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.
Prolyl 4-hydroxylase [procollagen-proline, 2-oxyglutarate 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 by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the alpha-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 beta-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 alpha-subunit does not have this C-terminal sequence, and thus one function of the beta-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 alpha-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 alpha-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.
Helaakoski
T
T
Collagen Research Unit, University of Oulu, Finland.
Vuori
K
K
Myllylä
R
R
Kivirikko
K I
KI
Pihlajaniemi
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D015183:Restriction Mapping; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D011393:Procollagen-Proline Dioxygenase