ISOLATION AND CHARACTERIZATION OF AN ALPHA-1,2-MANNOSIDASE CDNA FROM THE LEPIDOPTERAN INSECT-CELL LINE SF9

As part of our ongoing efforts to characterize the N-glycosylation pat hway of lepidopteran insect cells, we have isolated an alpha 1,2-manno sidase homolog from an Sf9 cDNA library. This cDNA contains an open re ading frame which encodes a 670 amino acid protein with a calculated m olecular weight of 75,225 Da. This protein has two potential N-glycosy lation sites, two consensus calcium binding sequences, and is predicte d to be a type II integral membrane protein with a 22 amino acid trans membrane domain (residues 31-52). The amino acid sequence of this prot ein is 35-57% identical to Drosophila, human, murine, and yeast alpha 1,2-mannosidases. A transcript of approximately 6 kilobases was detect ed by Northern blot analysis of Sf9 mRNA, Genomic Southern blots probe d with an intron-free fragment of the alpha 1,2-mannosidase gene indic ated that there are at least two copies or cross-hybridizing variants of this gene in the Sf9 genome. In vivo expression of the cDNA using a recombinant baculovirus produced a protein that released [H-3]mannose from [H-3]Man(9)GlcNAc. This activity required calcium, but not magne sium, and was inhibited by 1-deoxymannojirimycin. These results indica te that Sf9 cells encode and express an alpha 1,2-mannosidase with pro perties similar to those of other eukaryotic processing alpha 1,2-mann osidases.