Molecular cloning and functional characterization of a Lepidopteran insect beta4-N-acetylgalactosaminyltransferase with broad substrate specificity, a functional role in glycoprotein biosynthesis, and a potential functional role in glycolipid biosynthesis.
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.
Vadaie
Nadia
N
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, USA.
Jarvis
Donald L
DL
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006017:Glycolipids / Q000096:biosynthesis*; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D049452:Green Fluorescent Proteins; D006801:Humans; D007915:Lepidoptera / Q000201:enzymology* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D018411:Spodoptera / Q000201:enzymology; D013379:Substrate Specificity
D018076:DNA, Complementary; D006017:Glycolipids; D006023:Glycoproteins; D008164:Luminescent Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017350:N-Acetylgalactosaminyltransferases; D000116:Acetylgalactosamine