Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis 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 polypeptide that is identical to the beta subunit of prolyl 4-hydroxylases. 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. elegans PDI/beta polypeptides are 61% and 85% respectively, and those between 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-hydroxylase alpha 2 beta 2 tetramer with the human alpha subunit and an alpha beta dimer with the C. elegans alpha subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either alpha subunit. Removal of the 32-residue C-terminal extension from the C. elegans alpha subunit totally eliminated alpha beta dimer formation. The C. elegans PDI/beta polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans alpha subunits than did the human PDI/beta polypeptide, being particularly ineffective with the C. elegans alpha subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/beta polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/beta polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase alpha beta dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the K(m) for the hydroxylation of long polypeptide substrates.
Veijola
J
J
Collagen Research Unit, University of Oulu, Finland.
Annunen
P
P
Koivunen
P
P
Page
A P
AP
Pihlajaniemi
T
T
Kivirikko
K I
KI
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011393:Procollagen-Proline Dioxygenase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases