Co-expression of the alpha subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble alpha-subunit-BiP complexes have no prolyl 4-hydroxylase activity.
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, their beta subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-beta subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble alpha subunits in solution. We report here that expression of the alpha subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble alpha-subunit-BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the alpha subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the alpha subunit. Nevertheless the soluble alpha-subunit-BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-beta subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the alpha subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.
Veijola
J
J
Collagen Research Unit, University of Oulu, Finland.
Pihlajaniemi
T
T
Kivirikko
K I
KI
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D015870:Gene Expression; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera
D000906:Antibodies; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase