Jari Heikkinen

Lysyl Hydroxylase 3 Is a Multifunctional Protein Possessing Collagen Glucosyltransferase Activity

Lysyl hydroxylase (EC 1.14.11.4) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.

Expanding the lysyl hydroxylase toolbox: new insights into the localization and activities of lysyl hydroxylase 3 (LH3).

Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post‐translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine‐linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling. J. Cell. Physiol. 212: 323–329, 2007.

Lysyl Hydroxylase 3 (LH3) Modifies Proteins in the Extracellular Space, a Novel Mechanism for Matrix Remodeling

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple‐helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine‐linked carbohydrates in collagenous proteins.

Mutational analysis of the lysyl hydroxylase 1 gene (PLOD) in six unrelated patients with Ehlers‐Danlos syndrome type VI: Prenatal exclusion of this disorder in one family

Screening of full length cDNAs for lysyl hydroxylase 1 (LH1; also PLOD) amplified from dermal fibroblasts from six unrelated patients with the autosomal recessive disorder Ehlers‐Danlos syndrome type VI (EDS VI) has shown them to be both homozygous and compound heterozygous for mutations in the gene. These mutations, which were verified in genomic DNA, result in a deficiency of LH activity (<25% of normal) in the probands, who are clinically characterized by kyphoscoliosis and extensibility of skin and joints. Four novel mutations identified in these patients include a mutation of an inserted C in one homozygous patient (1702insC) and three point mutations resulting in premature termination codons (PTCs): Y142X, Q327X (in two patients), and R670X. In the family with the R670X mutation we have prenatally excluded EDS VI by the characterization of mutations and their allelic inheritance. We have identified two previously reported mutations in the new patients: a seven exon duplication (in two patients) and a point mutation that codes for a PTC, Y511X, (in two patients). Genotype analysis indicated that the Y511X mutation may originate from a common ancestral gene. Several alternative splicing pathways have been identified which bypass the PTCs and can also restore the open reading frame. Hum Mutat 16:90, 2000.

Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541-547.

Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear.

A nonsense codon of exon 14 reduces lysyl hydroxylase mRNA and leads to aberrant RNA splicing in a patient with Ehlers-Danlos syndrome type VI

Ehlers-Danlos syndrome type VI (EDSVI) is an autosomal recessively inherited connective tissue disease, characterized by kyphoscoliosis, muscular hypotonia and ocular manifestations. The cause of the syndrome is a deficiency in the activity of lysyl hydroxylase (LH), one of the enzymes involved in the post-translational modification of collagens. We describe here an unusual compound heterozygote British patient with EDSVI. Our investigations indicate that a maternally inherited nonsense mutation (Y511X) in exon 14 of the LH gene (PLOD1) results in a reduction of the mRNA level as well as a skipping of exon 14 sequences in the mRNA that produces a protein shortened by 38 amino acids. The transcription of the other allele of the LH gene is considerably reduced from the normal for reasons that are not yet known. As a consequence, the LH activity of the skin fibroblasts of the patient is markedly reduced.

Alu-alu recombination results in duplication of seven exons in the lysyl hydroxylase gene from a patient with the type VI variant of Ehlers-Danlos syndrome

The type VI variant of the Ehlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hypotonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS.

Mutational analysis of the lysyl hydroxylase 1 gene (PLOD) in six unrelated patients with Ehlers-Danlos syndrome type VI: Prenatal exclusion of this disorder in one family Communicated by: Darwin J. Prockop Online Citation: Human Mutation, Mutation in Brief #340(2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/340.pdf

Screening of full length cDNAs for lysyl hydroxylase 1 (LH1; also PLOD) amplified from dermal fibroblasts from six unrelated patients with the autosomal recessive disorder Ehlers-Danlos syndrome type VI (EDS VI) has shown them to be both homozygous and compound heterozygous for mutations in the gene. These mutations, which were verified in genomic DNA, result in a deficiency of LH activity (<25% of normal) in the probands, who are clinically characterized by kyphoscoliosis and extensibility of skin and joints. Four novel mutations identified in these patients include a mutation of an inserted C in one homozygous patient (1702insC) and three point mutations resulting in premature termination codons (PTCs): Y142X, Q327X (in two patients), and R670X. In the family with the R670X mutation we have prenatally excluded EDS VI by the characterization of mutations and their allelic inheritance. We have identified two previously reported mutations in the new patients: a seven exon duplication (in two patients) and a point mutation that codes for a PTC, Y511X, (in two patients). Genotype analysis indicated that the Y511X mutation may originate from a common ancestral gene. Several alternative splicing pathways have been identified which bypass the PTCs and can also restore the open reading frame.