Prolyl 4-hydroxylase : genomic cloning of the human and mouse α(II) subunit, tissue distribution of type I and II isoenzymes, and cloning and characterization of a novel prolyl 4-hydroxylase from Caenorhabditis elegans
1University of Oulu, Collagen Research Unit
2University of Oulu, Biocenter Oulu
3University of Oulu, Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology
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|Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Medical Biochemistry and Molecular Biology, on July 4th, 2002, at 12 noon.
Docent Mikko Lammi
Docent Sirkku Peltonen
The collagens are a family of extracellular matrix proteins with a widespread tissue distribution. Collagen biosynthesis requires the hydroxylation of a number of proline residues by prolyl 4-hydroxylase. This posttranslational modification is essential for the synthesis of all collagens, as 4-hydroxyproline deficient collagens cannot form stable triple helices at body temperature.
The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits were cloned and characterized in this study. The human and mouse genes are 34.6 and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The intron sizes vary from 48–49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but there are several motifs that may act as transcription factor binding sites. A novel mutually exclusively spliced exon 12a was identified in both genes. Both variants of the α(II) subunit were found to be expressed in a variety of tissues and both formed a fully active recombinant tetramer with the β subunit when expressed in insect cells.
Tissue distribution of the type I and type II prolyl 4-hydroxylase isoenzymes was studied in developing, mature, and malignant cells and tissues by immunofluorescence and Western blotting. The results indicate that the type I isoenzyme is the main form in many cell types. Skeletal myocytes and smooth muscle cells appeared to have the type I isoenzyme as their only prolyl 4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in chondrocytes. A strong signal for the type II enzyme was detected in cultured umbilical and capillary endothelial cells, whereas the type I isoenzyme could not be detected in these cells by immunostaining or Western blotting. Similar studies on primary chondro- and osteosarcomas and benign bone tumours indicated that the type I isoenzyme is the predominant form in both types of bone sarcoma, whereas the type II isoenzyme was more abundantly expressed in benign tumours. In chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant chondrocytes, whereas their malignant counterparts switched their expression pattern to that of the type I isoenzyme.
Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. This study reports the cloning and characterization of a third C. elegans α subunit isoform, PHY-3, which is much shorter than the previously characterized vertebrate and C. elegans α subunits. Nematodes homozygous for a phy-3 deletion were phenotypically wild type and fertile, but the 4-hydroxyproline content of their early embryos was reduced by about 90%. The expression of PHY-3 was found to be restricted to spermatheca of late larvae and adult nematode, indicating that PHY-3 is likely to be involved in the synthesis of collagens of the early embryo egg shells.
Acta Universitatis Ouluensis. D, Medica
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