Investigating the secretory pathway of the baculovirus-insect cell system using a secretory green fluorescent protein.

The secretory pathway is important in actively transporting proteins into the extracellular environment of eucaryotic cells. In this study a green fluorescent protein (GFP) mutant engineered to contain a secretion signal was used as a model protein in order to visualize the secretion process inside insect cells. Fluorescent microscopy indicated that significant amounts of secreted green fluorescent protein (sGFP) accumulated in High-Five, Trichoplusia ni, cells following infection with a baculovirus vector containing the gene under the polyhedrin promoter. Laser scanning confocal microscopy was used to reconstruct whole cell images of the infected High-Five cells at multiple days postinfection. While the protein was widely distributed at 2 days postinfection, certain intracellular regions appeared to contain higher or lower concentrations of the sGFP. A layer by layer examination indicated pockets in which sGFP was absent, and these appear to be vesicles that have recently released the sGFP or are not yet accumulating sGFP. By 3 days postinfection, the sGFP in some cells was concentrated in a number of widely dispersed globules, which may represent the vesicle remnants of a deteriorating secretory pathway. In contrast, nonsecreted GFP was more uniformly distributed in the cells than sGFP and did not accumulate in vesicles. In addition to GFP, the lectins wheat germ agglutinin (WGA) and concanavalin A (ConA), which have affinities for sugar residues, were used to examine the secretory pathway. The WGA, which is a Golgi marker, was distributed around the nucleus prior to infection but then was found to be polarized in one region of the cell following the baculovirus infection. The expansion of other cellular compartments following the baculovirus infection may have caused a change in intracellular distribution of the Golgi. While some of the sGFP was found to colocalize with the WGA label, much of the sGFP was outside this Golgi region. In contrast, ConA labeling, which was not as specific as WGA, was found throughout the cell both before and after infection similar to the sGFP distribution. These studies demonstrate that confocal visualization of fluorescent proteins can be used as an in vivo tool for examining secretory processing in insect cells.

Monitoring adenovirus infections with on-line and off-line methods.

Several known process monitoring methods were tested for their efficacy in the detection of adenovirus infections. The methods that we explored include several indirect indications of viral infections, including metabolic rate analysis, secondary gauges of respiration, cell size measurement, cell number and cell viability determination, and changes in capacitance. Direct indications of the adenovirus infection were also applied, including total viral particle and infectious particle measurements, as well as a flow cytometry method for detecting infected cells. All of the methods tested in the study provide some positive indication of an adenovirus infection. Many of the methods require repeated sampling, which may limit their utility in a manufacturing process. All of the indirect measures of viral infection may be limited by the fact that they do not uniquely identify an infection. The simplest monitoring methods appear to be detection of changes in respiration or the capacitance of the culture, both of which seem to provide a clear indication of an infection. Further work will be required to demonstrate that these indications are characteristic of only a successful and productive adenovirus infection.

Measuring the care needs of mothers of children with cancer: development of the FIN-PED.

This 2-phase study tested the Family Inventory of Needs-Pediatrics (FIN-PED), a 52-item instrument structured to include 2 subscales, the first measuring the importance of care needs and the second measuring the extent to which needs were met. In Phase I, an expert panel of 6 mothers of children with cancer rated the tool for clarity, apparent internal consistency, and content validity. All items met preset criteria for these assessments. In Phase II, 110 mothers rated the instrument for internal consistency reliability, stability over time, and internal construct validity. Both subscales achieved an estimated internal consistency of 0.94. Evidence of the instrument's stability over time was also achieved. Factor analysis resulted in 4 interpretable factors, suggesting that the tool is multidimensional.

Thioredoxin domain non-equivalence and anti-chaperone activity of protein disulfide isomerase mutants in vivo.

Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenbaugh, M. J. (1996) Protein Expression Purif. 7, 281-288). PDI is known to include catalytic active sites in two separate thioredoxin-like domains, one near the amino terminus and another near the carboxyl terminus. To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with cysteine to serine mutations at the first cysteine of one or both of the CGHC active site sequences. Trichoplusia ni insect cells were coinfected with a baculovirus vector coding for IgG in concert with either the wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl-terminal mutant (PDI-C), or mutant with both active sites altered (PDI-NC). Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells. To evaluate the effect of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with 35S-amino-acids for different periods, and the soluble immunoglobulins were immunoprecipitated from clarified cell lysates and culture medium using anti-IgG antibodies. Only coinfections with the wild-type PDI and PDI-N mutant led to increased immunoglobulin solubility and higher IgG secretion. In contrast, infection with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus. Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI protein were detected in soluble form. The capacity for PDI-N to increase immunoglobulin solubility whereas the PDI-C mutant lowered solubility indicates that the amino- and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the active site. Furthermore, mutations at the active site of the carboxyl-terminal thioredoxin domain result in PDI variants that can act as anti-chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).

Modeling assembly, aggregation, and chaperoning of immunoglobulin G production in insect cells.

A model for immunoglobulin G (IgG) production in the baculovirus-insect cell system was developed that incorporates polypeptide synthesis, oligomer assembly, protein aggregation, and protein secretion. In addition, the capacity of a chaperone to protect heavy and light chain polypeptides from protein aggregation was considered by including in vitro chaperone-peptide binding and dissociation kinetic constants from the literature. Model predictions were then compared to experiments in which the chaperone immunoglobulin heavy chain binding protein, BiP, was coexpressed by coinfecting insect cells with BiP-containing baculovirus. The model predicted a nearly twofold increase in intracellular and secreted IgG that was similar to the behavior observed experimentally after approximately 3 days of coexpressing heterologous IgG and BiP. However, immunoglobulin aggregation was still significant in both the model simulation and experiments, so the model was then used to predict the effect of strategies for improving IgG production even further. Increasing expression of the chaperone BiP by 10-fold over current experimental levels provided a 2.5-fold increase in secreted IgG production over IgG assembly without BiP. Alternatively, the expression of BiP earlier in the baculovirus infection cycle achieved a twofold increase in protein secretion without requiring excessive BiP production. The potential effect of cochaperones on BiP activity was considered by varying the BiP binding and release constants. The utilization of lower binding and release kinetic constants led to a severalfold increase in IgG secretion because the polypeptides were protected from aggregation for greater periods. An optimized strategy for chaperone action would include the rapid peptide binding of a BiP-ATP conformation along with the slow peptide release of a BiP-ligand conformation. However, even with an optimized chaperoning system, limitations in the secretion kinetics can result in the accumulation of intracellular IgG. Thus, the entire secretory pathway must be considered when enhanced secretion of heterologous proteins is desired.

Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells.

Structures of the N-linked oligosaccharide attached to the heavy chain of a heterologous murine IgG2a produced from Trichoplusia ni (TN-5B1-4, High Five) insect cells were characterized. Coexpression of the chaperone immunoglobulin heavy chain-binding protein (BiP) in the baculovirus-infected insect cells increased the soluble intracellular and secreted IgG level. This facilitated the detailed analysis of N-glycans from both intracellular and secreted IgG. Following purification of the immunoglobulins using Protein A-Sepharose, glycopeptides, prepared by trypsin-chymotrypsin digestion, were further digested with glycoamidase from sweet almond emulsin to obtain the oligosaccharide moieties. The resulting oligosaccharides were then reductively aminated with 2-aminopyridine and the structures identified by two-dimensional high performance liquid chromatography mapping (Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171, 73-90). The N-glycans obtained from the secreted IgG contain 35% complex type, some with terminal galactose residues at either alpha1, 3-Man or alpha1,6-Man branches of the Man3GlcNAc2 core. The remaining oligosaccharides detected in the secreted IgG were principally hybrid (30%) and paucimannosidic (35%) type N-glycans. Most (84%) of these secreted glycoforms contained fucose alpha1, 6-linked to the innermost GlcNAc residue and the presence of a potentially allergenic fucose alpha1,3-linked to the innermost GlcNAc residue was also detected. In contrast, the intracellular immunoglobulins included 50% high mannose-type N-glycans with lower levels of complex, hybrid, and paucimannosidic-type structures. Reverse phase one-dimensional high performance liquid chromatography analysis of the IgG N-glycans in the absence of heterologous BiP exhibited a similar distribution of intracellular and secreted glycoforms. These studies indicate that Trichoplusia ni TN-5B1-4 cells are capable of terminal galactosylation. However, the processing pathways in these cell lines appear to diverge from mammalian cells in the formation of paucimannosidic structures, in the presence of alpha1,3-fucose linkages, and in the absence of sialylation.