A novel strategy for secreting proteins: use of phosphatidylinositol-glycan-specific phospholipase D to release chimeric phosphatidylinositol-glycan anchored proteins.
Phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) specifically hydrolyzes the inositol-phosphate linkage in phosphatidylinositol-glycan (PI-G) anchored proteins. We recently deduced the primary structure of this enzyme and demonstrated specific enzymatic activity in transfected cells. Co-transfection of PI-G PLD with a natural PI-G anchored protein resulted in the secretion of the PI-G anchored protein via a PI-G PLD specific mechanism. We have taken advantage of these observations to develop an alternative system that may be useful for expressing and secreting proteins not amenable to secretion by conventional methods. Chimeric PI-G anchored proteins were constructed by transferring the COOH-terminal signal peptide for PI-G anchor attachment from placental alkaline phosphatase or from the low affinity IgG receptor, FcGRIIIB, to proteins that are not normally PI-G anchored. This process facilitates the cell surface expression of several proteins including the high affinity IgE receptor alpha subunit, FcERI alpha, which otherwise requires at least one other subunit for surface expression. Co-expression of these chimeric PI-G anchored proteins with PI-G PLD resulted in their secretion via a PI-G PLD specific mechanism.
Department of Molecular Genetics, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
B J
BJ
Scallon
H
H
Kado-Fong
M Y
MY
Nettleton
J P
JP
Kochan
D000595:Amino Acid Sequence; D000818:Animals; D017261:Glycosylphosphatidylinositols; D006801:Humans; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000737:chemistry*; D010739:Phospholipase D / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection / Q000235:genetics*
D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D011134:Polysaccharides; D011506:Proteins; D011994:Recombinant Proteins; D010739:Phospholipase D; C065017:glycoprotein phospholipase D