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D L Jarvis

Publications and source records attributed to D L Jarvis.

47 records · Page 3Linked to original sources
PubMed1989

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.

Animals↗
PubMed1988

Biochemical properties of SV40 large tumor antigen as a glycosylated protein.

The large tumor antigen (T-ag) of SV40 is a virus-encoded polypeptide that provides multiple biological activities required for virus replication and cellular transformation. T-ag is an exceptional model for the study of protein processing, because it displays a variety of chemical modifications and an unusual dual subcellular distribution. The cellular mechanisms responsible for the synthesis and processing of T-ag are unknown. With respect to glycosylation, this has been related to a lack of knowledge of the biochemical properties of T-ag as a glycoprotein. Several such properties are characterized here. We found that T-ag is glycosylated at multiple sites on the polypeptide chain. The oligosaccharides appear to belong to a single size class, molecular weight approximately 400, and the linkage between the polypeptide and the carbohydrate side chain is sensitive to beta-elimination under mild alkaline conditions. At least one glycosylation site was localized to the region between amino acids 1 and 272 (probably between residues 83 and 272), and at least one additional site was localized to a separate region, between amino acids 523 and 708. The results of cycloheximide experiments suggested that glycosylation of T-ag is a cotranslational event, and both the nuclear and the membrane-associated forms of T-ag appeared to be glycosylated. The results of these studies verify previous conclusions that the cellular secretory pathway is not involved in the glycosylation of T-ag; instead, a cytoplasmic mechanism might be involved.

Animals↗
PubMed1987

The cellular secretory pathway is not utilized for biosynthesis, modification, or intracellular transport of the simian virus 40 large tumor antigen.

Unlike most proteins, which are localized within a single subcellular compartment in the eucaryotic cell, the simian virus 40 (SV40) large tumor antigen (T-ag) is associated with both the nucleus and the plasma membrane. Current knowledge of protein processing would predict a role for the secretory pathway in the biosynthesis and transport of at least a subpopulation of T-ag to account for certain of its chemical modifications and for its ability to reach the cell surface. We have examined this prediction by using in vitro translation and translocation experiments. Preliminary experiments established that translation of T-ag was detectable with as little as 0.1 microgram of the total cytoplasmic RNA from SV40-infected cells. Therefore, by using a 100-fold excess of this RNA, the sensitivity of the assays was above the limits necessary to detect the theoretical fraction of RNA equivalent to the subpopulation of plasma-membrane-associated T-ag (2 to 5% of total T-ag). In contrast to a control rotavirus glycoprotein, the electrophoretic mobility of T-ag was not changed by the addition of microsomal vesicles to the in vitro translation mixture. Furthermore, T-ag did not undergo translocation in the presence of microsomal vesicles, as evidenced by its sensitivity to trypsin treatment and its absence in the purified vesicles. Identical results were obtained with either cytoplasmic RNA from SV40-infected cells or SV40 early RNA transcribed in vitro from a recombinant plasmid containing the SP6 promoter. SV40 early mRNA in infected cells was detected in association with free, but not with membrane-bound, polyribosomes. Finally, monensin, an inhibitor of Golgi function, failed to specifically prevent either glycosylation or cell surface expression of T-ag, although it did depress overall protein synthesis in TC-7 cells. We conclude from these observations that the constituent organelles of the secretory pathway are not involved in the biosynthesis, modification, or intracellular transport of T-ag. The initial step in the pathway of T-ag biosynthesis appears to be translation on free cytoplasmic polyribosomes. With the exclusion of the secretory pathway, we suggest that T-ag glycosylation, palmitylation, and transport to the plasma membrane are accomplished by previously unrecognized cellular mechanisms.

Animals↗
PubMed1986

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

Animals↗
PubMed1985

Modification of simian virus 40 large tumor antigen by glycosylation.

The SV40-encoded transforming protein, large tumor antigen (T-ag), is multifunctional. Chemical modifications of the T-ag polypeptide may be important for its multifunctional capacity. T-ag is additionally modified by glycosylation. T-ag was metabolically labeled in SV40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. The identity of glycosylated T-ag was established by immunoprecipitation with a variety of T-ag-specific antisera, including monoclonal antibodies. Incorporation of labeled sugar into T-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. Labeled monosaccharides could be preferentially removed from T-ag with a mixture of glycosidic enzymes. In addition, galactose was removed from purified T-ag by acid hydrolysis and identified as such by thin-layer chromatography. T-ag oligosaccharides were resistant to treatment with EndoH, and glycosylation was not inhibited by tunicamycin. Together, these data strongly suggest that T-ag is glycosylated. Several characteristics, including lack of mannose labeling, EndoH resistance, and tunicamycin resistance, suggest that T-ag is not an N-linked glycoprotein. Rather, these properties are more consistent with the identification of T-ag as an O-linked glycoprotein.

Acetylglucosaminidase↗
PubMed1984

Structural comparisons of wild-type and nuclear transport-defective simian virus 40 large tumor antigens.

PARA(nT) is a defective SV40-adenovirus 7 hybrid virus which contains the entire early region of the SV40 genome and codes for the synthesis of SV40 large tumor antigen (T-ag). A transport-defective variant of this hybrid, PARA(cT), encodes T-ag that is not transported to the nucleus, but accumulates in the cytoplasm. The structures of T-ags extracted from wild-type (WT) SV40-, PARA(nT)-, and PARA(cT)-infected cells were compared by peptide mapping. All three types of T-ag underwent considerable degradation when extracted using Tris-buffered Nonidet P-40 at pH 8.0. The addition of 200 microM leupeptin to the extraction buffer significantly inhibited this degradation. Comparison of methionine-containing tryptic peptides revealed no differences among the T-ags, suggesting that their primary structures are similar or identical. Phosphopeptide mapping revealed no differences between SV40- and PARA(nT)-encoded T-ags. In contrast, PARA(cT)-encoded T-ag lacked a prominent phosphopeptide that was present in both of the others. The possible relevance of this difference in phosphorylation to the transport defect is discussed.

Adenoviridae↗
PubMed1981

Modification of the poliovirus capsid by ultraviolet light.

Ultraviolet (UV) irradiation of type I poliovirus resulted in a modified (M) particle that had lost infectivity, lacked ability to adsorb to HeLa cells, lacked VP4, and reduced in S value. Additional irradiation resulted in the loss of VP2, further reduction in S value, and permeability of the capsid to RNAse, This particle (C) as well as M contain the genome. Acid pH (5.5-65) and sulfhydryl-reducing substances (dithiothreitol. reduced glutathione, and L-cysteine) inhibited UV-induced modification of the capsid. UV irradiation at alkaline pH (7.5-8.5) resulted in more extensive modification of the capsid than irradiation at neutral pH. Ionic compounds were found to inhibit the modifying reaction.

Adsorption↗
PubMedDate not supplied

Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity.

Four different insect cell lines that can be used as hosts for baculovirus infection were assayed for the presence of endogenous exoglycosidases. All four cell lines, derived from Spodoptera frugiperda, Trichoplusia ni, Bombyx mori, or Malacosoma disstria, contained N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, and sialidase activities. Exoglycosidase activities were found in cell lysates as well as cell-free supernatants from uninfected and wild-type baculovirus infected cells. Oligosaccharide analysis of cellular glycoproteins using lectins recognizing Gal beta 1, 3GalNAc, Gal beta 1, 4GlcNAc, and NeuAc alpha 2,6Gal demonstrated that only Gal beta 1,3GalNAc was present. The demonstration that these cells contain exoglycosidases raises the possibility that the oligosaccharides of baculovirus-expressed glycoproteins are subject to enzymatic degradation.

Acetylglucosaminidase↗
PubMedDate not supplied

Heterologous protein expression affects the death kinetics of baculovirus-infected insect cell cultures: a quantitative study by use of n-target theory.

The death of cultured insect cells after baculovirus infection is a time-dependent event. Without a quantitative model, it is difficult to characterize its kinetics. Our group has shown that the cell survival rate can be characterized by use of the n-target theory, which involves only two parameters: the number of hypothetical inactivation targets (n) and the first-order death rate (k). In this study, we used different recombinant viruses to examine the effect of heterologous protein expression on the cell survival rate. The proteins expressed were beta-galactosidase, human T-cell leukemia virus type I p40x, human interleukin-2, and human tissue plasminogen activator (tPA). The survival rate was affected by protein expression, but the n value remained constant if the protein expression level was high (above 30 mg/L). Low-level expression of secreted, glycosylated tPA resulted in a reduced n value, which was restored to the normal value when the tPA signal peptide and prosequence were deleted. In addition, if the n value was normal (10-11), the level of protein expression correlated negatively with the death rate. However, if the n value was reduced by unfavorable culture conditions or foreign protein expression, the expression level correlated positively with the death rate. A dimensionless plot with kt as the dimensionless time shows that alteration of the k value while retaining constant n is equivalent to a rescaling of time. Therefore, the survival curves with constant n reduce to a single curve on the dimensionless plot.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗