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)beta(2) tetramer

Prolyl 4-hydroxylase (proline 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, EC 5.3.4.1), Pie 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))(2) beta(2) 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)beta(2) 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 Chose of the type I enzyme, beet the K-i of the former fbr 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 K-m 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.