Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo.

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Abstract
Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (.alpha.2.beta.2). The .beta. subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the .alpha. subunit. One cDNA clone (.alpha.1) was isolated from a chicken embryo cDNA expression library in .lambda.gt11 by screening with anti-.alpha.-subunit polyclonal immunoglobulins. This .alpha.1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken .alpha.-subunit polypeptide verified that .alpha.1 cDNA encoded the .alpha. subunit. Polymerase chain reactions were used to extend the sequence of .alpha.1 cDNA encoded the .alpha. subunit. Polymerase chain reactions were used to extend the sequence of .alpha.1 cDNA toward the 5'' end of .alpha.-subunit mRNA. The mature .alpha. subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The complied amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the .alpha. subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the .alpha. subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the .alpha. subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.