Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Abstract

Prolyl 4‐hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4‐hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta‐subunit of prolyl 4‐hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non‐coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta‐subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4‐hydroxylase tetramer.

The protein disulphide-isomerase family: unravelling a string of folds

“…PDI can complement DsbA-deficient Escherichia coli, and increases the yield of heterologously expressed disulphide-containing protein in E. coli [21] and cultured insect cells [22] (DsbA is the E. coli periplasmic PDI homologue). PDI is also an essential structural subunit of the enzyme prolyl 4-hydroxylase (P4H) [23] and the microsomal triacylglycerol transfer protein (MTP) [24] in mammalian cells. Several other ER luminal proteins with high sequence identity to PDI have been identified (see below), and their putative functions have only recently started becoming apparent.…”

“…P4H, an ER luminal soluble enzyme that catalyses procollagen pro-α-chain prolyl hydroxylation, is a heterotetrameric (α # β # ) protein containing two PDI molecules as β-subunits [23]. Here, the function of PDI may be the retention or stabilization of the complex in the ER [109,110], although Wells et al [111] have shown that the β-subunit, as glutaredoxin, has dehydroascorbate reductase activity, and may therefore function in generating one of the cofactors of P4H, namely ascorbate.…”

Molecular cloning of the cDNA encoding a novel protein disulfide isomerase‐related protein (PDIR)

“…To identify new members of the PDI superfamily [6], we screened a human placental Agtl 1 cDNA library using human PDI cDNA [9,11] under conditions of low stringency. Through (Fig.…”

“…PDIR cDNA Human PDIR cDNA was cloned by screening a placental A, gtll cDNA library (Clontech) with a human PDI cDNA fragment [9,11], radiolabeled with [~-32p]dCTP (-220 TBq/mmol) (Amersham) using a random primer DNA labeling kit (Takara Shuzo). Hybridization proceeded at 37°C overnight in hybridization buffer [6 x SSC (1 x SSC: 0.15 M NaCI and 0,015 M sodium citrate, pH 7.0) containing 0.5% SDSa, 0.2% polyvinylpyrrolidone, 0.2% Ficoll 400, 0.2% BSA, and 20 #g/ml of sheared salmon sperm DNA], followed by a wash in 2 x SSC containing 0.1% SDSa at 37°C for 30 rain.…”

“…Mouse normal liver cells (BNL CL.2, ATCC No. TIB73) were grown in s-modified Eagle's minimum essential medium supplemented with 10% fetal calf serum for 12 h and treated with tunicamycin (10 yg/ml) or A23187 (7 mM) for 12 h. Total RNAs were purified from the cells as described [15] and 20ktg of each was Northern blotted using the radiolabeled PDIR insert, human PDI cDNA insert [9,11] and human fl-actin genomic fragment [14] as the probes, respectively. The hybridization conditions were as described above.…”