Abstract
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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-6756-7 |
| Date | 04 July 2002 |
| Creators | Nissi, R. (Ritva) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2002 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
Page generated in 0.0018 seconds