CLONING, BACULOVIRUS EXPRESSION, AND CHARACTERIZATION OF THE ALPHA-SUBUNIT OF PROLYL 4-HYDROXYLASE FROM THE NEMATODE CAENORHABDITIS-ELEGANS- THIS ALPHA-SUBUNIT FORMS AN ACTIVE ALPHA-BETA DIMER WITH THE HUMAN PROTEIN DISULFIDE-ISOMERASE BETA-SUBUNIT

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydro xyproline in collagens. The vertebrate enzyme is an alpha(2) beta(2) t etramer, the beta subunit of which is identical to protein disulfide-i somerase (PDI). We report here on the cloning of the catalytically imp ortant alpha subunit from Caenorhabditis elegans. This polypeptide con sists of 542 amino acids and a signal peptide of 16 additional residue s. The C. elegans alpha subunit is 25 amino acids longer than the huma n alpha subunit, mainly because of a 32-amino-acid C-terminal extensio n present only in the former. The overall amino acid sequence identity between these two alpha subunits is 45%, a 127-amino acid region clos e to the C terminus being especially well conserved. When the C. elega ns alpha subunit was expressed together with the human PDI/beta subuni t in insect cells by baculovirus vectors, an active prolyl 4-hydroxyla se was formed, but surprisingly this C. elegans/human enzyme appeared to be an alpha beta dimer. The specific activity of this C. elegans/hu man enzyme was comparable with that of the human enzyme, and most of t he other catalytic properties were also highly similar. Nevertheless, the C. elegans/human enzyme was not inhibited by poly(L-proline). The data indicate that the multifunctional PDI/beta subunit can form an ac tive prolyl 4-hydroxylase with alpha subunits having marked difference s in their amino acid sequences.