Glycobiology vol 7 no 1 pp. 113-127, 1997
© 1997
research-article |
Isolation and characterization of a class II 
-mannosidase cDNA from lepidopteran insect cells
Department of Entomology and Center for Advanced Invertebrate Molecular Sciences, Texas A&M University College Station, TX 77843, USA
1Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia Athens, GA 30602, USA
2To whom correspondence should be addressed
Received on June 20, 1996; revised on July 25, 1996; accepted on July 25, 1996
Lepidopteran insect cells are used routinely as hosts for foreign glycoprotein expression by recombinant baculoviruses, but the precise nature of their N-glycosylation pathway remains poorly defined. These cells clearly have processing glucosidases and mannosidases that can convert precursors to Man3GlcNAc2 structures and fucosyltransferases that can add fucose to the oligosaccharide core. However, their ability to extend these structures to produce complex side chains like those found in mammalian cells remains to be determined. To begin to examine this pathway at the molecular genetic level, we isolated and characterized a class II 
-mannosidase (-mannosidase II) cDNA from Sf9, a lepidopteran insect cell line. In mammalian cells, this enzyme catalyzes the committed step in the pathway converting N-linked carbohydrates to complex forms. Degenerate primers against conserved regions in known class H ot-mannosidase protein sequences were used to generate an -mannosidase II-specific PCR product from Sf9 cell DNA. Sequence information from this product was used to isolate a partial cDNA clone, the 5' end was isolated by ligation-anchored PCR, and the full length -mannosidase II cDNA was assembled. This cDNA contained a long open reading frame predicted to encode an 1130 amino acid protein with 37% identity to human Golgi -mannosidase II and with a type II membrane topology, a feature of all known Golgi processing enzymes. Southern blotting indicated that -mannosidase II is a single copy gene in Sf9 cells. Other Lepidoptera had related -mannosidase II genes, but there was variation among different genera, and the Sf9 -mannosidase II cDNA did not cross-hybridize with DNA from animals outside Lepidoptera. Steady-state levels of -mannosidase II RNA were low in uninfected Sf9 cells and even lower after baculovirus infection. The in vitro-translated Sf9 -mannosidase II protein had the expected size and was translocated and N-glycosylated by microsomal membranes. Expression of the Sf9 -mannosidase II cDNA in the baculovirus system produced large amounts of a protein with the expected size and swainsonine-sensitive -mannosidase II activity towards an aryl--mannoside substrate. These results demonstrate that Sf9 cells encode and express an -mannosidase II with properties similar to those of the mammalian enzyme.
class II -mannosidase
cDNA
lepidopteran insect cells
baculovirus expression system
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Vadaie and D. L. Jarvis Molecular Cloning and Functional Characterization of a Lepidopteran Insect {beta}4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis J. Biol. Chem., August 6, 2004; 279(32): 33501 - 33518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. O. Washburn, E. Y. Chan, L. E. Volkman, J. J. Aumiller, and D. L. Jarvis Early Synthesis of Budded Virus Envelope Fusion Protein GP64 Enhances Autographa californica Multicapsid Nucleopolyhedrovirus Virulence in Orally Infected Heliothis virescens J. Virol., December 6, 2002; 77(1): 280 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. Francis, L. Paquin, C. Weinkauf, and D. L. Jarvis Biosynthesis and processing of Spodoptera frugiperda{alpha}-mannosidase III Glycobiology, June 1, 2002; 12(6): 369 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Watanabe, T. Kokuho, H. Takahashi, M. Takahashi, T. Kubota, and S. Inumaru Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under beta -N-Acetylglucosaminidase Inhibition J. Biol. Chem., February 8, 2002; 277(7): 5090 - 5093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ohyama, P. L. Smith, K. Angata, M. N. Fukuda, J. B. Lowe, and M. Fukuda Molecular Cloning and Expression of GDP-D-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells J. Biol. Chem., June 5, 1998; 273(23): 14582 - 14587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.A. Kukuruzinska and K. Lennon Protein N-Glycosylation: Molecular Genetics and Functional Significance Critical Reviews in Oral Biology & Medicine, January 1, 1998; 9(4): 415 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Kawar, K. Karaveg, K. W. Moremen, and D. L. Jarvis Insect Cells Encode a Class II alpha -Mannosidase with Unique Properties J. Biol. Chem., May 4, 2001; 276(19): 16335 - 16340. [Abstract] [Full Text] [PDF]
|
|