Abstract
Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-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 beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(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 beta-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 beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.
Highlights
Osaminidases belong to the glycosyl hydrolase (GH)2 3, GH20, or GH84 family [1,2,3,4]
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
In addition to the above-mentioned studies, a microsomal membrane-associated hexosaminidase activity in lepidopteran insect cells that can catalyze such a reaction has been reported [24], and the importance of a cellular hexosaminidase activity in N-glycan processing in insect cells was supported by the finding of an inverse relationship between the level of cellular hexosaminidase activity and the level of GlcNAc-containing N-glycans in glycoproteins expressed in S. frugiperda Sf9 cells and Estigmene acrea cells [25]
Summary
Materials—The following materials were obtained from the sources indicated: -galactosidase (jack bean), -N-acetylglucosaminidase (jack bean), ␣-L-fucosidase (bovine kidney), 2-aminopyridine, borane-dimethylamine complex, human apotransferrin, human IgG, bovine pancreatic ribonuclease B, 4-methylumbelliferyl 2-acetamido-2-deoxy--D-glucopyranoside (MU-GlcNAc), 4-methylumbelliferyl 2-acetamido-2-deoxy--D-galactopyranoside (MU-GalNAc), MU-GlcNAc6SO4, and Nonidet P-40 (Sigma); tri-N-acetylchitotriose (Seikagaku America, East Falmouth, MA); 2-acetamido-1,2dideoxynojirimycin (Toronto Research Chemicals Inc., North York, Ontario, Canada); Sephacryl S-200 HR (Amersham Biosciences); Shim-pack CLC-ODS column (6 ϫ 150 mm; Shimadzu Scientific Instruments, Columbia, MD); Amide-80 column (2 ϫ 250 mm; Tosoh Biosep LLC, Montgomeryville, PA); Sf-900 II SFM medium (Invitrogen); and peptide N-glycosidase F (ProZyme, San Leandro, CA). When PA-derivatized oligosaccharides were used as substrates, the reaction mixture contained substrates (500 pmol in 5 l), an appropriately diluted enzyme solution (10 l), and 0.1 M sodium citrate/phosphate buffer (pH 5.5; 10 l). After washing the column with 3 bed volumes of the same buffer, bound protein was eluted with 10 mM sodium citrate/phosphate buffer (pH 7.0) containing 1 M D-GlcNAc at a flow rate of 5 ml/min by pumping the elution buffer in the reverse direction, and 5-ml fractions were collected. The medium was removed by aspiration; 1 ml of the transfection mixture containing the pIB/V5-His-Sf hex plasmid or the control pIB/V5-His plasmid (prepared as described above) was added; and the plates were rocked at six side-to-side motions/min at room temperature. An unrooted phylogenetic tree was generated using the neighborjoining method [36], and 1000 bootstrapping trials were performed
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