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Lysyl hydroxylases:studies on recombinant lysyl hydroxylases and mouse lines lacking lysyl hydroxylase 1 or lysyl hydroxylase 3Takaluoma, K. (Kati) 15 May 2007 (has links)
Abstract
Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures.
In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend.
In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos.
The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.
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Lysyl hydroxylases:characterization of mouse lysyl hydroxylases and generation of genetically modified lysyl hydroxylase 3 mouse linesRuotsalainen, H. (Heli) 31 May 2005 (has links)
Abstract
Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine, 2-oxyglutarate, 5-dioxygenase, Plod) catalyzes the hydroxylation of certain lysine residues in collagens and in other proteins with collagenous domains. Three lysyl hydroxylase isoforms have been cloned from human and rat. The importance of lysyl hydroxylase 1 in collagen biosynthesis is demonstrated by the heritable disorder, Ehlers-Danlos syndrome type VI, which is characterized by joint laxity, progressive scoliosis, muscle hypotonia, scleral fragility and rupture of the ocular globe. An alternatively spliced form of lysyl hydroxylase 2 seems to function as a telopeptide lysyl hydroxylase. Lysyl hydroxylase 3 has three enzyme activities, lysyl hydroxylase, hydroxylysyl galactosyltransferase (EC 2.4.1.50), and galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66) activities that have been demonstrated earlier with in vitro experiments.
In this thesis study, the cDNAs of mouse lysyl hydroxylase isoforms 1, 2 and 3 were cloned and characterized and the gene structures of lysyl hydroxylase 2, Plod2, and lysyl hydroxylase 3, Plod3, were determined. Mouse lysyl hydroxylase isoforms were found to be highly homologous to the corresponding human isoforms and they were approximately 60% identical with each other. The mouse Plod3 gene has 19 exons as do the human PLOD1 and PLOD3 genes, and mouse Plod2, like the human PLOD2, has 20 exons including one alternatively spliced extra exon. The mouse isoforms were also found to have distinct tissue distributions. Phylogenetic analysis revealed that the lysyl hydroxylase genes have evolved from an ancestral gene through two gene duplication events. Lysyl hydroxylase 3 was demonstrated to be the oldest isoform, which is further supported by the association of glycosyltransferase activities with lysyl hydroxylase 3 and with the only lysyl hydroxylase of Caenorhabditis elegans.
The roles of the different enzyme activities of lysyl hydroxylase 3 were determined in vivo by generating three genetically modified lysyl hydroxylase 3 mouse lines. The analysis of these mouse lines demonstrated that lysyl hydroxylase 3 possesses at least lysyl hydroxylase and glucosyltransferase activities in vivo and it functions as the main, if not the only glucosyltransferase during embryogenesis. The absence of lysyl hydroxylase 3 and, especially, its glucosyltransferase activity results in the abnormal glycosylation of type IV collagen, and thus causes a severe basement membrane defect leading to death during early development. By contrast, lysyl hydroxylase activity had no effect on embryonic development, but caused changes in the structure of the epidermal basement membrane and changes in collagen fibril organization and probably in their interactions.
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Expression of lysyl hydroxylases and characterization of a novel disorder caused by mutations in the lysyl hydroxylase 3 geneSalo, A. (Antti) 18 August 2009 (has links)
Abstract
Collagens and collagenous proteins undergo several post-translational modifications that are important for their structure and functions. Lysine hydroxylation produces hydroxylysines, which are important for collagen cross-link formation and provide attachment sites for galactose and glucosylgalactose. Glycosylated hydroxylysines are crucial for embryonic development and the assembly of certain collagen types. They may also facilitate interactions between collagen and adjacent molecules as well as control the diameter of collagen fibrils. Lysine hydroxylation is catalyzed by three lysyl hydroxylases (LH1, LH2 and LH3). In addition to lysyl hydroxylase activity, LH3 possesses collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities.
In this study, polyclonal antibodies against the lysyl hydroxylase isoforms were produced for protein level studies to localize the expression and understand the functions of the different isoenzymes. The results indicated ubiquitous expression during embryonic development compared to the more restricted, cell and tissue specific expression patterns observed in adult mouse tissues. Differences were seen also in the alternative splicing of LH2 during embryogenesis and between tissue types. Analyses of the subcellular localization revealed that LH3 is also present in extracellular space. Tissue and cell specific differences were noted in the distribution of LH3 between cellular compartments. Substrate analysis suggested an additional and novel role for LH3 as an enzyme catalyzing lysine modifications of collagenous proteins in the extracellular space.
The importance of LH1 and LH2 has been highlighted in Ehlers-Danlos type VI and Bruck syndromes, respectively. In this study, the lysyl hydroxylase 3 gene was linked to a heritable disorder for the first time. Urinary screening revealed a patient that lacked a glucosylgalactosyl derivative of a pyridinium cross-link. The GGT activity levels measured from the patient’s serum and lymphoblastoid cells were also reduced, which suggested a defect in the lysyl hydroxylase 3 gene. Genetic analyses revealed two mutations, one in each allele of LH3 in this compound heterozygous patient. Recombinant mutant proteins showed defects in lysyl hydroxylase and collagen glycosyltransferase activities, respectively. In conclusion, it was shown that a defect in LH3 catalyzed modifications leads to a novel disorder, which shares features with many other connective tissue disorders.
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