Structure and regulation of prolyl 4 hydroxylase complementary dna clones for the human alpha subunits and beta subunits genomic clones for the human alpha subunit and messenger rna levels of both subunits in differentiating mouse f9 cells

Helaakoski, T.

Acta Universitatis Ouluensis Series A Scientiae Rerum Naturalium 213: 3

1990


Accession: 007826875

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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 .alpha.2.beta.2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like sequences by the hydroxylation of proline residues in peptide linkages. In the present work cDNA clones for the .alpha.- and .beta.-subunits and genomic clones for the .alpha.-subunit of human prolyl 4-hydroxylase were isolated and characterized. Furthermore, changes in mRNA levels for both types of subunit were studied in differentiating mouse teratocarcinoma F9 cells. The characterization of cDNA clones for the .beta.-subunit of human prolyl 4-hydroxylase indicated that they encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. The size of the corresponding mRNA is about 2.5 kilobases (kb). The carboxy-terminal end of the .beta.-subunit contains a sequence -Lys-Asp-Glu-Leu which is necessary for the retention of a polypeptide in the lumen of the endoplasmic reticulum. The .alpha.-subunit cDNA clones did not have this carboxy-terminal sequence and thus one function of the .beta.-subunit appears to be to retain the enzyme tetramer within this cell organelle. The human .beta.-subunit sequences were found to be very similar to those reported for rat protein disulphide isomerase (EC 5.3.4.1) the degree of homology being 84% at the nucleotide level and 94% at the amino acid level. Southern blot analyses indicated the presence of only one gene containing these sequences. One polypeptide thus appears to possess two different enzymatic functions depending on whether it is present in cells in a monomer form or in the prolyl 4-hydroxylase tetramer. The characterization of cDNA clones for the .alpha.-subunit of human prolyl 4-hydroxylase indicated that they encode a polypeptide of 517 amino acids with a signal peptide of 17 amino acids. The size of the corresponding mRNA is about 3 kb. Two types of cDNA clone coding for the .alpha.-subunit were identified, differing over a stretch of 64 nucleotides (nt). 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 analysis and characterization of human genomic clones and genomic DNA indicated the presence of only one gene coding for the two types of mRNA which thus appears to result from mutually exclusive alternative splicing of transcripts of a single gene. These mutually exclusively sequences are coded by two consecutive homologous 71 base pair (bp) exons. The overall identity between these exons is 61% at the nucleotide level and 58% at the level of the coded amino acids. The gene for the human .alpha.-subunit is more than 58 kb in length and consists of 15 exons. Mouse teratocarcinoma stem cells differentiate in the presence of retinoic acid, dibutyryl cyclic AMP and isobutyl methylxanthine into parietal endoderm-like cells. This differentiation was found to be associated with a marked increase of up to 50-fold in the concentrations of the mRNAs for the .alpha.- and .beta.-subunits of prolyl 4-hydroxylase. The time courses and magnitudes of the increases were similar to those observed with mRNA for the .alpha.1-chain of type IV collagen in the same experiments. The concentration of the .alpha.-subunit mRNA in the partially and fully differentiated F9 cells was about 30% of that of the .beta.-subunit mRNA.