Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts.

Abstract

Prolyl 4-hydroxylase [procollagen-proline, 2-oxoglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an a212 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the a-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the f8-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The a-subunit does not have this C-terminal sequence, and thus one function of the fl-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the a-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the a-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.Prolyl 4-hydroxylase [procollagen-proline, 2-oxoglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2] catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. This enzyme plays a central role in collagen synthesis, as the 4-hydroxyproline residues formed in the reaction are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules. The active prolyl 4-hydroxylase is a tetramer (a2/32) with a molecular weight of -240,000 and consisting of two different types of enzymically inactive monomer with molecular weights of =64,000 (a-subunit) and -60,000 (,3-subunit) (for a recent review, see ref. 1). Complete cDNA-derived amino acid sequences have recently been determined for the 13-subunit of human (2) and chicken (3, 4) prolyl 4-hydroxylases. Surprisingly, this 13-subunit has been found to be identical to the enzyme protein disulfide isomerase (2, 5, 6) and a major cellular thyroid hormone binding protein (7,8) and highly similar to a glycosylation site binding protein of oligosaccharyl transferase (9).The a-subunit of prolyl 4-hydroxylase probably contributes a major part of the ...

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

“…The medium and cells were then analyzed as above by SDS/ PAGE followed by Coomassie staining. As in the case of the ,( subunit, no secretion ofthe a-subunit polypeptides into the culture medium was found, although the a subunits have no Lys-Asp-Glu-Leu (KDEL) retention signal for endoplasmic reticulum luminal proteins (4,29). In contrast to the data obtained with the (8 subunit, only trace amounts of the a subunits were solubilized from the cell homogenate with 0.1% Triton X-100, and only small amounts even with 1% Triton X-100; treatment with 1% SDS was needed to release the a-subunit polypeptides (Fig.…”

“…We expected initially that the a subunit, when expressed alone, might be secreted into the medium, as it has no carboxyl-terminal KDEL sequence (4,32), but no such secretion was detected. The reason for this lack of secretion appears to be the marked tendency of the a subunit to form insoluble aggregates in the absence of the (B subunit (17,18), as the use of 1% SDS was required to obtain an efficient extraction.…”

“…The two types of mRNA were present in human skin fibroblasts in approximately equal amounts, which suggested that the prolyl 4-hydroxylase a2132 tetramer may contain one a subunit of each type (2,4). The , subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1) (5-7), a cellular thyroid hormone-binding protein (8,9), a component of the microsomal triacylglycerol transfer protein complex (10,11), a dehydroascorbate reductase (12), and an endoplasmic reticulum luminal polypeptide uniquely binding various peptides (13,14).…”

“…The resulting Pst I-Pst I and Pst I-BamHI fragments containing 61 bp of the 5' untranslated sequence, the whole coding region, and 551 bp of the 3' untranslated sequence were cloned to the Pst I-BamHI site of the baculovirus transfer vector pVL1392 (22). The baculovirus transfer vector pVLa59 was similarly constructed from pVL1392 and another cDNA clone, PA-59 (4) Proc. Nadl.…”

“…Molecular cloning of the a subunit of human prolyl 4-hydroxylase has identified two types of cDNA clone that differ in a stretch of 64 base pairs (bp) due to mutually exclusive alternative splicing (4). The two types of mRNA were present in human skin fibroblasts in approximately equal amounts, which suggested that the prolyl 4-hydroxylase a2132 tetramer may contain one a subunit of each type (2,4).…”