cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.
Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.
Rached
E
E
Laboratorium für Biochemie II, Eidgenössischen Technischen Hochschule, ETH-Zentrum, Zurich, Switzerland.
Hooper
N M
NM
James
P
P
Semenza
G
G
Turner
A J
AJ
Mantei
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D006017:Glycolipids / Q000502:physiology*; D017261:Glycosylphosphatidylinositols; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D009865:Oocytes / Q000502:physiology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000502:physiology*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine; D014981:Xenopus
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011092:Polyethylene Glycols; D017830:Octoxynol; D004247:DNA; C010615:Nonidet P-40; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D004150:Dipeptidases; C038937:dipeptidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase