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.

Johanna Veijola,Pia Annunen,Taina Pihlajaniemi,Kari I Kivirikko,Tarja Helaakoski,Johanna Myllyharju

doi:10.1074/jbc.272.28.17342

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.

Highlights

Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens
All the data so far available on the existence of the type II prolyl 4-hydroxylase tetramer are based on insect cell coexpression experiments, but we demonstrate that this enzyme is present in cultured human fibroblasts and represents about 30% of their total prolyl 4-hydroxylase activity
We studied whether the ␣(I) and ␣(II) subunits can form a mixed ␣(I)␣(II)␤2 tetramer, and whether any differences are found between the type I and II enzymes in their Km values for various peptide substrates, as the two mouse enzymes differ so markedly from each other with respect to inhibition by poly(L-proline)

Summary

Introduction

Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The ␣ subunit of prolyl 4-hydroxylase cloned from the nematode Caenorhabditis elegans [10] has been found to have features of both types of mouse ␣ subunit, suggesting that C. elegans may have only one type of ␣ subunit [9] This forms active prolyl 4-hydroxylase in insect cell coexpression experiments with either the C. elegans or the human PDI/␤ polypeptide, but surprisingly, the enzymes containing the C. elegans ␣ subunit are ␣␤ dimers [10, 11]. All the data so far available on the existence of the type II prolyl 4-hydroxylase tetramer are based on insect cell coexpression experiments, but we demonstrate that this enzyme is present in cultured human fibroblasts and represents about 30% of their total prolyl 4-hydroxylase activity. A new affinity purification procedure was developed that is based on the use of a histidine tag in the N terminus of the PDI/␤ polypeptide, and this makes it possible to obtain large amounts of any form of the recombinant enzyme by very simple steps

Methods

Results

Conclusion