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-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI). We report here on the cloning of the catalytically important alpha subunit from Caenorhabditis elegans. This polypeptide consists of 542 amino acids and signal peptide of 16 additional residues. The C. elegans alpha subunit is 25 amino acids longer than the human alpha subunit, mainly because of a 32-amino-acid C-terminal extension present only in the former. The overall amino acid sequence identity between these two alpha subunits is 45%, a 127-amino acid region close to the C terminus being especially well conserved. When the C. elegans alpha subunit was expressed together with the human PDI/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, but surprisingly this C. elegans/human enzyme appeared to be an alpha beta dimer. The specific activity of this C. elegans/human enzyme was comparable with that of the human enzyme, and most of the 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 active prolyl 4-hydroxylase with alpha subunits having marked differences in their amino acid sequences.
Veijola
J
J
Collagen Research Unit, University of Oulu, Finland.
Koivunen
P
P
Annunen
P
P
Pihlajaniemi
T
T
Kivirikko
K I
KI
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase