Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.
Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Vuori
K
K
Collagen Research Unit, University of Oulu, Finland.
Pihlajaniemi
T
T
Myllylä
R
R
Kivirikko
K I
KI
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D009036:Moths; D016297:Mutagenesis, Site-Directed; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases