O-glycosylation potential of lepidopteran insect cell lines

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.     

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.     

Establishment and characterization of insect cell lines from 10 lepidopteran species

Summary Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer,Ostrinia nubilalis, a plutellid, the diamondback moth,Plutella xylostella, as well as eight noctuids: the black cutworm,Agrotis ipsilon, the celery looper,Anagrapha falcifera, the velvetbean caterpillar,Anticarsia gemmatalis, the corn earworm,Helicoverpa zea, the tobacco budworm,Heliothis virescens, the beet armyworm,Spodoptera exigua, the fall armyworm,Spodoptera frugiperda, and the cabbage looper,Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines fromA. ipsilon andH. virescens being adapted to suspension culture, using shaker flasks. The potential use for these cell lines in baculovirus production is discussed. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion sex, age, marital status, or handicap.     

Replication of an entomopoxvirus in two lepidopteran cell lines

Pseudaletia separata entomopoxvirus replicated in two lepidopteran cell lines, SIE-MSH-805-F and BM-N. Microscopic examination, and the virus passage tests, of infected cultures indicated that the virus replicated more readily in the former cell line. Virus release by exocytosis occurred in both cell lines. A sequence of virus morphogenesis in the cultured cells was described, based on electron microscopic observations of thin sections. The nucleus of infected cells contained spherical inclusions, and the cytoplasm contained virions, immature virus forms, spheroids, and spindles. A portion of the virions in the cytoplasm was occluded within spheroids, which were often associated with crystallogenic matrix. Virions acquired a coat prior to their occlusion.     

Baculovirus infection influences host protein expression in two established insect cell lines

We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus-host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and semi-permissive to H. zea single nucleopolyhedrovirus (HzSNPV). Hz-AM1 cells are non-permissive to AcMNPV. We challenged each cell line with baculovirus infection and after 24 h determined protein identities by MALDI TOF/TOF mass spectrometry. For Hv-AM1 cells, 21 proteins were identified, and for Hz-AM1 cells, 19 proteins were newly identified (with 8 others having been previously identified). In the permissive relationship, 18 of the proteins changed in expression by 70% or more in AcMNPV infected Hv-AM1 cells as compared with non-infected controls; 12 were significantly decreased and 6 cellular proteins were significantly increased. We also identified 3 virus-specific proteins. In the semi-permissive infections, eight proteins decreased by 2-fold or more. Non-permissive interactions did not lead to substantial changes in host cell protein expression. We hypothesize that some of these proteins act in determining host cell specificity for baculoviruses.     

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA

Summary Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R², IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.     

Modulation of trehalase activity in two insect cell lines by virus infection and trehalose

Trehalase (EC 3.2.1.28), an important glycosidase involved in regulating trehalose levels and metabolic energy in insects, was measured in cell lines from fall army worm, Spodoptera frugiperda and salt marsh caterpillar, Estigmene acrea, treated with either glucose or trehalose in the presence or absence of Tipula Iridescent Virus (TIV), a cytoplasmic deoxyribovirus. In medium containing 15-35 mM trehalose, both of these cells increased their trehalase activities by 4.5 to 8x the basal levels from cells in glucose medium. Trehalase activity was rapidly reduced after cells were exposed to TIV. Maximum loss in activity (70-90%), occurring about the same time as peak viral DNA synthesis, was significantly delayed when cells were pre-incubated with 30 mM trehalose. These experiments demonstrate the potential utility of trehalase as a marker for monitoring stresses induced by viral infection and changes in nutrition.     

雷帕霉素对二种鳞翅目昆虫细胞自噬和凋亡的影响

以2种鳞翅目昆虫细胞为材料,采用雷帕霉素进行处理,初步研究自噬作用与昆虫细胞凋亡的关系。结果表明:雷帕霉素能够提高家蚕细胞系BMN-e细胞的自噬水平,并能诱导BMN-e细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤能抑制雷帕霉素诱导的BMN-e细胞凋亡。相反,雷帕霉素虽能诱导斜纹夜蛾细胞系SL-HP细胞的自噬水平提高,但不能诱导斜纹夜蛾Spodoptera litura(Fabricius)细胞发生凋亡;雷帕霉素的预处理能抑制放线菌素D诱导的斜纹夜蛾细胞系SL-HP细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤对放线菌素D诱导的细胞凋亡没有影响。因此家蚕Bombyx mori细胞自噬水平的提高与细胞凋亡具有正相关性,而斜纹夜蛾细胞自噬水平的提高与细胞凋亡不相关,相反还对细胞凋亡的诱导具有一定的抑制作用。     

Induction, selection and antibacterial activity of the antibacterial peptides from lepidopteran insect cultured cell lines

We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coli DH_5α to stimulate the antibacterial peptide followed by antibacterial activity assay, induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment. The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest, and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation. A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 cells in 16 hours after stimulation. Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus, Escherichia coli K₁₂D₃₁ and Salmonella derby. It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli K₁₂D₃₁ and Salmonella derby. __________ Translated from Journal of Central China Normal University (Natural Sciences), 2006, 40(2): 240–243 [译自: 华中师范大学学报 (自然科学版]     

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.     

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: Large scale production of the E1 protein

The two envelope glycoproteins of rubella virus (RV), El of 58 kDa and E2 of 42–47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10−1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.     

Antioxidant defense systems of two lipidopteran insect cell lines

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O(2)(*-)) to hydrogen peroxide (H(2)O(2)) and ascorbate peroxidase (APOX) for reducing the resulting H(2)O(2) to H(2)O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (approximately two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H(2)O(2) to H(2)O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H(2)O(2). Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

Distinct N-glycan fucosylation potentials of three lepidopteran cell lines.

The fucosyltransferase activities of three insect cell lines, MB-0503 (from Mamestra brassicae), BM-N (from Bombyx mori) and Sf-9 (from Spodoptera frugiperda), were investigated and compared with that of honeybee venom glands. Cell extracts and venom gland extracts were incubated with GDP-[14C]fucose and glycopeptides isolated from human IgG and from bovine fibrin. The labeled oligosaccharide products were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, fluorescence marked with 2-aminopyridine and analyzed both by reversed-phase and size-fractionation HPLC. They were identified by their elution positions before and after exoglycosidase treatment in comparison with standard oligosaccharides. These experiments revealed distinct fucosylation potentials in the three cell lines tested. While MB-0503 cells, like honeybee venom glands, are able to transfer fucose into alpha 1-3 and alpha 1-6 linkage to the innermost N-acetylglucosamine, only alpha 1-6-fucosyl linkages were detected with BM-N and Sf-9 cells.     

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.     

Enhancement of 5-Aminolevulinic acid-induced oxidative stress on two cancer cell lines by gold nanoparticles

Abstract

5-Aminolevulinic acid (5-ALA) and its methyl ester (5-ALA-Me) at mM concentration levels induce oxidative stress via the production of reactive oxygen species (ROS). Human cancer cell lines (MCF-7 and HepG2) incubated in the dark in the simultaneous presence of 5.0 mM or more 5-ALA or 5-ALA-Me (for MCF-7) and 7 µg/mL of 15 nm citrate capped gold nanoparticles (AuNPs) were damaged more seriously compared to those in the presence of the levulinic acid alone. Damage is visible in electron micrographs which reveal similar morphology both in the presence or absence of AuNPs. Cytotoxicity was observed irrespective of the presence of serum and medium. Production of ROS in cell free samples containing 5-ALA-Me was monitored by EPR as the DMPO-OH spin adduct and also showed a catalytic effect of AuNPs. Both SOD and CAT inhibited the production of ROS and also reduced cytotoxicity in the cell samples. These observations can be explained by initial attack on the cell membrane by ROS produced in the medium outside the cell and provide insight into possible uses of 5-ALA in cancer chemotherapy.     

Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators

Background

Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone.

Results

The antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA.

Conclusion

Recombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.