BACULOVIRUS EXPRESSION OF 2 PROTEIN DISULFIDE-ISOMERASE ISOFORMS FROMCAENORHABDITIS-ELEGANS AND CHARACTERIZATION OF PROLYL 4-HYDROXYLASES CONTAINING ONE OF THESE POLYPEPTIDES AS THEIR BETA-SUBUNIT

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional po lypeptide that is identical to the beta subunit of prolyl 4-hydroxylas es. We report here on the cloning and expression of the Caenorhabditis elegans PDI/beta polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. el egans PDI/beta polypeptides are 61% and 85% respectively, and those be tween the C. elegans PDI/beta polypeptide and the PDI isoform 46 % and 73 %. The isoform differs from the PDI/beta and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/beta polypeptide forms an active prolyl 4-hydr oxylase alpha(2) beta(2) tetramer with the human a subunit and an alph a beta dimer with the C. elegans a subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either ct, subunit. Remov al of the 32-residue C-terminal extension from the C. elegans alpha su bunit totally eliminated alpha beta dimer formation. The C. elegans PD I/beta polypeptide formed less prolyl 4-hydroxylase with both the huma n and C. elegans alpha subunits than did the human PDI/beta polypeptid e, being particularly ineffective with the C. elegans a subunit. Exper iments with hybrid polypeptides in which the C-terminal regions had be en exchanged between the human and C. elegans PDI/beta polypeptides in dicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formatio n between the human and C. elegans PDI/beta polypeptides. The catalyti c properties of the C. elegans prolyl 4-hydroxylase alpha beta dimer w ere very similar to those of the vertebrate type II prolyl 4-hydroxyla se tetramer, including the K-m for the hydroxylation of long polypepti de substrates.