Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain.
A mutant of human gamma-glutamyl transpeptidase (EC 2.3.2.2, a membrane-bound enzyme of importance in glutathione metabolism) that differs from the wild type by deletion of the putative signal peptide/anchor domain (amino acid residues 1-27) was expressed in insect cells using a baculovirus system. In contrast to the wild-type enzyme--which, as expected, was mainly cell-associated--the mutant enzyme was secreted into the medium. The mutant and wild-type enzymes were purified and found to exhibit virtually identical catalytic properties. The mutant enzyme was glycosylated and processed into two subunits, as found for the wild-type enzyme. Brefeldin A inhibited secretion of the mutant enzyme and led to its accumulation in cells. The findings indicate that gamma-glutamyl transpeptidase can be targeted to the endoplasmic reticulum in a manner that does not involve function of an amino-terminal "signal/anchor" domain and that this domain is involved primarily in a membrane anchoring function. Another region of the enzyme may function as a signal domain.
Ikeda
Y
Y
Department of Biochemistry, Cornell University Medical College, New York, NY 10021.
Fujii
J
J
Taniguchi
N
N
Meister
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000032:analysis; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D017931:DNA Primers; D006023:Glycoproteins; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase