Expression of a glycosyl phosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a gl ycosylphosphatidylinositol (GPI) anchor in insects, In this study, me repor t the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encode s a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify a ny putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and tran siently expressed in an insect cell Line derived from Spodoptera frugiperda (Sf21 cells), Immunoblot analysis with anti-APN antiserum showed that APN1 a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but no t endogenous Sf21 proteins, indicating that APN1a has an intact glycosylpho sphatidylinositol anchor, These results are evidence that Sf21 cells synthe size few, if any, endogenous GPI-linked proteins. Immunofluorescence staini ng showed that the expressed APN1a was located on the surface of Sf21 cells , (C) 1999 Academic Press.