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.

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. Hydroxylysine and its glycosylations are post-translational modifications involved in collagen biosynthesis. There are several comprehensive reviews, which discuss collagen biosynthesis and diseases linked to collagens (reviewed in Kielty et al., 1993;Kadler, 1994;Bateman et al., 1996; Kivirikko, 2001, 2004). The focus of the current review is to update the recent discovery that LH3 has multiple activities in the modification of lysyl residues in vivo, the importance of the various activities of LH3 during embryonic development, and the localization of functional LH3 both in the ER and in the extracellular space. The hydroxylation of lysine is one of the modifications (reviewed in Kivirikko et al., 1992;Kielty et al., 1993;Kadler, 1994;Prockop and Kivirikko, 1995;Bateman et al., 1996;Myllyharju and Kivirikko, 2001), which is characteristic of collagens. Hydroxylysyl residues in fibrillar collagens participate in the formation of collagen cross-links that connect molecules to each other and stabilize the extracellular matrix. The hydroxylysyl residues within the telopeptide region of the collagen molecule are converted to hydroxyallysines, which subsequently react with lysyl or hydroxylysyl residues within the triple helix to form deoxypyridinoline and pyridinoline cross-links, respectively (Robins and Brady, 2002;Eyre and Wu, 2005...

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.

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. Collagen biosynthesis contains many co-and post-translational modifications that are unique to collagenous proteins. These modifications include hydroxylation of lysyl residues and their further glycosylation to galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues. Lysyl hydroxylase (LH 2 ; E.C. 1.14.11.4) catalyzes the hydroxylation of lysyl residues in the Y-position of the repeating collagen Gly-X-Y triplets (1, 2), and three lysyl hydroxylase isoforms have been characterized: LH1, LH2, and LH3 (3-10).LH3 is a multifunctional enzyme containing, in addition to lysyl hydroxylase activity, collagen galactosyltransferase (E.C. 2.4.1.50) and glucosyltransferase (E.C. 2.4.1.66) activities in vitro (11-13). LH3 is functional in both the endoplasmic reticulum and extracellular space (14), and loss of LH3 leads to early embryonic lethality (15, 16). We have reported recently that LH3 is also multifunctional in vivo and that especially the glucosyltransferase activity of LH3 is essential for normal embryonic development (16).The amount of lysine hydroxylation and glycosylation varies between different collagen types. Type IV and VI collagens are highly hydroxylated and glycosylated, whereas fibrillar collagens, especially types I and III, have much lower levels of these modifications (1, 17, 18). The hydroxylysyl residues have an important role in collagen cross-linking (19,20), but the function of the glycosylation of the hydroxylysyl residues is still poorly understood. The role of glycosylated hydroxylysines in the formation and morphology of collagen fibrils has been debated (21, 22), and we have shown that they are necessary for the correct basement membrane localization of type IV collagen (16).We produced previously three different mouse lines with a manipulated LH3 gene (16). The mice with no or a very low level of the LH3 protein, thus lacking the lysyl hydroxylase and glucosyltransferase activities of LH3, had an embryonic lethal phenotype. The mouse line (LH mutant) with a point mutation that specifically destroys the LH activity of LH3 without affecting glycosyltransferase activities (11) was viable, but showed ultrastructural alterations (16). In this study, we used LH3 knock-out and LH mutant mice and fibroblasts derived from them as a model to investigate the functions of glycosylated hydroxylysines of collagens. This work provides new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion of type IV collagen and in the tetramerization and *

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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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