Site-directed mutagenesis of conserved cysteine residues in porcine membrane dipeptidase. Cys 361 alone is involved in disulfide-linked dimerization.
Membrane dipeptidase (EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine membrane dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residues per subunit, six of which are conserved in the human enzyme. Chemical modification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues was replaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus, cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme) and to the membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that membrane dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that of the wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type, is released from COS-1 cell membranes by trypsin and by an endogenous serine protease.
Keynan
S
S
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Habgood
N T
NT
Hooper
N M
NM
Turner
A J
AJ
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D015377:Cilastatin / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D004150:Dipeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney; D008871:Microvilli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D013053:Spectrophotometry; D013552:Swine; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D004220:Disulfides; D017261:Glycosylphosphatidylinositols; D015377:Cilastatin; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010738:Type C Phospholipases; D004150:Dipeptidases; C038937:dipeptidase; D014357:Trypsin; D003545:Cysteine