Donald L. Jarvis

Baculovirus as versatile vectors for protein expression in insect and mammalian cells.

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce "mammalianized" recombinant glycoproteins.

Baculovirus expression vectors are frequently used to express glycoproteins, a subclass of proteins that includes many products with therapeutic value. The insect cells that serve as hosts for baculovirus vector infection are capable of transferring oligosaccharide side chains (glycans) to the same sites in recombinant proteins as those that are used for native protein N-glycosylation in mammalian cells. However, while mammalian cells produce compositionally more complex N-glycans containing terminal sialic acids, insect cells mostly produce simpler N-glycans with terminal mannose residues. This structural difference between insect and mammalian N-glycans compromises the in vivo bioactivity of glycoproteins and can potentially induce allergenic reactions in humans. These features obviously compromise the biomedical value of recombinant glycoproteins produced in the baculovirus expression vector system. Thus, much effort has been expended to characterize the potential and limits of N-glycosylation in insect cell systems. Discoveries from this research have led to the engineering of insect N-glycosylation pathways for assembly of mammalian-style glycans on baculovirus-expressed glycoproteins. This chapter summarizes our knowledge of insect N-glycosylation pathways and describes efforts to engineer baculovirus vectors and insect cell lines to overcome the limits of insect cell glycosylation. In addition, we consider other possible strategies for improving glycosylation in insect cells.

Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production.

The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains.

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Baculovirus-insect cell expression systems.

In the early 1980s, the first-published reports of baculovirus-mediated foreign gene expression stimulated great interest in the use of baculovirus-insect cell systems for recombinant protein production. Initially, this system appeared to be the first that would be able to provide the high production levels associated with bacterial systems and the eukaryotic protein processing capabilities associated with mammalian systems. Experience and an increased understanding of basic insect cell biology have shown that these early expectations were not completely realistic. Nevertheless, baculovirus-insect cell expression systems have the capacity to produce many recombinant proteins at high levels and they also provide significant eukaryotic protein processing capabilities. Furthermore, important technological advances over the past 20 years have improved upon the original methods developed for the isolation of baculovirus expression vectors, which were inefficient, required at least some specialized expertise and, therefore, induced some frustration among those who used the original baculovirus-insect cell expression system. Today, virtually any investigator with basic molecular biology training can relatively quickly and efficiently isolate a recombinant baculovirus vector and use it to produce their favorite protein in an insect cell culture. This chapter will begin with background information on the basic baculovirus-insect cell expression system and will then focus on recent developments that have greatly facilitated the ability of an average investigator to take advantage of its attributes.

Engineering N-glycosylation pathways in the baculovirus-insect cell system

The inability to produce eukaryotic glycoproteins with complex N-linked glycans is a major limitation of the baculovirus-insect cell expression system. Recent studies have demonstrated that metabolic engineering can be used to extend the glycoprotein processing capabilities of lepidopteran insect cells. This approach is being used to develop new baculovirus-insect cell expression systems that can produce more authentic recombinant glycoproteins and obtain new information on insect N-glycosylation pathways.

A fused lobes Gene Encodes the Processing β-N-Acetylglucosaminidase in Sf9 Cells

Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual β-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Manα6(GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing β-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing β-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum β-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing β-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing β-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing β-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum β-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation.

Novel Baculovirus Expression Vectors That Provide Sialylation of Recombinant Glycoproteins in Lepidopteran Insect Cells

ABSTRACT This report describes novel baculovirus vectors designed to express mammalian β1,4-galactosyltransferase and α2,6-sialyltransferase genes at early times after infection. Sf9 cells infected with these viral vectors, unlike cells infected with a wild-type baculovirus, produced a sialylated viral glycoprotein during the late phase of infection. Thus, the two mammalian glycosyltransferases encoded by these viral vectors are necessary and sufficient for sialylation of a foreign glycoprotein in insect cells under the conditions used in this study. While some of the new baculovirus vectors described in this study produced less, one produced wild-type levels of infectious budded virus progeny.

Early Synthesis of Budded Virus Envelope Fusion Protein GP64 Enhances Autographa californica Multicapsid Nucleopolyhedrovirus Virulence in Orally Infected Heliothis virescens

ABSTRACT Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the type species of the Nucleopolyhedrovirus genus (Baculoviridae family), has two highly unusual traits shared by several baculovirus species. First, the occlusion-derived virus (ODV) that establishes primary infection in the midgut following its ingestion by host larvae contains multiple nucleocapsids, all of which enter the same midgut cell. Second, GP64, the envelope fusion protein of the budded virus (BV) that spreads infection beyond the midgut, is synthesized both early and late during infection. We tested the hypothesis that, together, these two traits enable parental ODV nucleocapsids to bud from infected midgut cells, essentially as BV, to establish secondary infections prior to completion of viral replication within the midgut. This “pass-through” strategy would enable the virus to counter the hosts principal defense, sloughing of infected midgut cells, by accelerating the onset of systemic infections. To test this hypothesis, we created an AcMNPV recombinant, AcLate21/20-64HB, that can express gp64 only during the late phase of infection (coincident with the other structural proteins). We then compared the virulence of this virus to that of a control recombinant virus that expresses gp64 in a wild-type manner. We found that when administered orally, the control virus was far more virulent and established secondary infection earlier than AcLate21/20-64HB, but when administered intrahemocoelically, infectivity and virulence of the two recombinants were identical. Our results demonstrate that early gp64 expression is a key component of a unique and highly adaptive baculovirus infection strategy.

The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

Background: ppGalNAc transferases, which initiate O-glycosylation, possess a poorly understood lectin domain. Results: The lectin domain directs glycosylation in an N- or C- terminal direction in an isoform-specific manner. Conclusion: Unanticipated isoform-specific directionality was revealed for modification of glycopeptide substrates. Significance: A novel mechanism of controlling of mucin type O-glycosylation has been discovered based on tethered lectin domains specifying N- or C-terminal modification of glycopeptide substrates. Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.