Comparison of the N-linked glycosylation of human β1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae

Abstract

N-Glycosylation of human beta1,3N-acetylglucosaminyltransferase 2 (beta3GnT2) is essential for its biological function. beta3GnT2 fused to GFP(uv) (GFP(uv)-beta3GnT2) was produced by non-virus expression systems in stably transformed insect cells and silkworm larvae using a recombinant BmNPV bacmid, and purified for analysis of N-glycosylation. The N-glycan structure of beta3GnT2 was identified by glycoamidase A digestion, labeling with 2-aminopyridine (PA), and HPLC mapping. The paucimannosidic N-glycan structure (73.2%) was predominant in stably transformed Trichoplusia ni cells. In contrast, N-glycan with Gal (21.3%) and GlcNAc (16.2%) terminal residues linked to Manalpha(1,3) branch were detected on beta3GnT2 expressed in silkworm larvae. The presence of terminal Gal and bisecting GlcNAc residues such as Galbeta1, 4GlcNAcbeta1, 2Manalpha1,3(GlcNAcbeta1,4)(Manalpha1,6)Manbeta1, 4GlcNAc is not typical structure for lepidopteran insect N-glycosylation. Although allergenic alpha1,3-fucose residues have been found in T. ni cells, only alpha1,6-fucose residues were attached to the beta3GnT2 glycan in silkworm larvae. Therefore, silkworm larvae might be a useful host for producing human glycoproteins.

Improved secretion of molecular chaperone‐assisted human IgG in silkworm, and no alterations in their N‐linked glycan structures

“…5 In the case of b1,3N-acetylglucosaminyltransferase 2 expressed in silkworm larvae, N-linked glycans with a terminal Gal and bisecting GlcNAc residues were detected. 43 However, for IgG1 expression, paucimannosidic N-linked structures were predominant in the present study. Antibodies are N-glycosylated on the C H 2 domain of the Fc fragment at the Asn297 residue.…”

“…46,47 In addition, increased sialylation can result in decreased binding to certain immobilized or cell surface expressed antigens. 43 On the other hand, the effect of high-mannose type Fc glycans on the serum half-life of IgGs can vary according to the antibody concerned, with the mechanism not yet resolved. 45,48,49 Therefore, if the glycosylation pathways could be improved by the coexpression of N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase, silkworm larvae would prove a useful host system for producing human antibodies.…”

Proteolytic Activation of Human Cathepsin A

“…Mass Spectrometry-Insect cell-expressed cathepsin A glycoprotein was subjected to enzymatic deglycosylation with endoglycosidase H and peptide-N-glycosidase F. However, the carbohydrate was resistant to enzymatic removal (presumably because of fucosylation) (30,31), leading to poor results in electrospray ionization experiments. The glycosylated protein was subsequently used for Matrix-assisted laser desorption/ionization (MALDI) analysis on a Bruker OmniFlex MALDI-TOF MS in linear mode to determine masses.…”

Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity

“…With this system, we have successfully produced cellular, mitochondrial, and membrane proteins with proper folding and posttranslational modifications (Fig. S3, Supporting Information) (Dojima et al 2009;Du et al 2009;Hwang et al 2014).…”