Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Abstract

Prolyl 4-hydroxylase (EC 1.14.11.2), an a2132 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important a subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The 13 subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the a and 13 subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodopterafrugiperda insect cells by baculovirus vectors. When the 13 subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the a subunit was expressed alone, only traces of the a subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These a subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both a-and 1subunit-producing viruses, an enzyme tetramer was formed, but signifcant amounts of a and 13 subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the a subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.Prolyl 4-hydroxylase (EC 1.14.11.2), an enzyme residing in the lumen of the endoplasmic reticulum, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by the hydroxylation of proline residues in peptide linkages. This cotranslational and posttranslational modification plays a crucial role in collagen synthesis, as the 4-hydroxyproline residues formed are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules. The active prolyl 4-hydroxylase is an a2P32 tetramer consisting of two types of inactive monomer with mo-

Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates

“…This method makes it possible to measure HIF-P4H activities under true initial velocity conditions and using saturating peptide substrate concentrations, and therefore the values obtained are often somewhat different from those obtained in assays involving capture of hydroxylated HIF-␣ or a fragment of this by pVHL. (Pro-Pro-Gly) 10 was used as the substrate for a purified recombinant human type I C-P4H (23 (24) and fibroblasts from a healthy control were cultured in Dulbecco's medium with 10% fetal bovine serum. Medium samples were collected for analysis of the production of VEGF and Epo (see below) and total cell extracts were obtained by lysing the cells in 150 mM NaCl, 0.1% SDS, and 20 mM Tris-HCl, pH 6.8.…”

Three Binding Sites in Protein-disulfide Isomerase Cooperate in Collagen Prolyl 4-Hydroxylase Tetramer Assembly

“…The resultant viral pools were collected 4 days later, amplified twice, and used for recombinant protein production. Other recombinant baculoviruses used in this work coded for the human C-P4H ␣(I) and ␣(II) subunits, the human PDI/␤ subunit (6,9), and the C. elegans ␣ subunit PHY-1 (32). Sf9 cells were cultured in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) at 27°C as monolayers.…”

“…an Escherichia coli expression vector encoding mature human PDI with an N-terminal His tag in-frame with the cloned gene; pVL␤, an insect expression vector encoding full-length PDI; and a vector encoding ERpaPDIbbЈaЈc in an insect expression vector had been generated previously (6,18,22). PDI mutants were generated on these vectors using site-directed mutagenesis performed using the QuikChange TM site-directed mutagenesis kit (Stratagene) as recommended by the manufacturer.…”

Recombinant Human Collagen and Biomimetic Variants Using a De Novo Gene Optimized for Modular Assembly

“…Although there are two isoforms for human prolyl-4-hydroxylase R, no kinetic differences have been found between the two isoforms. 40 We chose type 2 (p4Ha2) for our system because it had already been successfully expressed to hydroxylate a human collagen III fragment in S. cereVisiae. 28 Plasmid YEplac195 41 is a 2 µ-based vector with a URA3 selection marker.…”