrotranslocation of CFTRDeltaF508 via the derlin-1 complex. Cell.2008, 133(6):1080-1092
[61]Ye Y, Shibata Y, Kikkert M, et al. Inaugural Article:Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane. Proc Natl Acad Sci U S A.2005,102(40):14132-14138
[62]Ye Y, Shibata Y, Yun C, et al. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature.2004,429(6994):841-847
[63]Li G, Zhao G, Zhou X, et al. The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor. Proc Natl Acad Sci U S A.2006,103(22):8348-8353
[64]Ballar P, Zhong Y, Nagahama M, et al. Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation. J Biol Chem.2007,282(47):33908-33914.
[65]Ye Y, Meyer HH, Rapoport TA. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature.2001,414(6864):652-656
[66]Bays NW, Hampton RY. Cdc48-Ufdl-Npl4:stuck in the middle with Ub. Curr Biol.2002, 12(10):R366-R371
[67]Ye Y, Meyer HH, Rapoport TA. Function of the p97-Ufdl-Npl4 complex in retrotranslocation from the ER to the cytosol:dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J Cell Biol.2003,162(1):71-84
[68]Meyer HH, Wang Y, Warren G Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufdl-Npl4. EMBO J.2002,21(21):5645-5652
[69]DeLaBarre B, Brunger AT. Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains. Nat Struct Biol.2003,10(10):856-863
[70]Pye VE, Beuron F, Keetch CA, et al. Structural insights into the p97-Ufdl-Npl4 complex. Proc Natl Acad Sci U S A.2007,104(2):467-472
[71]Park S, Isaacson R, Kim HT, et al. Ufdl exhibits the AAA-ATPase fold with two distinct ubiquitin interaction sites. Structure.2005,13(7):995-1005
[72]Protzel C, Kakies C, Kleist B, et al. Down-regulation of the metastasis suppressor protein KAI1/CD82 correlates with occurrence of metastasis, prognosis and presence of HPV DNA in human penile squamous cell carcinoma. Virchows Arch.2008,452(4):369-375
[73]Abe M, Sugiura T, Takahashi M, et al. A novel function of CD82/KAI-1 on E-cadherin-mediated homophilic cellular adhesion of cancer cells. Cancer Lett.2008, 266(2):163-170
[74]Briese J, Schulte HM, Sajin M, et al. Correlations between reduced expression of the metastasis suppressor gene KAI-1 and accumulation of p53 in uterine carcinomas and sarcomas. Virchows Arch.2008,453(1):89-96
[75]Xu JH, Guo XZ, Ren LN, et al. KAI1 is a potential target for anti-metastasis in pancreatic cancer cells. World J Gastroenterol.2008,14(7):1126-1132
[76]Yang JM, Peng ZH, Si SH, et al. KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97-H cells in vitro and in animal models. Liver Int.2008, 28(1):132-139
[77]Voeltz GK, Prinz WA, Shibata Y, et al. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell.2006,124(3):573-586
[78]Rizzuto R, Brini M, Murgia M, et al. Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science.1993,262(5134):744-747.
[79]Szabadkai G, Bianchi K, Varnai P, et al. Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J Cell Biol.2006,175(6):901-911
[80]Ross CA, Meldolesi J, Milner TA, et al. Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature.1989,339(6224):468-470
[81]Pearce MM, Wang Y, Kelley GG, et al. SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors and other substrates in mammalian cells. J Biol Chem.2007,282(28):20104-20115
[82]Le PU, Guay G, Altschuler Y, et al. Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J Biol Chem.2002,277(5):3371-3379
[83]Tsutsumi S, Hogan V, Nabi IR, et al. Overexpression of the autocrine motility factor/phosphoglucose isomerase induces transformation and survival of NIH-3T3 fibroblasts. Cancer Res.2003,63(1):242-249
[84]Kojic LD, Wiseman SM, Ghaidi F, et al. Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase:a potential drug delivery route for tumor cells. PLoS One. 2008,3(10):e3597
[85]Haga A, Funasaka T, Niinaka Y, et al. Autocrine motility factor signaling induces tumor apoptotic resistance by regulations Apaf-1 and Caspase-9 apoptosome expression. Int J Cancer.2003,107(5):707-714
[86]Yanagawa T, Watanabe H, Takeuchi T, et al. Overexpression of autocrine motility factor in metastatic tumor cells:possible association with augmented expression of KIF3A and GDI-beta. Lab Invest.2004,84(4):513-522
[87]Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motility factor signaling enhances pancreatic cancer metastasis. Clin Cancer Res.2004,10(22):7775-7784
[88]Silletti S, Paku S, Raz A. Autocrine motility factor and the extracellular matrix. II. Degradation or remodeling of substratum components directs the motile response of tumor cells. Int J Cancer.1998,76(1):129-135
[89]Yu FL, Liao MH, Lee JW, et al. Induction of hepatoma cells migration by phosphoglucose isomerase/autocrine motility factor through the upregulation of matrix metalloproteinase-3. Biochem Biophys Res Commun.2004,314(1):76-82
[90]Haga A, Funasaka T, Deyashiki Y, et al. Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway. FEBS Lett.2008,582(13):1877-1882
[91]Tsutsumi S, Gupta SK, Hogan V, et al. Activation of small GTPase Rho is required for autocrine motility factor signaling. Cancer Res.2002,62(15):4484-4490
[92]Wang W, Yang LY, Yang ZL, et al. Elevated expression of autocrine motility factor receptor correlates with overexpression of RhoC and indicates poor prognosis in hepatocellular carcinoma. Dig Dis Sci.2007,52(3):770-775
[93]Talukder AH, Bagheri-Yarmand R, et al. Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors. Clin Cancer Res.2002,8(10):3285-3289
[94]Dobashi Y, Watanabe H, Sato Y, et al. Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas. J Pathol.2006,210(4):431-440
[95]Onishi Y, Tsukada K, Yokota J, et al. Overexpression of autocrine motility factor receptor (AMFR) in NIH3T3 fibroblasts induces cell transformation. Clin Exp Metastasis.2003, 20(1):51-58
[96]Otto T, Birchmeier W, Schmidt U, et al. Inverse relation of E-cadherin and autocrine motility factor receptor expression as a prognostic factor in patients with bladder carcinomas. Cancer Res.1994,54(12):3120-3123
[97]Simard D, Nabi IR. Inverse relation of autocrine motility factor receptor and E-cadherin expression following MDCK epithelial cell transformation. Biochem Biophys Res Commun. 1996,219(1):122-127
[98]Nabi IR, Watanabe H, Raz A. Autocrine motility factor and its receptor:role in cell locomotion and metastasis. Cancer Metastasis Rev.1992,11(1):5-20
[99]Silletti S, Raz A. Regulation of autocrine motility factor receptor expression in tumor cell locomotion and metastasis. Curr Top Microbiol Immunol.1996,213 (Pt 2):137-169
[100]Torimura T, Ueno T, Kin M, et al. Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of betal integrins. Hepatology. 2001,34(1):62-71
[101]Funasaka T, Hu H, Yanagawa T, et al. Down-regulation of phosphoglucose isomerase/autocrine motility factor results in mesenchymal-to-epithelial transition of human lung fibrosarcoma cells. Cancer Res.2007,67(9):4236-4243
[102]Funasaka T, Haga A, Raz A, et al. Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility. Biochem Biophys Res Commun.2001, 285(1):118-128
[103]Funasaka T, Haga A, Raz A, et al. Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid. Biochem Biophys Res Commun.2002,293(1):192-200
[104]Gong W, Jiang Y, Wang L, et al. Expression of autocrine motility factor correlates with the angiogenic phenotype of and poor prognosis for human gastric cancer. Clin Cancer Res. 2005, 11(16):5778-5783