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 pathway of lepidopteran insect cells, we have isolated an alpha 1,2-mannosidase homolog from an Sf9 cDNA library. This cDNA contains an open reading frame which encodes a 670 amino acid protein with a calculated molecular weight of 75,225 Da. This protein has two potential N-glycosylation sites, two consensus calcium binding sequences, and is predicted to be a type II integral membrane protein with a 22 amino acid transmembrane domain (residues 31-52). The amino acid sequence of this protein is 35-57% identical to Drosophila, human, murine, and yeast alpha 1,2-mannosidases. A transcript of approximately 6 kilobases was detected by Northern blot analysis of Sf9 mRNA. Genomic Southern blots probed with an intron-free fragment of the alpha 1,2-mannosidase gene indicated 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 [3H]mannose from [3H]Man9GlcNAc. This activity required calcium, but not magnesium, and was inhibited by 1-deoxymannojirimycin. These results indicate that Sf9 cells encode and express an alpha 1,2-mannosidase with properties similar to those of other eukaryotic processing alpha 1,2-mannosidases.
Kawar
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Department of Entomology, Texas A&M University, College Station 77843, USA.
Herscovics
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Jarvis
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D018076:DNA, Complementary / Q000302:isolation & purification*; D004330:Drosophila / Q000201:enzymology; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017385:Sequence Homology; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase