Baculovirus-infected Spodoptera Frugiperda Cells Research Articles

Systematic Mutagenesis of Potential Glycosylation Sites of Lysosomal Acid Lipase

Lysosomal acid lipase (LAL; EC 3.1.1.13) is a key enzyme in the intracellular lipid metabolism. It hydrolyzes exogenous triglycerides and cholesterol esters taken up by various cell types. LAL has six potential N-glycosylation sites and one potential O-glycosylation site. Elimination of each of the six Asn-(X)-Ser/Thr sites by site-directed mutagenesis and expression in baculovirus-infected Spodoptera frugiperda cells resulted in two single-mutant enzymes without lipolytic activities (N134Q and N246Q) and four mutants with preserved activities. The two inactive mutants were not detectable on immunoblot analysis, indicating that they were not secreted. Six double mutants in all possible combinations except for the two inactive single mutants were produced and expressed. Double mutants in combination with the N9 glycosylation site showed reduced activities as compared to the other mutants or the wild-type enzyme. Kinetic data of LAL glycosylation mutants indicate that substrate affinity of N9Q was not changed, but k (cat) of N9 mutants was reduced distinctly compared to the wild-type enzyme. Peanut agglutinin lectin did not recognize LAL, demonstrating that the protein has no core1 structure (Galbeta 1-3 GalNAc) of O-glycosylation. These data indicate that at least two of the six N-glycosylation sites are used in native lipase. N134 and N246 were found to be essential for LAL activity. We conclude that glycosylation plays an important role in the formation of functional LAL.

P53 autoantibodies in sera from Danish ovarian cancer patients and their correlation with clinical data and prognosis.

The p53 gene, a tumour suppressor gene located on the short arm of chromosome 17 (17p13), is frequently mutated in various human tumours. Accumulation of p53 protein in neoplastic cells and its release following tumour necrosis can lead to development of circulating autoantibodies (AAb) against p53. Earlier studies of ovarian cancer (OC) patients reported different frequencies of p53 AAb and conclusions regarding the clinical and prognostic value of these AAb have not been in agreement. We therefore analysed for the presence of p53 AAb in a total of 227 preoperative serum samples from 193 OC patients and 34 patients with ovarian borderline tumours, and, in addition, serum samples from 86 healthy controls. An enzyme-linked immunosorbent assay (ELISA) was used to measure serum IgG antibodies against p53. The p53 protein used in the assay was produced as a hexahistidine-tagged fusion protein by baculovirus-infected Spodoptera frugiperda cells. Cut-off values for p53 AAb were evaluated, and correlations of p53 AAb with clinical-, biochemical data and survival were examined. We found a low sensitivity for p53 AAb alone, and no major additional effect of the detection rate of CA125 was found. No significant associations were found between p53 AAb and clinical stage, age, histological subtype and radicality after primary surgery. In contrast, we found significantly elevated CA125 levels in p53 AAb-positive patients compared to lower CA125 levels in p53 AAb-negative patients (p=0.003). No significant differences were found between p53 AAb-positive and p53 AAb-negative patients in the univariate and multivariate survival analyses. In conclusion, in a screening study for OC serum p53 AAb levels are of no diagnostic value, even if combined with the tumour marker CA125. The presence of increased serum p53 AAb in patients with diagnosed OC could not be correlated with any clinical data and preoperative serum p53 AAb status had no evident value.

Cloning and expression of a novel juvenile hormone-metabolizing epoxide hydrolase during larval-pupal metamorphosis of the cabbage looper, Trichoplusia ni.

A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity. TmEH-1 was 1887 base pairs in length with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH III epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH III epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH III as compared to the general EH substrates, cis- and trans-stilbene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.

Histidine Tagging Both Allows Convenient Single-step Purification of Bovine Rhodopsin and Exerts Ionic Strength-dependent Effects on Its Photochemistry

For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15-20 pmol/10(6) cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of > or = 35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/A500 nm = 2.5 +/- 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 +/- 1 nm) compared to wild type rhodopsin (498 +/- 1 nm). The pK alpha of the Meta I<==>Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho-->Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x.

A study of uninfected and baculovirus-infected Spodoptera frugiperda cells in T- and spinner flasks

Insect cells have been propagated in monolayers in T-flasks or in suspension culture in spinner flasks, the latter being conducted over a range of spinner speeds. In both configurations, the cells were also infected with either wild or recombinant β-galactosidase baculovirus at MOI of 0.1, 1 and 10. The strength of both uninfected and infected cells was also measured by a micro-manipulation technique. No significant difference in growth rate was obtained between monolayer culture and suspension culture at the spinner rate which was optimum for growth. This optimum was quite sharp. At the lowest speeds cells settled, whilst above the optimum speed the spinner action led to significant cell damage. The maximum infectivity was obtained at this optimum speed which also gave maximum survival after infection. There were significant changes of cell survival and infection, even over relatively small changes of speed, and presumably energy dissipation rate. As changes in growth in turbine-agitated bioreactors have been shown to be much less, even when the energy inputs varied by two orders of magnitude, these findings throw doubt on the usefulness of spinner flasks for assessing “shear” sensitivity of cell lines. The percentage of infected cells and β-galactosidase production were significantly lower in the monolayer culture compared to that in the suspension culture at MOI values below 10 pfu/cell. This difference is explained as being due to the reduced movement of released virus particles from infected to non-infected cells in the T-flasks.

Overproduction and purification of Mycobacterium tuberculosis chaperonin 10.

The overexpression of the Mycobacterium tuberculosis chaperonin 10 (Cpn10)-encoding gene was accomplished using baculovirus expression vectors. The product was immunoreactive with a Cpn10 monoclonal antibody (mAb) and had an electrophoretic mobility identical to authentic Cpn10. The baculovirus system was most successful in terms of reaching nearly the full expression potential of the system. Recombinant Cpn10 was purified from recombinant baculovirus-infected Spodoptera frugiperda cells by isoelectrofocussing and size-exclusion chromatography. The baculovirus vector and purification methodology described represent a very powerful system for the large-scale production of the M. tuberculosis Cpn10 which may allow us to undertake structure-function analysis.

Characterization of the Replication of Plasmids Containing hr Sequences in Baculovirus-Infected Spodoptera frugiperda Cells

The replication of a series of plasmids, each containing one or more of 6 homologous regions ( hrs) from the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) genome, was investigated in AcMNPV-infected Spodoptera frugiperda cells. Although a construct containing hr2 showed the most efficient replication, the sequences flanking the hr region appeared to influence the levels of replication. Plasmids with non- hr-containing AcMNPV inserts showed greatly reduced replication relative to hr-containing plasmids. A variety of features of hr -containing plasmid replication were characterized. These included the time course of plasmid replication and the influence of plasmid concentration and multiplicity of infection of the helper AcMNPV on replication efficiency. It was found that replicated plasmid DNA was detectable by 36 hr p.i. and plateaued by 60 hr p.i. Under our experimental conditions, plasmid levels of 0.5-2 μg/1.25 × 10 6 cells and multiplicities of infection of 0.1 to 10 produced optimal levels of plasmid replication. The transfection procedure was shown to delay viral DNA replication by about 12 hr. Experiments directed toward determining the form of the replicated hr -containing plasmid DNA were conducted using partial restriction enzyme digestion. The results indicated that the AcMNPV-replicated plasmids were composed of high molecular weight concatemers.

Breaching the conformational integrity of the catalytic triad of the serine protease plasmin: localized disruption of a side chain of His-603 strongly inhibits the amidolytic activity of human plasmin.

Site-directed mutagenesis has been used to construct a cDNA that encodes a recombinant variant human plasminogen (hPg) containing a Pro-611-->Ile mutation (MrhPg). The mutein was expressed in recombinant baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE), and purified. After activation of this zymogen to its corresponding form of the serine protease plasmin (MrhPm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteration of the spatial relationships of the active-site His-603 to its partners of the catalytic triad, Asp-646 and Ser-741. Partial amidolytic activity of MrhPm was restored as a consequence of imidazole addition to the assay medium, due to an increase in the catalytic constant kcat of the enzyme. The serine protease inhibitor, diisopropylphosphofluoridate, when preincubated with MrhPm, did not inhibit restoration of its amidolytic activity with imidazole, whereas diisopropylphosphofluoridate did inhibit the amidolytic activity of MrhPm in the presence of imidazole. This result implies that His-603 directly influences the nucleophilic character of Ser-741. When imidazole as pretreated with alpha-N-tosyl-L-lysine chloromethyl ketone, the ability of this imidazole solution to restore amidolytic activity to MrhPm was eliminated, suggesting that N alpha-(p-tosyl)lysine chloromethyl ketone directs into the binding pocket a derivatized form of imidazole, which is ineffective as an His-603 substitute. These results indicate that the conformational reorientation of His-603 results in a malfunctional catalytic triad in the serine protease MrhPm, thus leading to an inactive enzyme despite the presence of all three essential amino acids of the catalytic triad. Addition of extramolecular imidazole restores a portion of the amidolytic activity of this mutant enzyme. These data also argue for an enzyme mechanism in which the active-center His-603 residue directly influences the nucleophilicity of the active-site Ser 741 residue.

.alpha.-Mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells

Incubation of a Spodoptera frugiperda (IPLB-SF-21AE) cell extract with the oligosaccharide Man9GlcNAc2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant Asn residue in the nascent protein, results in its trimming to Man6GlcNAc2, an intermediate that is relatively stable to further alpha-D-mannosidase action in these cells. On the other hand, incubation of a similar extract from cells that had been infected for various times with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) or a recombinant baculovirus (r-BAC)/human plasminogen (HPg) construct employed for expression of HPg led to rapid trimming of Man6GlcNAc2 to Man5GlcNAc2 and Man3GlcNAc2. These latter reactions displayed temporal effects, in that an enhancement of this latter trimming process occurred as a function of the time of infection of the cells with the wild-type and recombinant viral constructs. We have previously demonstrated that the nature of the oligosaccharide assembled on Asn289 of HPg expressed in several lepidopteran insect cell lines was dependent on the time of infection of the cells with r-BAC/HPg and that the amount of complex glycan found on this recombinant protein increased with an increase in infection times [Davidson, D. J., & Castellino, F. J. (1991) Biochemistry 30, 6167-6174].(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells.

As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies.

Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells

Proper assembly of nucleocapsids of the baculovirus Autographa californica nuclear polyhedrosis virus is prevented by cytochalasin D, a drug that interferes with actin microfilament function. To investigate the involvement of microfilaments in A. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. Tetramethylrhodamine isothiocyanate-labeled phalloidin was used to label microfilaments, and monoclonal antibody was used to label p39, the major viral capsid protein. Three microfilament arrangements were found in infected cells. During uptake of virus, thick cables were formed. These were insensitive to cycloheximide, indicating that this configuration was a rearrangement of preexisting cellular actin mediated by a component of the viral inoculum. At the time of cell rounding and before viral DNA replication, ventral aggregates of actin were observed. These were sensitive to cycloheximide but not to aphidicolin, indicating that an early viral gene mediated this actin rearrangement. Ventral aggregates did not result from the rounding process itself. Uninfected cells prerounded with colchicine did not form ventral aggregates. Cells prerounded with colchicine and then infected did form aggregates. At the time of exponential production of progency virus, microfilaments were found in the nucleus surrounding the virogenic stroma. In this area (where nucleocapsid assembly is known to take place) microfilaments colocalized with p39. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis identified p39 among proteins retained on an f-actin affinity column. We postulate that microfilaments in the nucleus provide a scaffold to position capsids for proper assembly and filling with DNA.

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.

Expression of snowshoe hare bunyavirus S RNA coding proteins by recombinant baculoviruses

Recombinant baculoviruses have been constructed that express the two snowshoe hare (SSH) bunyavirus proteins coded in overlapping reading frames of the SSH S viral-complementary RNA species (namely the nucleoprotein, N, and the nonstructural protein, NS s). The 26.5 kDa N protein, which is read from the first AUG of the mRNA containing the SSH S sequence, was expressed at a high level (estimated to be ca 40% of the stained cellular proteins in recombinant baculovirusinfected Spodoptera frugiperda cells). This level of expression was much higher than that of the 10.5 kDa NS s protein made at the same time (estimated to be < 1% of the stained proteins), presumably due in part to lower levels of translation initiation from the second AUG (19 nucleotides downstream). Ba131 nuclease digestion was used to delete the first ATG of the SSH DNA sequence in the baculovirus transfer vector and BamHI was used to remove downstream N coding sequences. A second recombinant baculovirus was constructed from the products that only expressed the SSH NS s protein. The yield of NS s protein was estimated to be of the order of ca 2% of the stained cellular proteins. A third recombinant transfer vector made from the products of the Bal31 digestion, fortuitously possessed a new ATG 8–10 nucleotides upstream of the NS s ATG. A recombinant virus derived from this vector synthesized essentially similar quantities (ca 2% each) of both the NS s protein and a 16.7 kDa N-related product.