ABSTRACT
A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm.
Subject(s)
Baculoviridae/genetics , Bombyx/genetics , Genetic Vectors , Glycoproteins/biosynthesis , Polysaccharides/metabolism , Protein Engineering/methods , Recombinant Proteins , Animals , Baculoviridae/metabolism , Bombyx/metabolism , Cells, Cultured , Cloning, Molecular , Galactose/metabolism , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Gene Expression , Genetic Vectors/genetics , Genetic Vectors/metabolism , Glycoproteins/isolation & purification , Glycoproteins/metabolism , Glycosylation , Humans , Mannose/metabolism , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Protein Processing, Post-Translational/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sialyltransferases/genetics , Sialyltransferases/metabolismABSTRACT
The baculovirus-silkworm recombinant protein expression system is an excellent method for achieving high-level expression and post-translational modifications, especially glycosylation. However, the presence of paucimannosidic-type N-glycan in glycoproteins restricts their clinical use. Paucimannosidic-type N-glycan is produced by insect-specific membrane-binding-type ß-N-acetylglucosaminidase (GlcNAcase). In the silkworm, BmGlcNAcase1, BmGlcNAcase2, and BmFDL are membrane-binding-type GlcNAcases. We investigated the localization of these GlcNAcases and found that BmFDL and BmGlcNAcase2 were mainly located in the fat body and hemolymph, respectively. The fat body is the main tissue of recombinant protein expression by baculovirus, and many glycoproteins are secreted into the hemolymph. These results suggest that inhibition of BmFDL and BmGlcNAcase2 could increase GlcNAc-type N-glycan levels. We therefore injected a GlcNAcase inhibitor into silkworms to investigate changes in the N-glycan structure of the glycoprotein expressed by baculovirus; modest levels of GlcNAc-type N-glycan were observed (0.8% of total N-glycan). Next, we generated a transgenic silkworm in which RNA interference (RNAi) reduced the BmFDL transcript level and enzyme activity to 25% and 50%, respectively, of that of the control silkworm. The proportion of GlcNAc-type N-glycan increased to 4.3% in the RNAi-transgenic silkworm. We conclude that the structure of N-glycan can be changed by inhibiting the GlcNAcases in silkworm.
Subject(s)
Acetylglucosaminidase/antagonists & inhibitors , Acetylglucosaminidase/metabolism , Bombyx/enzymology , Glycoproteins/biosynthesis , Glycoproteins/chemistry , Polysaccharides/chemistry , Protein Processing, Post-Translational , Acetylglucosaminidase/isolation & purification , Animals , Animals, Genetically Modified , Baculoviridae/genetics , Bombyx/genetics , Bombyx/metabolism , Fat Body/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Hemolymph/metabolism , Polysaccharides/metabolism , Protein Transport , RNA Interference , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.
