β-(1→4)-Galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence

Abdul-Rahman, Badarulhisam; Ailor, Erik; Jarvis, Donald L.; Betenbaugh, Michael J.; Lee, Yuan Chuan

doi:10.1016/s0008-6215(02)00260-4

Cited by 26 publications

(15 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…24). In addition, the level of ␤1,4-galactosyltransferase in the Golgi is much higher in mammalian cells (74), serving to cap the N-glycans and to protect them from the action of hexosaminidases. Indeed, we have observed that overexpression of this galactosyltransferase protects the exposed GlcNAc residue from cellular hexosaminidases (7).…”

Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a ␤-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a ␤-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the ␤(1,2)-linked N-acetylglucosamine from the Man␣(1,3) branch compared with the Man␣(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal ␤-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing ␤-Nacetylhexosaminidase acting on N-glycans from Sf9 cells.␤-N-Acetylhexosaminidase (hexosaminidase, EC 3.2.1.52) catalyzes the hydrolysis of nonreducing terminal N-acetyl-Dhexosamine residues in N-acetyl-␤-D-hexosaminides. Hexosaminidases belong to the glycosyl hydrolase (GH) 2 3, GH20, or GH84 family (1-4). Of these, family 20 hexosaminidases include mammalian lysosomal hexosaminidases, fungal exochitinases, bacterial chitobiases, and insect chitinolytic hexosaminidases.The hexosaminidase activity of insects and insect cells is of particular interest because of the role that the enzyme may play in altering the structures of N-glycans generated by these cells. The N-glycan synthesis pathway in insects differs from that in mammals in that insects and insect cells produce appreciable amounts of paucimannosidic glycans (reviewed in Ref. 5). The intracellular N-glycan processing pathway in the endoplasmic reticulum of insects has been observed to include the addition of a Glc 3 Man 9 GlcNAc 2 group to the acceptor Asn residue, followed by the subsequent trimming of the initial oligosaccharide to generate Man 5 GlcNAc 2 . Insect cells also contain significant levels of N-acetylglucosaminyltransferase I, which adds a GlcNAc residue to the Man␣(1,3) branch, followed by the removal of two Man residues to produce the GlcNAc␤(1,2)Man␣(1,3)(Man(1,6))-Man␤(1,4)GlcNAc␤(1,4)GlcNAc structure. Unlike ma...

show abstract

Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Moreover, the baculovirus infection eventually results in cell death and lysis in 4 -5 days, thus often leading to immature posttranslational modification, release of proteases, and limitation of the batch production process. To circumvent these problems, cell engineering by cloning essential genes for the desired posttranslational modifications into the insect cells has been reported (Abdul-Rahman et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Hsu

Wang

et al. 2004

Biotech & Bioengineering

View full text Add to dashboard Cite

Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.

show abstract

“…On the other hand, galactosylated N-glycans are found only rarely in glycoproteins expressed in insect cells. The activity of b4GalT in insect cells has been reported to be very low (38)(39)(40)(41). For instance, Tn-5B1-4 cells contain only 10z of the GalT activity detected in CHO cells, and Sf9 cells contain no detectable levels of the GalT activity (41).…”

Section: Processing Enzymes In the Golgimentioning

confidence: 99%

“…The activity of b4GalT in insect cells has been reported to be very low (38)(39)(40)(41). For instance, Tn-5B1-4 cells contain only 10z of the GalT activity detected in CHO cells, and Sf9 cells contain no detectable levels of the GalT activity (41). Interestingly, T. ni (42) and Drosophila (43) possess orthologous genes to human b4GalTs; however, the encoded gene products do not catalyze e‹cient transfer of Gal from UDP-Gal to acceptor substrates.…”

Section: Processing Enzymes In the Golgimentioning

confidence: 99%

Humanization of recombinant glycoproteins expressed in insect cells

Tomiya¹

2009

TIGG

View full text Add to dashboard Cite

Many of the most valuable recombinant DNA products, including monoclonal antibodies, are secreted glycoproteins, which contain N-glycans. The biochemical nature of these attached glycans depends on the characteristics of the host cell used, the protein synthesized, and the culture environment. The capacity to produce functional glycoproteins with desired glycosylation patterns depends on the presence of proper cellular enzymatic machinery required for the processing of N-glycans. It is well recongnized that expression of therapeutic proteins using insect cells in combination with the baculovirus-expression system has potential advantages in terms of the ease of large scale, low cost production of heterologous glycoproteins. However, the glycoforms expressed in several commonly used insect cell lines are markedly diŠerent from those expressed in human cells. As N-glycans often aŠect the in vivo biological activity, pharmacokinetics, and several other properties of glycoproteins, the glycoforms produced in this system have become a major concern in the last few years. Accordingly, considerable eŠort has been invested in the past decade in order to gain a better understanding of the processing of N-glycans in insect cells that generate diŠerent glycoforms, and also to overcome the bottlenecks to producing glycoproteins with humanized Nglycans that are more suitable for therapeutic use.

show abstract

β-(1→4)-Galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence

Cited by 26 publications

References 26 publications

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Humanization of recombinant glycoproteins expressed in insect cells

Contact Info

Product

Resources

About