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.

Background: MPV17 is a mitochondrial inner membrane protein with unknown function.Results: Recombinant human MPV17 shows highly regulated channel-forming activity; the mitochondrial membrane potential and the reactive oxygen species formation were elevated in embryonic fibroblasts from Mpv17 Ϫ/Ϫ mice.

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 *

BackgroundGlycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.Methods/Principal FindingsWe hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl−∼0.31), while the other two types of channels are slightly selective for cations (P K+/P Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore.Conclusions/SignificanceThese results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.