Truncation attenuates molecular chaperoning and apoptosis inhibition by p26, a small heat shock protein from Artemia franciscana

The small heat shock proteins (sHSPs), which prevent irreversible protein denaturation and inhibit apoptosis, consist of an amino-terminus, the canonical α-crystallin domain, and a carboxy-terminal extension. It remains difficult, however, to define sHSP structure-function relationships and with this in mind p26, an sHSP from the crustacean Artemia franciscana, was truncated by deletion mutagenesis. Wild-type p26 cDNA and three truncated variants inserted into the eukaryotic expression vector pcDNA3.1/HisC were used to generate stably transfected 293H cells. p26 shielded transfected cells against death upon exposure to heat and oxidative stress. Truncation reduced chaperone activity, with cells synthesizing the p26 α-crystallin domain being the least resistant. Wild-type p26 inhibited apoptosis in transfected cells, with protection against oxidation-generated apoptosis being more effective than that against heat-induced apoptosis. Truncation reduced p26 apoptotic inhibitory activity, with the α-crystallin domain again being the least effective. The results show that a crustacean sHSP functions effectively in mammalian cells, demonstrating interchangeability of these proteins between distantly related organisms and indicating similarities in their mechanisms of action. Moreover, maximal activity was observed for full-length p26, indicating that structural elements required for chaperone activity and apoptosis inhibition reside throughout the protein.     

Development of recombinant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> producing virus-like particles of a fish nervous necrosis virus

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.     

Expression of Streptococcus mutans wall-associated protein A gene in Chinese hamster ovary cells: prospect for a dental caries DNA vaccine.

The Streptococcus mutans strain GS-5 wall-associated protein A (Wap-A) is a precursor to the extracellular antigen A (AgA), a recognized candidate dental caries vaccine. The full-length wapA gene (wapA-E) and a C-terminal truncated version (wapA-G) encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The resulting constructs were propagated in the Escherichia coli Top10. To investigate the expression of the S. mutans genes in mammalian cells, the above constructs were used to transfect Chinese hamster ovary (CHO) cells in the presence of the cationic lipid pfx-8. Transient expression of the wapA-E and wapA-G genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis using a rabbit antiserum to S. mutans cell wall. Immunochemical staining of the transfected CHO cells showed expression of WapA mainly in the cells and budding vesicles, whereas AgA was found mainly in the transfected cells and extracellular medium. The expression of S. mutans proteins in CHO cells, in either vesicles or soluble form, suggested an antibody response to the above DNA constructs. Work is under way to test the efficacy of these as DNA vaccines against S. mutans.     

Antitumor effects of a xenogeneic survivin bone marrow derived dendritic cell vaccine against murine GL261 gliomas

Survivin is a member of the inhibitor of apoptosis protein family. Gliomas and many other tumors express survivin at high levels; whereas, normal fully differentiated cells generally do not. Therefore, survivin represents a tumor-specific target for cancer vaccine therapy. It has been shown that it is possible to produce a MHC-I-restricted cellular immunologic response to survivin vaccines. To study differences in immunogenicity between murine and human survivin proteins, we vaccinated C57BL/6 mice with bone marrow dendritic cells (BMDC) transfected with expression vectors containing the murine and human survivin genes. Mice vaccinated with BMDCs expressing a truncated human survivin protein developed cytotoxic T lymphocyte to subcutaneous GL261 glioma cells and exhibited prolonged tumor-free survival compared to mice vaccinated with BMDCs transfected with vector alone (P<0.01). While mice challenged with intracerebral GL261 cells had increased survival, no cures were observed. In contrast, vaccinated mice that fully resisted subcutaneous tumor challenge were rendered resistant to intracerebral GL261 re-challenge. BMDCs transfected with the full-length human survivin molecule were significantly more effective at prolonging survival than BMDCs expressing the full-length murine survivin gene (P=0.0175). Therefore, xenogeneic differences between human and murine sequences might be exploited to develop more immunogenic tumor vaccines.     

幽门螺杆菌毒素蛋白CagA诱导的蛋白的差异表达分析及其基因在胃癌组织中的表达

为了研究胃癌细胞中幽门螺杆菌(Hp)毒素蛋白CagA诱导的蛋白差异表达及其基因在人胃癌组织中的表达,用Hp感染胃癌细胞系SGC 7901和AGS及用含CagA基因的表达载体稳定转染SGC 7901细胞, 构建3组实验模型.提取各组细胞的总蛋白进行双向凝胶电泳,筛选3组重叠的差异表达蛋白质斑点进行质谱鉴定.共获得135个差异表达的蛋白质,其中上调蛋白质73个,下调蛋白质62个. 鉴定出10个差异表达蛋白质, 其中有6个差异表达蛋白是首次发现,它们主要参与细胞的能量代谢和信号转导等.最后定量检测了这10个差异表达蛋白基因在人胃癌组织中的表达, 发现有4个基因高表达和1个基因低表达. 本结果将为研究幽门螺杆菌感染引起胃癌的分子机制提供新的线索.     

Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains

The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains.     

Constitutive c-myb expression in K562 cells inhibits induced erythroid differentiation but not tetradecanoyl phorbol acetate-induced megakaryocytic differentiation.

K562 cells were stably transfected with a plasmid vector constitutively expressing a full-length human c-myb gene. Parental cells possess the dual potential of inducibility of cellular differentiation along two lineages, i.e., erythroid and megakaryocytic. The resulting lineage is dependent on the inducing agent, with a number of compounds being competent to various degrees for inducing erythroid differentiation, while the tumor promoter tetradecanoyl phorbol acetate (TPA) induces a macrophage-like morphology with enhanced expression of proteins associated with megakaryocytes. Exogeneous expression of c-myb in transfected cell lines abrogated erythroid differentiation induced by cadaverine or cytosine arabinoside as assessed by hemoglobin production. However, TPA-induced megakaryocytic differentiation was left intact, as assessed by cell morphology, cytochemical staining, and the expression of the megakaryocytic antigens. These results indicate that c-Myb and protein kinase C play important roles in cellular differentiation of K562 cells and suggest that agents which directly modulate protein kinase C can induce differentiation in spite of constitutively high levels of c-Myb.     

Activities of the EM10 protein from Echinococcus multilocularis in cultured mammalian cells demonstrate functional relationships to ERM family members

The ezrin-radixin-moesin (ERM) homolog EM10 is expressed by the larval stage of the parasite E. multilocularis and shows 46.9% overall identity in the primary structure with human ezrin. To determine whether EM10 has similar activities to ERM proteins, we investigated properties of the protein expressed in mammalian cells. In particular, we transiently expressed haemagglutinin-tagged (HA-tagged) versions of the full-length EM10 as well as the amino- and the carboxy-terminal halves of EM10 in HtTA-1 cells. In addition we stably transfected NIH-3T3 cells with untagged full-length EM10. The data demonstrate that EM10 polypeptides behave like their corresponding portions of radixin when transiently expressed in mammalian cells. The full-length and amino-terminal EM10 polypeptides were localized to cortical structures. Cells expressing the carboxy-terminal polypeptide of EM10 showed long actin-filled protrusions. Cells expressing full-length EM10 showed a reduction in endogenous moesin-staining at cortical structures. In stably transfected NIH-3T3 cells EM10 was not crisply localized but rather was diffuse throughout the cytoplasm. These cells showed a conspicuous loss of stress-fibers, a phenotype that was not seen in analogous experiments with ERM proteins. The results demonstrate both similarities and differences between the functional properties of EM10 and ERM proteins expressed in vertebrate cells.     

The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain

The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.     

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.     

Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.     

Down-regulation of heat shock protein 27 in neuronal cells and non-neuronal cells expressing mutant ataxin-3

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. Unstable CAG trinucleotide repeat expansion in the MJD gene has been identified as the pathologic mutation of MJD. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD with 78 CAG repeats were established. Compared with the parental cells, cells expressing mutant ataxin-3 displayed normal morphology for over 80 generations. Less than 1% of the transfected cells contained nuclear aggregates under basal conditions, indicating that this cellular model represented an early disease stage. While t-butyl hydroperoxide (TBH) was used to assess the oxidative tolerance of cells, the results demonstrated that the transfected cells were more susceptible to low concentrations of TBH than the parental cells. Most interestingly, from 2D gel electrophoresis analysis, we identified that the expression of heat shock protein 27 (HSP27), known as a suppressor of poly(Q)-mediated cell death, dramatically decreased in SK-N-SH cells stably transfected with full-length mutant MJD. The same reduction of HSP27 was further confirmed in lymphoblastoid cells from MJD patients. Our results demonstrated that both neuronal and non-neuronal cells with expanded full-length ataxin-3 revealed reduced protein expression of HSP27. We propose that the reduction of HSP27 in the early stage of the disease plays an important role during cell death process in MJD.     

The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain.

The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined. Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal. Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein. These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources. Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies. A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry. The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells. Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation. Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells. Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells. Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells. Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.     

Overexpressed human mitochondrial thioredoxin confers resistance to oxidant-induced apoptosis in human osteosarcoma cells

Oxidative damage to mitochondria is a central mechanism of apoptosis induced by many toxic chemicals. Thioredoxin family proteins share a conserved Cys-X-X-Cys motif at their active center and play important roles in control of cellular redox state and protection against oxidative damage. In addition to the well studied cytosolic and extracellular form (Trx1), rat and avian mitochondrial forms of thioredoxin (mtTrx) have been reported. In this study, we cloned the full-length human mtTrx cDNA and performed localization and functional studies in 143B human osteosarcoma cells. The coding sequence of human mtTrx consists of a region with homology to Trx1 as well as a putative mitochondrial localization signal (MLS) at its N terminus. In stably transfected cell lines, mtTrx had a mitochondrial localization as measured by subcellular fractionation studies and by confocal fluorescence microscopy. Deletion of the MLS rendered mtTrx to be solely expressed in the cytosolic fraction. On SDS-PAGE, transfected mtTrx had the same apparent molecular weight as the MLS truncated form, indicating that the leader sequence is cleaved during or after mitochondrial import. Treatment with the oxidant tert-butylhydroperoxide induced apoptosis in 143B cells. This oxidant-induced apoptosis was inhibited by overexpressing the full-length mtTrx in 143B cells. Thus, human mtTrx is a member of the thioredoxin family of proteins localized to mitochondria and may play important roles in protection against oxidant-induced apoptosis.     

The use of chimeric gene constructs to express a bacterial endoglucanase in mammalian cells.

The synthesis and secretion of a truncated Clostridium thermocellum endoglucanase (EGE') encoded by the celE' gene was investigated in Chinese hamster ovary (CHO) cells. Fusion genes consisting of the human growth hormone (hGH) gene and celE', transcribed from the SV40 early enhancer/promoter, were constructed and stably transfected into CHO cells. A gene consisting of celE' inserted into the first exon of the hGH gene resulted in the synthesis of truncated proteins (less than or equal to 22 kDa) lacking endoglucanase activity. Cloning celE' into the second exon of the hGH gene, resulted in the synthesis and secretion of a 50 kDa protein with endoglucanase activity. A 50 kDa protein was also synthesised by cells transfected with celE' cloned into the fifth exon of the hGH gene. However, despite a 5-fold increase in enzyme activity compared to the exon 2 transfected cell line less than 40% of the protein was secreted. Constructs devoid of introns, in which celE' was fused to the SV40 early promoter and to the rabbit beta-globin polyadenylation sequence resulted in a 2-18-fold increase in endoglucanase activity compared to the constructs containing introns. In addition more than 75% of the synthesised protein was secreted. Analyses of EGE' encoded mRNA from the transfected cell lines suggests that the presence of introns results in the aberrant splicing of message by the use of cryptic splice sites in the celE' gene. These results demonstrate that introns are not required for the efficient expression of a bacterial endoglucanase in mammalian cells, rather introns appear to reduce expression of the encoded protein.     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

KDM3A全长及截短质粒构建及其在细胞中的定位探究

赖氨酸去甲基化酶3A(lysine demethylase 3A,KDM3A)是组蛋白去甲基化酶家族成员之一,通过特异性对组蛋白H3K9甲基化进行去甲基化的作用调控基因转录。目前研究证实KDM3A在肝癌、雌激素受体(estrogen receptorα,ERα)阳性乳腺癌及前列腺癌等肿瘤中发挥促癌作用,但其具体作用机制仍有待更深层次的研究。为深入解析KDM3A在各种肿瘤中的作用及其分子机制,本研究利用分子生物学方法构建了KDM3A基因的全长和截短基因表达质粒,酶切鉴定及测序证实KDM3A全长及截短基因的表达质粒构建成功。在此基础上,再将构建成功的上述多种表达质粒分别转染到HEK293细胞和MCF7细胞中,进行Western blotting和免疫荧光共聚焦实验,进一步对各重组表达质粒在培养的哺乳动物细胞中的蛋白表达及蛋白细胞定位进行初步探究。Western blotting结果显示KDM3A全长基因表达的蛋白约147 kD,其他KDM3A截短基因表达的蛋白均对应其特异性的蛋白条带;免疫荧光共聚焦实验结果显示KDM3A全长质粒以及pN1、pN2、p N3、pΔ截短质粒表达的蛋白分布在细胞核,pC1、pC2截短质粒表达的蛋白分布在细胞质,pC3截短质粒表达的蛋白分别分布于细胞核与细胞质。本实验为后续深入研究KDM3A在肿瘤中的作用及分子机制提供了实验基础。     

High level expression of monomeric and dimeric human alpha1,3-fucosyltransferase V

alpha3/4-Fucosyltransferases play a crucial role in inflammatory processes and tumor metastasis. While several human fucosyltransferases (FucTs) with different acceptor substrate specificities have been identified, the design of specific inhibitors for therapeutic approaches is hampered by the lack of structural information. In this study, we evaluated the expression of different constructs of human fucosyltransferase V to generate the large amounts required for structural studies. The truncated constructs lacking the transmembrane region and the cytosolic N-terminus, were expressed in baculovirus-infected Trichoplusia ni (Tn) insect cells and in two non-lytic expression systems, stably transfected human HEK 293 and T. ni cells. Since secretion of some glycosyltransferases is controlled by formation of dimeric molecules via disulfide bonds, one of the fucosyltransferase V constructs contained the N-terminal cysteine residue 64 for dimerization, whereas this residue was replaced in the other construct by serine. In both human and insect cells dimerization did not prove to be essential for efficient expression and secretion. On the basis of enzymatic activity, the yield of secreted fucosyltransferase V was approximately 10-fold higher in stably transfected insect cells than in HEK 293 cells. In particular the monomeric form of the enzyme provides a valuable tool for structural analyses to elucidate the fine specifity of fucosyltransferase V-mediated fucosylation of Lewis type glycans.