title: Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.
abstract: Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha2beta2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered alpha(II) subunit from human sources. The mRNA for the alpha(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (alpha(II))2beta2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5-15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed alpha(I)alpha(II)beta2 tetramer. PDI/beta polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/beta polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but the Ki of the former for poly(L-proline) was about 200-1000 times that of the latter. In agreement with this, a minor difference, about 3-6-fold, was found between the two enzymes in the Km values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.
authors.affiliation: Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland.
authors.forename: P
authors.initials: P
authors.lastname: Annunen
authors.forename: T
authors.initials: T
authors.lastname: Helaakoski
authors.forename: J
authors.initials: J
authors.lastname: Myllyharju
authors.forename: J
authors.initials: J
authors.lastname: Veijola
authors.forename: T
authors.initials: T
authors.lastname: Pihlajaniemi
authors.forename: K I
authors.initials: KI
authors.lastname: Kivirikko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002642:Chick Embryo; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D006639:Histidine; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D006639:Histidine; D011393:Procollagen-Proline Dioxygenase