Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells.
Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.
Aumiller
Jared J
JJ
Department of Molecular Biology, University of Wyoming, Laramie, 82071, USA.
Hollister
Jason R
JR
Jarvis
Donald L
DL
D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D006056:Golgi Apparatus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000502:physiology; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology
D019476:Insect Proteins; D000118:Acetylglucosaminidase