Cloning of the Human Prolyl 4-Hydroxylase α Subunit Isoform α(II) and Characterization of the Type II Enzyme Tetramer

Prolyl 4-hydroxylase (proline hydroxylase, EC1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an α2β2tetramer, the β subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered α(II) subunit from human sources. The mRNA for the α(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (α(II))2β2 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 α(I)α(II)β2 tetramer. PDI/β polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/β 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 theK i 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 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.