SITE-DIRECTED MUTAGENESIS OF HUMAN PROTEIN DISULFIDE 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 alpha2beta2 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. The data further demonstrate that one function of the PDI/beta-subunit in prolyl 4-hydroxylase is to retain the enzyme within the endoplasmic reticulum, as deletion of the C-terminal -KDEL sequence of this polypeptide led to secretion of considerable amounts of both the free polypeptide and the active enzyme tetramer, whereas no secretion of either of these was found when the -KDEL sequence was present, and little or no secretion was found when the sequence had been modified to -HDEL.