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

Abstract

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...

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

“…Further analysis of the Sfhex gene product, which is identical to the gene product we designated SfGlcNAcase-3, confirmed that the SfGlcNAcase-3/SfHex gene product lacks the ␣3 branch specificity of the processing enzyme activity originally described by Altmann et al (9). However, because this enzyme had a 2-5-fold higher preference for the terminal N-acetylglucosamine residue on the ␣3 branch of an N-glycan substrate, Tomiya et al (15) concluded that the SfGlcNAcase-3/SfHex gene encodes the processing ␤-N-acetylglucosaminidase of Sf9 cells.…”

“…Accordingly, we concluded that none of these S. frugiperda genes encoded the N-glycan processing enzyme but more likely encoded broad-spectrum ␤-N-acetylglucosaminidases involved in N-glycan and chitin degradation. In a similar study, Tomiya et al (15) also molecularly cloned two allelic variants of an Sf9 cell ␤-N-acetylglucosaminidase gene, which they termed Sfhex. Further analysis of the Sfhex gene product, which is identical to the gene product we designated SfGlcNAcase-3, confirmed that the SfGlcNAcase-3/SfHex gene product lacks the ␣3 branch specificity of the processing enzyme activity originally described by Altmann et al (9).…”

“…Subsequently, two laboratory groups independently reported molecular cloning of genes encoding ␤-N-acetylglucosaminidases from Sf9 cells, which are a clonal derivative of the IPLB-Sf21AE cell line (15,16). Our group described the isolation of three ␤-N-acetylglucosaminidase genes from Sf9 cells, which were designated SfGlcNAcase-1, -2, -3 (16).…”

Humanization of recombinant glycoproteins expressed in insect cells

“…It was also suggested that, other than some resident Golgi proteins, any glycoprotein leaving the Golgi apparatus, i.e., the vast majority of glycoproteins passing through the secretion pathway, is susceptible to the action of DmFDL. Our group has proposed a similar view that another GlcNAcase (Sfhex-/SfGlcNAcase-3) in Sf9 cells with relatively broad speciˆc-ity may contribute to the generation of paucimannosidic glycans (46). However, the extent of the contribution of each of these enzymes to the degradation of N-glycans is not yet clear.…”

“…On the basis of the known structural data, the relative activity of GnT I and II in insect cells, and the requirement of the GlcNAcb1,2 linked to Mana1,3-branch for the actions of a-mannosidase II and fucosyltransferase(s), they suggested that these structures originally carried GlcNAc on the Mana1,3-branch, which was then removed by GlcNAcase in insect cells,. Recently, several groups have succeeded in cloning the GlcNAcase from Drosophila (37) and Sf9 cells (45)(46)(47). GlcNAcase from Drosophila (DmFDL) and Sf9 cells (SfFDL) have exhibited exclusive activity toward GlcNAc linked to the Mana1,3-branch, and mutant fruit ‰ies lacking DmFDL produce increased amount of N-glycans containing GlcNAc on the Mana1,3-branch.…”