Prolyl 4-hydroxylase: Molecular Cloning and the Primary Structure of the α Subunit from Chicken Embryo

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 (α 2β 2). The β 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 α subunit. One cDNA clone (α 1) was isolated from a chicken embryo cDNA expression library in λ gt11 by screening with anti-α -subunit polyclonal immunoglobulins. This α 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 α -subunit polypeptide verified that α 1 cDNA encoded the α subunit. Polymerase chain reactions were used to extend the sequence of α 1 cDNA toward the 5′ end of α -subunit mRNA. The mature α subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the α 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 α subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the α subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.