家蚕微孢子虫极管蛋白1 (NbPTP1) 在果蝇S2细胞中的表达与糖基化修饰

极管蛋白(Polar tube protein)是极管的主要成分,能特异性定位于微孢子虫极管,在微孢子虫侵染宿主过程中发挥重要作用。文中分析了家蚕微孢子虫极管蛋白1中潜在的O-、N-糖基化修饰位点,克隆了家蚕微孢子虫极管蛋白1全基因序列,并将其插入带有V5和His标签的真核表达载体pMT/Bip/V5-His A中,成功构建了pMT/Bip/V5-His A-NbPTP1重组质粒,经转染果蝇S2细胞后,发现NbPTP1基因能在果蝇细胞中高效表达。此外,Lectin blotting和β-消除反应分析结果表明:果蝇S2细胞内表达的NbPTP1具有O-糖基化修饰特征。以上结果为研究NbPTP1的糖基化修饰特征与其功能之间的关系提供了基础,有助于揭示微孢子虫侵染机制,建立可行有效的微孢子虫病诊断和防治措施。     

Lectin Binding Assays for In-Process Monitoring of Sialylation in Protein Production

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.     

Identification of a Microsporidian Polar Tube Protein Reactive Monoclonal Antibody

ABSTRACT. The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.     

The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnaires''s disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.     

Y-box结合蛋白功能及对肿瘤发生的影响

Y-box结合蛋白家族成员是一类高度保守的顺式作用元件, 广泛存在于原核及真核生物细胞中。它是一种多功能蛋白, 与转录调节、翻译调控、mRNA选择性剪接、DNA的修复、细胞增殖和再生等有关。Y-box结合蛋白的氨基酸序列包含3个结构域: 氨基酸N末端, 亲水结构域C末端, 冷休克结构域(cold shock domain CSD), 保守的冷休克结构域决定了Y-box结合蛋白的大部分功能。最近研究发现, 定位于细胞核中的YB-1蛋白在局部晚期非小细胞肺癌的预防上可作为新的靶位点, YB-1蛋白还可通过对抑癌基因p53启动子抑制起负调控作用, 此外, YB-1蛋白在PI3K/Akt信号通路中也起到重要的作用, 这些研究都为肿瘤的治疗提供了新的线索和启示。文章就Y-box结合蛋白功能及其对肿瘤发生的影响等方面进行概述。     

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia)

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed‐sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.     

DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

The highly purified DNA Pol- from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase- showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231–6237].The catalytic polypeptide, DNA polymerase- of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase- from embryonic chicken brain (ECB) contains an -galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase- reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567–573] by the treatment with methyl--galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (M _r = 186 kDa) with an -galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA.Pol- as determined by immunostaining with the polymerase--specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA- and a complete separation of polymerase complex and primase.     

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.     

光间受体视黄类物质结合蛋白基因(IRBP)在棉鼠亚科物种中的序列变异及其系统发育意义

以光间受体视黄类物质结合蛋白基因(IRBP)为研究对象,从GenBank中下载了棉鼠亚科66个种的IRBP序列,分析IRBP的结构及其在棉鼠亚科的进化关系.G+C含量高于A+T,密码子第3位点的G+C含量高达61.2%,并且呈现出较低的核苷酸多样性(3.786%).IRBP在棉鼠亚科没有经历正向达尔文选择.以IRBP构建的系统发育树和以往研究的结果总体上是一致的,但由于本文样本数量的增加,发现Oryzomyini还存在第3个分支,不同于以往研究只划分了2个分支.同时系统发育树的结构表明Ichthyomyini和Sigmodontini很可能不是两个独立的族.另外和前人以细胞色素b基因构建的系统发育树相比较,树的结构总体上也是一致的.这些都表明IRBP在棉鼠亚科中有较好的应用效果,但要更好地解决此亚科存在的系统发育问题,重点在于增加用于分析的物种数量.     

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu³⁵⁶ to Thr³⁷⁰) of the JD in the presence of calcium (Ca²⁺). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.     

Structure of the Non-Catalytic Domain of the Protein Disulfide Isomerase-Related Protein (PDIR) Reveals Function in Protein Binding

Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein (also known as PDIA5) is a specialized member that participates in the folding of α₁-antitrypsin and N-linked glycoproteins. Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates.     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的     

Role of Oxysterol Binding Protein in Hepatitis C Virus infection

Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (ΔPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.Hepatitis C virus (HCV) infection is one of the leading causes of chronic hepatitis. HCV infection is associated with cirrhosis, steatosis, and hepatocellular carcinoma (33). The HCV RNA genome of ∼9.6 kb is translated via an internal ribosome entry site element on the rough endoplasmic reticulum (ER) as a polyprotein precursor of about 3,010 amino acids that is co- and posttranslationally processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins (33). HCV replicates within ribonucleoprotein (RNP) complexes associated with modified ER membranous structures (15). Recent work implicated lipid droplets that emanate from the ER as sites of RNA replication (28, 44). Almost all of the HCV NS proteins along with a variety of cellular factors are associated with the RNP complexes engaged in viral RNA replication (37). It is likely that these NS proteins not only participate in replication process but also are involved in the various steps of virion morphogenesis and assembly. Membrane-associated RNP complexes are generally composed of viral proteins, replicating RNA, host proteins, and altered cellular membranes (1). In this respect, a growing body of evidence implicates the functional role of NS5A in early steps of virion assembly and morphogenesis (3, 27, 45). NS5A is a phosphoprotein that migrates in sodium dodecyl sulfate gels as 56-kDa (basally phosphorylated) and 58-kDa (hyperphosphorylated) forms of proteins. The C-terminal domain III region of NS5A and the phosphorylated residue (Ser⁴⁵⁷) are important for virion maturation (3, 27, 45). NS5A domain III contains the binding site for viral core protein, indicating the possible involvement of NS5A protein in virus assembly (27). NS5A anchors to the ER membrane by an N-terminal hydrophobic α-helix, and this attachment is needed for its key role(s) in viral replication (10). Studies suggest that phosphorylation of NS5A plays a functional role in viral replication (12). The hyperphosphorylated NS5A reduces its interaction with the human vesicle-associated membrane protein-associated protein A (VAP-A) (12). VAP-A binds both NS5A and NS5B (13, 17). These associations are important for RNA replication (13, 17).HCV alters lipid homeostasis to benefit its infectious processes. Host lipids and their synthesis affect viral infectious process (21, 40, 51, 57). HCV RNA replication can be induced by saturated and monounsaturated fatty acids and inhibited by polyunsaturated fatty acids (18, 21). HCV gene expression induces lipogenesis by stimulating the activation of the sterol regulatory element binding proteins, the master regulators of lipid/fatty acid biosynthetic pathways (51). Reagents that interfere with host lipid biosynthetic pathways abrogate viral replication (21, 57). It has been suggested that HCV utilizes the very-low-density lipoprotein (VLDL) secretion pathway for its viral particle release (14, 19). These studies collectively suggest that host lipid metabolism plays a key role in the viral life cycle including replication, virion assembly, and secretion (56).In the present study, we focus on the functional role of oxysterol binding protein (OSBP) that was identified by proteomic analysis as one of the host factors associated with the HCV RNP complexes. OSBP belongs to a family of the OSBP-related proteins. Originally discovered as a major cytosolic receptor for oxidized cholesterols, it undergoes translocation from the cytosolic/vesicular compartment to the Golgi apparatus upon ligand (hydroxycholesterol) binding (38). OSBP also binds to VAP-A via its FFAT motif (53). Golgi apparatus translocation of OSBP is regulated by the pleckstrin homology (PH) domain. This domain also harbors binding sites for phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PI4,5P₂) (25). OSBP and OSBP-related proteins are implicated in cholesterol homeostasis, phospholipid metabolism, vesicular transport, and cell signaling (55). OSBP functions as sterol sensor that regulates the transport of ceramide from the ER to the Golgi apparatus for de novo synthesis of sphingomyelin by coordinated action with ceramide transport protein (CERT) (36). OSBP also functions as a scaffolding protein for two phosphatases (phosphatase 2A/HePTP) (49). This complex regulates the activity of extracellular signal-regulate kinase. This cytosolic 440-kDa complex disassembles by the addition of 25-hydroxycholesterol (25-HC) or depletion of cholesterol, both of which cause OSBP translocation to the Golgi compartment (49). Thus, in addition to its role in intracellular trafficking, OSBP appears to regulate cell signaling. We investigated the functional significance of OSBP association with HCV RNP complexes. RNA interference studies support a functional role of OSBP in virion morphogenesis and release process. The OSBP PH domain deletion mutant (ΔPH) failed to localize to the Golgi apparatus and caused an inhibition of the HCV particle release. Our work described herein also demonstrates that the association of OSBP with NS5A may also contribute to the overall HCV maturation process.     

The Structure of Myristoylated Mason-Pfizer Monkey Virus Matrix Protein and the Role of Phosphatidylinositol-(4,5)-Bisphosphate in Its Membrane Binding

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C₈ fatty acid chains was monitored by observation of concentration‐dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in ³¹P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein–phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a ¹³C-filtered/¹³C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P₂ binding was not strong enough for triggering of the myristoyl‐switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.     

Cloning and Sequencing of a Lectin Protein Gene from the Roots of Sophora flavescens (in English)

A lectin protein（SFL） with molecular weight about 32 kD which markedly agglutinated rabbit and human red blood cells was purified from the roots of Sophora flavescens Ait. This protein, and apparently inhibited the growth of Fusarium vasinfectum Atk., Gibberella saubinetii (Mont.) Sacc., and Piricularia oryzae Cav. A set of degenerate PCR primer was synthesized according to the N-terminal sequence of the purified protein. The full-length cDNA coding the lectin was cloned by RT-PCR and 5′-RACE and sequenced (GenBank AF285121). The deduced amino acid sequence indicates that a preprotein with 284 amino acid residues is firstly translated and then processed to a mature protein with 254 amino acids. A N-Glycosylation site is the Asn 182 residue.      

A Survey of Lectin Binding in Paramecium1

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA₆₀) and agglutinin (RCA₁₂₀), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.