Identification of membrane anchoring site of human renal dipeptidase and construction and expression of a cDNA for its secretory form.
The chemical properties of human renal dipeptidase (hrDP) purified from the membrane fraction of kidney have been characterized. When treated with phosphatidylinositol-specific phospholipase C, hrDP was released from renal membrane fractions. After digestion with trypsin, carboxyl-terminal peptide was isolated employing anhydrotrypsin-agarose column chromatography and reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide was identified at positions 363-369 in the primary structure deduced from the cDNA sequence (Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., Nishihara, T., And Nakazato, H. (1990) J. Biol. Chem. 265, 3992-3995). Further examination of the chemical composion of the peptide showed that it contained, respectively, 2, 1, 5, 1, and 1 mol of ethanolamine, glucosamine, mannose, inositol, and phosphate in addition to amino acids. These results suggest that the mature hrDP molecule lacks the carboxyl-terminal hydrophobic peptide extension predicted from the cDNA sequence and is anchored at Ser369 via glycosylphosphatidylinositol to the membrane. To characterize further the action of the enzyme, we have established expression systems for both secretory and membrane anchored forms of hrDP using COS-1 cells and found that both recombinant forms were as active as natural enzyme. Our expression system made it possible to prepare large amounts of soluble enzyme, and will contribute toward elucidation of the physiological roles of the enzyme.
Adachi
H
H
Suntory Institute for Biomedical Research, Osaka, Japan.
Katayama
T
T
Inuzuka
C
C
Oikawa
S
S
Tsujimoto
M
M
Nakazato
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004150:Dipeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010957:Plasmids; D014162:Transfection
D010446:Peptide Fragments; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D004150:Dipeptidases; C038937:dipeptidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase