Isolation and characterization of a class II alpha-mannosidase cDNA from lepidopteran insect cells.
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 alpha-mannosidase (alpha-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 II alpha-mannosidase protein sequences were used to generate an alpha-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 alpha-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 alpha-mannosidase II and with a type II membrane topology, a feature of all known Golgi processing enzymes. Southern blotting indicated that alpha-mannosidase II is a single copy gene in Sf9 cells. Other Lepidoptera had related alpha-mannosidase II genes, but there was variation among different genera, and the Sf9 alpha-mannosidase II cDNA did not cross-hybridize with DNA from animals outside Lepidoptera. Steady-state levels of alpha-mannosidase II RNA were low in uninfected Sf9 cells and even lower after baculovirus infection. The in vitro-translated Sf9 alpha-mannosidase II protein had the expected size and was translocated and N-glycosylated by microsomal membranes. Expression of the Sf9 alpha-mannosidase II cDNA in the baculovirus system produced large amounts of a protein with the expected size and swainsonine-sensitive alpha-mannosidase II activity towards an aryl-alpha-mannoside substrate. These results demonstrate that Sf9 cells encode and express an alpha-mannosidase II with properties similar to those of the mammalian enzyme.
Jarvis
D L
DL
Department of Entomology, Texas A&M University, College Station 77843, USA.
Bohlmeyer
D A
DA
Liao
Y F
YF
Lomax
K K
KK
Merkle
R K
RK
Weinkauf
C
C
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008361:Mannosidases / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014158:Transcription, Genetic; D043323:alpha-Mannosidase
D018076:DNA, Complementary; D008361:Mannosidases; D043323:alpha-Mannosidase