Lysyl hydroxylases:characterization of mouse lysyl hydroxylases and generation of genetically modified lysyl hydroxylase 3 mouse lines

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