Expression of a glycosylphosphatidylinositol-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 glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes 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 any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a 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 not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.
Luo
K
K
Department of Entomology, University of Georgia, Athens, Georgia 30602, USA.
McLachlin
J R
JR
Brown
M R
MR
Adang
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018826:CD13 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D066298:In Vitro Techniques; D018402:Manduca / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D018826:CD13 Antigens