A fused lobes gene encodes the processing beta-N-acetylglucosaminidase in Sf9 cells.
Manalpha6(Manalpha3)Manbeta4GlcNAcbeta4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual beta-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Manalpha6(GlcNAcbeta2Manalpha3)Manbeta4GlcNAcbeta4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing beta-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing beta-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum beta-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing beta-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing beta-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing beta-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum beta-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation.
Geisler
Christoph
C
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, USA.
Aumiller
Jared J
JJ
Jarvis
Donald L
DL
D000118:Acetylglucosaminidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002686:Chitin / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010802:Phylogeny; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D029721:Drosophila Proteins; D011134:Polysaccharides; D012330:RNA, Double-Stranded; D002686:Chitin; D000118:Acetylglucosaminidase; C509916:fdl protein, Drosophila