Adenoviral Gene Transfer Restores Lysyl Hydroxylase Activity in Type VI Ehlers-Danlos Syndrome

Type VI Ehlers-Danlos syndrome is a disease characterized by disturbed lysine hydroxylation of collagen. The disease is caused by mutations in lysyl hydroxylase 1 gene and it affects several organs including the cardiovascular system, the joint and musculoskeletal system, and the skin. The skin of type VI Ehlers-Danlos syndrome patients is hyperelastic, scars easily, and heals slowly and poorly. We hypothesized that providing functional lysyl hydroxylase 1 gene to the fibroblasts in and around wounds in these patients would improve healing. In this study we tested the feasibility of transfer of the lysyl hydroxylase 1 gene into fibroblasts derived from rats and a type VI Ehlers-Danlos syndrome patient (in vitro) and into rat skin (in vivo). We first cloned human lysyl hydroxylase 1 cDNA into a recombinant adenoviral vector (Ad5RSV-LH). Transfection of human type VI Ehlers-Danlos syndrome fibroblasts (about 20% of normal lysyl hydroxylase 1 activity) with the vector increased lysyl hydroxylase 1 activity in these cells to near or greater levels than that of wild type, unaffected fibroblasts. The adenoviral vector successfully transfected rat fibroblasts producing both β-galactosidase and lysyl hydroxylase 1 gene activity. We next expanded our studies to a rodent model. Intradermal injections of the vector to the abdominal skin of rats produced lysyl hydroxylase 1 mRNA and elevated lysyl hydroxylase 1 activity, in vivo. These data suggest the feasibility of gene replacement therapy to modify skin wound healing in type VI Ehlers-Danlos syndrome patients

Retrieval-independent localization of lysyl hydroxylase in the endoplasmic reticulum via a peptide fold in its iron-binding domain.

Lysyl hydroxylase (LH) is a peripheral membrane protein in the lumen of the endoplasmic reticulum (ER) that catalyses hydroxylation of lysine residues in collagenous sequences. Previously, we have mapped its primary ER localization motif within a 40-amino acid segment at its C-terminus. Here, we have characterized this localization mechanism in more detail, and our results indicate that this segment confers ER residency in a KDEL-receptor-independent manner, and without any apparent recycling of the enzyme between the Golgi apparatus and the ER. In addition, we show that a rather long peptide region, rather than a specific peptide sequence per se, is required for efficient retention of a reporter protein in the ER. Accordingly, the minimal retention motif was found to require the last 32 C-terminal amino acids, and sequential substitution of all five charged residues within this critical segment interfered only marginally with the retention or association of the enzyme with the ER membranes. Moreover, our fold-recognition and structure-prediction analyses suggested that this critical peptide segment forms an extended loop within LH's iron-binding domain, and that this loop is exposed and readily accessible for binding. Collectively, our results define a novel retrieval-independent retention mechanism in the ER

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

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

Oligomer distribution of recombinant MBL-A is altered in LH3^−/− knockout MEF cells.

<p>(A) Recombinant rat MBL-A produced in LH3 manipulated MEFs was separated under non-reducing and non-heat-denaturing conditions on SDS-PAGE and immunoblotted. Different oligomeric pattern was seen in MBL-A produced in LH3^−/− knockout MEFs compared with the other samples. The migration positions of covalent oligomeric forms of MBL-A are indicated on the right. Band marked with # represents four-chain covalent species of polypeptides. Representative samples are shown. (B) Quantification of MBL-A bands from immunoblots confirmed the change in the oligomer distribution of MBL-A produced in LH3^−/− knockout MEFs. Intensity of MBL-A band around molecular marker 150 kDa (marked with #) was quantified instead of the 2×3 band in LH3^−/− knockout MEFs. The values represent the average ± SD of six to eight experiments. (C) The oligomeric forms of recombinant rat MBL-A produced in LH3 manipulated MEFs were separated with a gel filtration chromatography and quantified from immunoblots. The gel filtration elution profile of MBL-A produced in LH3^−/− knockout MEFs also differed from wild type. Double transfection with MBL-A and LH3 constructs normalized the elution profile in LH3^−/− knockout MEFs. Equal volumes of concentrated cell culture media were used in all analysis. The average elution profiles of four to five experiments are shown. The elution positions of molecular weight markers are indicated. Abbreviations: WT = wild type; KO = LH3^−/− knockout; LH3 = full length LH3; LH3-N = amino-terminal fragment of LH3; MUT = LH mutant.</p

LH mutant mice form similar MBL-A oligomers as wild type mice.

<p>(A) In serum of 2 months old female and male LH mutant (MUT) and wild type (WT) mice, MBL-A was mainly seen as tetramers, trimers and dimers, when serum samples were separated under non-reducing and non-heat-denaturing conditions on SDS-PAGE and immunoblotted. Only small amount of monomers (1×3) was present in serum. The migration positions of covalent oligomeric forms of MBL-A are indicated on the right. Band marked with 2 represent two-chain covalent species of polypeptides. Representative samples are shown. (B) The distribution of the MBL-A oligomers was unchanged in serum of 2 months old female LH mutant mice compared with wild type (n = 4 WT, 4 MUT). Intensity of MBL-A oligomers was quantified from immunoblots and the level of oligomeric form was calculated as a proportion of total intensity of MBL-A. The values represent the average ± SD of the serum samples. (C) The elution profile of MBL-A in serum of 1 year old LH mutant mice was quite similar to the elution profile of wild type mice. The oligomeric forms were separated with gel filtration chromatography and quantified from immunoblots. The average elution profiles of three mice (WT and MUT) are shown. The elution positions of molecular weight markers are indicated.</p

Mass spectrometry identification of peptides and modifications from tryptic digests of recombinant rat MBL-A.

<p>Abbreviations: WT = wild type; KO = LH3^−/− knockout; LH3 = full length LH3; LH3-N = amino-terminal fragment of LH3; MUT = LH mutant; Hyl = hydroxylysine; Gal = galactosyl; Glc = glucosyl; Hyp = hydroxyproline.</p><p>Glc-Gal-Hyl 340 Da, Gal-Hyl 178 Da, Hyl/Hyp 16 Da.</p><p>Mass spectrometry identification of peptides and modifications from tryptic digests of recombinant rat MBL-A.</p