Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

Nadia Vadaie,Donald L Jarvis

doi:10.1074/jbc.m404925200

Abstract

A degenerate PCR approach was used to isolate a lepidopteran insect cDNA encoding a beta4-galactosyl-transferase family member. The isolation and initial identification of this cDNA was based on bioinformatics, but its identification as a beta4-galactosyltransferase family member was experimentally confirmed. The newly identified beta4-galactosyltransferase family member had unusually broad donor and acceptor substrate specificities in vitro, as transferred galactose, N-acetylglucosamine, and N-acetylgalactosamine to carbohydrate, glycoprotein, and glycolipid acceptors. However, the enzyme preferentially utilized N-acetylgalactosamine as the donor for all three acceptors, and its derived amino acid sequence was closely related to a known N-acetylgalactosaminyltransferase. These data suggested that the newly isolated cDNA encodes a beta4-N-acetylgalactosaminyltransferase that functions in insect cell glycoprotein biosynthesis, glycolipid biosynthesis, or both. The remainder of this study focused on the role of this enzyme in N-glycoprotein biosynthesis. The results showed that the purified enzyme transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to a synthetic N-glycan in vitro. The structure of the reaction product was confirmed by chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses. Co-expression of the new cDNA product in insect cells with an N-glycoprotein reporter showed that it transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to this N-glycoprotein in vivo. Confocal microscopy showed that a GFP-tagged version of the enzyme was localized in the insect cell Golgi apparatus. In summary, this study demonstrated that lepidopteran insect cells encode and express a beta4-N-acetylgalactosaminyltransferase that functions in N-glycoprotein biosynthesis and perhaps in glycolipid biosynthesis, as well. The isolation and characterization of this gene and its product contribute to our basic understanding of insect protein N-glycosylation pathways and to the growing body of evidence that insects can produce glycoproteins with complex N-glycans.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY601103
Isolation and Characterization of an Insect ␤4-Galactosyltransferase Family Member—The cDNA described in this study was isolated on the basis of short, conserved amino acid sequences in ␤4-galactosyltransferase family members, which were noted in the literature by several different investigators during the late 1990s [75,76,77,78,79,80]
The ␤4-galactosyltransferase family members had to be expressed early in infection so each would be available intracellularly prior to expression of the reporter, which occurred later, under polyhedrin control. The results of these analyses clearly showed that the glutathione S-transferase (GST)-SfManI produced by cells co-infected with AcP(ϩ)IETn␤4GalNAcT reacted with Wisteria floribunda agglutinin (WFA), a lectin specific for ␤4-linked N-acetylgalactosamine residues (Fig. 12B), but not with Ricinus communis agglutinin (RCA), a lectin specific for ␤4-linked galactose residues (Fig. 12C)

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY601103. A relatively smaller number of reports have focused on insect genes encoding the glycosyltransferases mediating elongation of N-glycan termini One reason for this is that insects are generally considered to lack functional levels of these enzymes. Putative N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase genes have been identified in the D. melanogaster data bases [44], and the biochemical functions of the putative fly N-acetylglucosaminyltransferase I [45] and sialyltransferase [46] gene products have been experimentally confirmed in published studies These and other relatively recent observations support the hypothesis that insect cells have a branched protein Nglycosylation pathway, which includes a previously unrecognized repertoire of terminal glycosyltransferases that can mediate N-glycan elongation [26, 47]

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Conclusion