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.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.

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.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF ؉/؊ ) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF ؉/؊ , may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.Lysyl hydroxylase (LH) 2 catalyzes the post-translational formation of hydroxylysines in -X-Lys-Gly-sequences in collagens and other proteins with collagen-like domains (1, 2). Three lysyl hydroxylase isoforms (LH1, LH2, and LH3) have been identified from human, mouse, rat, and zebrafish (3-10). In addition, LH2 has two alternatively spliced forms, LH2a (short) and LH2b (long) (11). Several mutations of the LH1 gene (PLOD1) have been identified in patients with Ehlers-Danlos syndrome (EDS) type VIA, a heritable disorder characterized by kyphoscoliosis, joint laxity, skin fragility, and muscle hypotonia (12, 13). EDS VIA data indicate that LH1 hydroxylates helical crosslinking lysines of type I collagen in bone and type II collagen in cartilage (14,15). Patients with Bruck syndrome, characterized by joint contractures, fragile bones, and osteoporosis, have mutations in the LH2 gene (PLOD2) that result in the complete absence of telopeptide hydroxylysine residues in bone collagens (16,17). In addition, overexpression of LH2b has been linked to an increase in the hydroxylysine content of collagen telopeptides seen in fibrotic disorders (18).LH3 differs from the other lysyl hydroxylase isoforms in that it possesses, in addition to lysyl hydroxylase activity (6, 19), hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase (GGT) activities (20 -22), thus LH3 is able to catalyze the formation of glucosylgalactosylhydroxylysine residues. Recent studies show that LH3 is located not only in the endoplasmic reticulum but also in the extracellular space, and that the GGT activity in serum originates from LH3 (23). Furthermore, LH3 knock-out studies in mice demonstrate that the loss of LH3 leads to embryonic lethality due to disruption of the formation of basement membranes (24,25). Analyses of LH3 knock-out embryos and embryonic fibroblasts indicate that the ...

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