A Connective Tissue Disorder Caused by Mutations of the Lysyl Hydroxylase 3 Gene

Abstract

Lysyl hydroxylase 3 (LH3, encoded by PLOD3) is a multifunctional enzyme capable of catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues producing either monosaccharide (Gal) or disaccharide (Glc-Gal) derivatives, reactions that form part of the many posttranslational modifications required during collagen biosynthesis. Animal studies have confirmed the importance of LH3, particularly in biosynthesis of the highly glycosylated type IV and VI collagens, but to date, the functional significance in vivo of this enzyme in man is predominantly unknown. We report here a human disorder of LH3 presenting as a compound heterozygote with recessive inheritance. One mutation dramatically reduced the sugar-transfer activity of LH3, whereas another abrogated lysyl hydroxylase activity; these changes were accompanied by reduced LH3 protein levels in cells. The disorder has a unique phenotype causing severe morbidity as a result of features that overlap with a number of known collagen disorders.

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

“…Thus, we replaced the type XVIII collagen trimerization domain with the NC2 domain of human type XIX collagen. The 5Ј-NcoI-His 6 -thioredoxin-thrombin cleavage site BamHItype III collagen (775-1026) (wild-type, G910A/V, or G1018A/ V)-(GPP) 7 -XmaI fragment was recloned into pET23-HisTrx_gpp7-NC2XIXB (31) using the cloning sites NcoI and XmaI. As a result the sequence encoding the type XVIII trimerization domain was replaced with the human type XIX collagen NC2 domain containing the last COL2 domain tripeptide Gly-Ile-Pro (residues 1007-1040 of collagen type XIX human protein, accession number CAI42716 version CAI42716.1 GI:57208771).…”

“…Cloning, Expression, and Purification of the C-terminal Quarter Fragment of Human Type III Collagen with (GPP) 7 and a Short NC2 Domain of Type XIX Collagen-Human cDNA clone of COL3A1 was kindly gifted by Prof. Takako Sasaki (Oita University, Oita, Japan). The C-terminal quarter fragment, the residue 775-1026 of the major triple helical region was PCRamplified using two oligonucleotides 5Ј-TGC GGA TCC GGA GCT CCA GGC CCA CTT G-3Ј (forward primer, the BamHI site is underlined) and 5Ј-AGA GGG CCC AGG GGC ACC AGG AGG TCC A-3Ј (reverse primer, the ApaI site is underlined).…”

“…The natural collagenous domain forms the correctly staggered collagen triple helical structure. Importantly, the (GPP) 7 -NC2XIX portion of this chimeric molecule is thermally more stable than the collagenous domain. This allows us to thermally unfold the collagenous domain located N-terminally while keeping the C termini in a correctly pre-staggered trimeric conformation (locked by the (GPP) 7 -NC2XIX portion).…”

Recombinant expression of hydroxylated human collagen in Escherichia coli

“…Hyl also serves as acceptor for the attachment of collagenspecific glycans (Schegg et al 2009). Defects of lysyl hydroxylation lead to diseases such as Ehlers-Danlos type-VI (Hyland et al 1992), Bruck syndrome (van der Slot et al 2003), and skeletal dysplasia (Salo et al 2008), demonstrating the biological importance of this post-translational modification.…”

Missense Mutations That Cause Bruck Syndrome Affect Enzymatic Activity, Folding, and Oligomerization of Lysyl Hydroxylase 2

“…This glycosyltransferase activity has been shown to be important for the proper assembly and secretion of collagens IV and VI (22) and for cell growth and viability (23). A patient with a human connective tissue disorder caused by mutations in the gene for LH3 was reported very recently to be a compound heterozygote with mutations leading to a marked reduction in the glycosyltransferase and LH activities of LH3 and a decrease in the amount of LH3 polypeptide (24). The patient has several congenital connective tissue malformations that overlap with a number of other collagen disorders (24).…”