Structure and Expression of the Human Lysyl Hydroxylase Gene (PLOD): Introns 9 and 16 Contain Alu Sequences at the Sites of Recombination in Ehlers-Danlos Syndrome Type VI Patients

Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. This enzyme activity is known to be reduced in patients with the type VI variant of the Ehlers-Danlos syndrome, and the first mutations in the lysyl hydroxylase gene (PLOD) have recently been identified. We have now isolated genomic clones for human lysyl hydroxylase and determined the complete structure of the gene, which contains 19 exons and a 5′ flanking region with characteristics shared by housekeeping genes. The constitutive expression of the gene in different tissues further suggests that lysyl hydroxylase has an essential function. We have sequenced the introns of the gene in the region where many mutations and rearrangements analyzed to date are concentrated. Intron 9 and intron 16 show extensive homology resulting from the many Alu sequences found in these introns. Intron 9 contains five and intron 16 eight Alu sequences. The high homology and many short identical or complementary sequences in these introns generate many potential recombination sites with the gene. The delineation of the structure of the lysyl hydroxylase gene contributes significantly to our understanding of the rearrangements in the genome of Ehlers-Danlos type VI patients.     

A case of Ehlers Danlos syndrome type VI

Ehlers Danlos type VI is a rare autosomal recessive connective tissue disease involving primarily the skin and joints. The main feature of the condition is neonatal hypotonia and rare complications are ruptures of arteries and the eye globe. A 4 year old girl with a typical clinical presentation and molecular diagnosis of EDS VI is presented. Sequencing of PLOD1 gene revealed a homozygous deletion in exon 13 (c.1362delC), leading to a frameshift and truncation of the lysyl hydroxylase, an enzyme necessary for collagen biosynthesis. Early diagnosis allowed treatment with high doses of ascorbic acid in order to prevent complications, genetic counseling of the family and prenatal diagnosis of an unaffected embryo.     

Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome--an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13

We present clinical, radiological, biochemical, and genetic findings on six patients from two consanguineous families that show EDS-like features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin, hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature, osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl pyridinoline (LP/HP), of approximately 1 as opposed to approximately 6 in EDS VI or approximately 0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass spectrometry of the alpha1(I)- and alpha2(I)-chain-derived peptides of collagen type I and involved at least collagen types I and II. A genome-wide SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a membrane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn(2+) content inside the endoplasmic reticulum competes with Fe(2+), a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These data suggest an entity that we have designated "spondylocheiro dysplastic form of EDS (SCD-EDS)" to indicate a generalized skeletal dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like features.     

Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. A deficiency in this enzyme activity is known to exist in patients with the type VI variant of the Ehlers-Danlos syndrome, but no amino acid sequence data have been available for the wildtype or mutated human enzyme from any source. We report the isolation and characterization of cDNA clones for lysyl hydroxylase from a human placenta lambda gt11 cDNA library. The cDNA clones cover almost all of the 3.2-kb mRNA, including all the coding sequences. These clones encode a polypeptide of 709 amino acid residues and a signal peptide of 18 amino acids. The human coding sequences are 72% identical to the recently reported chick sequences at the nucleotide level and 76% identical at the amino acid level. The C-terminal region is especially well conserved, a 139-amino-acid region, residues 588-727 (C-terminus), being 94% identical between the two species and a 76-amino-acid region, residues 639-715, 99% identical. These comparisons, together with other recent data, suggest that lysyl hydroxylase may contain functionally significant sequences especially in its C-terminal region. The human lysyl hydroxylase gene (PLOD) was mapped to chromosome 1 by Southern blot analysis of human-mouse somatic cell hybrids, to the 1p34----1pter region by using cell hybrids that contain various translocations of human chromosome 1, and by in situ hybridization to 1p36.2----1p36.3. This gene is thus not physically linked to those for the alpha and beta subunits of prolyl 4-hydroxylase, which are located on chromosomes 10 and 17, respectively.     

Preferential hydroxylation of type IV collagen by lysyl hydroxylase from Ehlers-Danlos syndrome type VI fibroblasts

Normal and Ehlers-Danlos syndrome type VI human skin and cornea fibroblasts were assayed for lysyl hydroxylase activity using two different collagen types as substrates. The enzyme from normal fibroblasts hydroxylated type I collagen more readily than type IV collagen. In the diseased cells the enzyme activity was significantly reduced, and the residual activity was preferentially directed towards type IV collagen. This suggests the existence of isoenzymes of lysyl hydroxylase or an alteration in the Ehlers-Danlos syndrome type VI that affects the binding of type I collagen more than that of type IV collagen.     

Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.     

Characterization of cDNAs for mouse lysyl hydroxylase 1, 2 and 3, their phylogenetic analysis and tissue-specific expression in the mouse.

We report on the isolation and characterization of cDNA clones for mouse lysyl hydroxylases 1, 2 and 3 (LH1, LH2, LH3). Phylogenetic analysis using nine lysyl hydroxylase sequences from five species indicates that the isoforms are derived from an ancestral gene by two duplication events, isoforms 1 and 2 being more closely related and having resulted from a more recent duplication than isoform 3. Expression of the isoforms is highly regulated in adult mouse tissues. LH1 is strongly expressed in the liver, heart, lung, skeletal muscle and kidney tissue, LH2 expression is high in the heart, lung, kidney, eye, ovary and placenta, whereas LH3 expression is high in the heart, lung, liver and testis tissue.     

Genotyping and prenatal assessment of collagen lysyl hydroxylase deficiency in a family with Ehlers-Danlos syndrome type VI.

Collagen lysyl and prolyl hydroxylase activities were measured in cultured fibroblasts from a child with clinical features of Ehlers-Danlos syndrome. Lysyl-to-prolyl hydroxylase activity ratios in cells from the proband, mother, father, and control were .24, .86, .52, and 1.00, respectively, providing a biochemical diagnosis of Ehlers-Danlos syndrome type VI and indicating an autosomal recessive mode of inheritance in this family. Prenatal assessment of lysyl hydroxylase deficiency was requested and accomplished for the first time during a subsequent pregnancy in the family. A series of control cultures established lysyl hydroxylase activity to be similar in cultured amniotic fluid cells (AF and F cells) and in cultured dermal fibroblasts. Cultured F and AF cells from the monitored pregnancy had enzyme activity similar to controls, indicating that the fetus should not be affected by lysyl hydroxylase deficiency. This finding was confirmed by demonstration of normal lysyl hydroxylase activity in fibroblasts cultured from the newborn baby. These studies show that cells cultured from second trimester amniotic fluid have collagen lysyl hydroxylase activity similar to that in dermal fibroblasts, making prenatal diagnosis of lysyl hydroxylase deficiency possible.     

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.     

A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.     

Biochemical and genetic analysis of variant mouse hepatoma cells defective in the induction of benzo(a)pyrene-metabolizing enzyme activity

We have analyzed wild type mouse hepatoma (Hepa 1c1c7) cells and variant cells which are defective in the induction of benzo(a)pyrene-metabolizing enzyme activity. One type of variant has no detectable basal or inducible aryl hydrocarbon hydroxylase activity. This class contains apparently normal cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin, but is unable to translocate the inducer-receptor complex to the nucleus. The second type of variant has low levels of basal and inducible aryl hydrocarbon hydroxylase activity. This class contains cytosolic receptors which are decreased either in their number or in their ability to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin; translocation of the inducer-receptor complex to the nucleus is apparently normal. Cell fusions indicate that both variant phenotypes are recessive with respect to wild type. Complementation analyses indicate that the defects are located on different genes.     

The let-268 locus of Caenorhabditis elegans encodes a procollagen lysyl hydroxylase that is essential for type IV collagen secretion

Basement membranes are thin sheets of specialized extracellular matrix molecules that are important for supplying mechanical support and for providing an interactive surface for cell morphology. Prior to secretion and assembly, basement membrane molecules undergo intracellular processing, which is essential for their function. We have identified several mutations in a procollagen processing enzyme, lysyl hydroxylase (let-268). The Caenorhabditis elegans lysyl hydroxylase is highly similar to the vertebrate lysyl hydroxylase, containing all essential motifs required for enzymatic activity, and is the only lysyl hydroxylase found in the C. elegans sequenced genome. In the absence of C. elegans lysyl hydroxylase, type IV collagen is expressed; however, it is retained within the type IV collagen-producing cells. This observation indicates that in let-268 mutants the processing and secretion of type IV collagen is disrupted. Our examination of the body wall muscle in these mutant animals reveals normal myofilament assembly prior to contraction. However, once body wall muscle contraction commences the muscle cells separate from the underlying epidermal layer (the hypodermis) and the myofilaments become disorganized. These observations indicate that type IV collagen is required in the basement membrane for mechanical support and not for organogenesis of the body wall muscle.     

Processes of fungal proteome evolution and gain of function: gene duplication and domain rearrangement

During evolution, organisms have gained functional complexity mainly by modifying and improving existing functioning systems rather than creating new ones ab initio. Here we explore the interplay between two processes which during evolution have had major roles in the acquisition of new functions: gene duplication and protein domain rearrangements. We consider four possible evolutionary scenarios: gene families that have undergone none of these event types; only gene duplication; only domain rearrangement, or both events. We characterize each of the four evolutionary scenarios by functional attributes. Our analysis of ten fungal genomes indicates that at least for the fungi clade, species significantly appear to gain complexity by gene duplication accompanied by the expansion of existing domain architectures via rearrangements. We show that paralogs gaining new domain architectures via duplication tend to adopt new functions compared to paralogs that preserve their domain architectures. We conclude that evolution of protein families through gene duplication and domain rearrangement is correlated with their functional properties. We suggest that in general, new functions are acquired via the integration of gene duplication and domain rearrangements rather than each process acting independently.     

Characterization and regulation of sex-specific mouse steroid hydroxylase genes

We characterized the genes of the male-specific mouse steroid 16 alpha-hydroxylase (C-P-45016 alpha) and the female-specific mouse steroid 15 alpha-hydroxylase (P-45015 alpha) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15 alpha-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.     

Two patterns of genome organization in mammals: the chromosomal distribution of duplicate genes in human and mouse

Gene duplication occurs repeatedly in the evolution of genomes, and the rearrangement of genomic segments has also occurred repeatedly over the evolution of eukaryotes. We studied the interaction of these two factors in mammalian evolution by comparing the chromosomal distribution of multigene families in human and mouse. In both species, gene families tended to be confined to a single chromosome to a greater extent than expected by chance. The average number of families shared between chromosomes was nearly 60% higher in mouse than in human, and human chromosomes rarely shared large numbers of gene families with more than one or two other chromosomes, whereas mouse chromosomes frequently did so. A higher proportion of duplicate gene pairs on the same chromosome originated from recent duplications in human than in mouse, whereas a higher proportion of duplicate gene pairs on separate chromosomes arose from ancient duplications in human than in mouse. These observations are most easily explained by the hypotheses that (1) most gene duplications arise in tandem and are subsequently separated by segmental rearrangement events, and (2) that the process of segmental rearrangement has occurred at a higher rate in the lineage of mouse than in that of human.     

Partial gene duplication involving exon-Alu interchange results in lipoprotein lipase deficiency.

Major structural rearrangements are uncommon causes of mutation in human genetic diseases. We have previously described that a significant proportion of unrelated patients of western European descent who are deficient in lipoprotein lipase (LPL) activity have a major structural rearrangement in the LPL gene. Here we report the detailed characterization of this mutation. We show that this rearrangement is due to a duplication of approximately 2 kb which results from juxtaposition of intron 6 to a partially duplicated exon 6. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint in intron 6 is associated with the simple repeat found at the 3' end of an Alu element. The breakpoint within exon 6 shows no homology to this simple repeat. This result both suggests that this interchange arose as a nonhomologous recombination event and shows that such events resulting in duplication which occur in normal gene evolution may also lead to genetic disease. Cloning of the junction fragment has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with LPL deficiency.     

Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.