Mice lacking exon 3 of perlecan (Hspg2) gene were generated by gene targeting. Exon deletion does not alter the expression or the reading frame but causes loss of attachment sites for three heparan sulfate (HS) side chains. Hspg2 D3/D3 mice are viable and fertile but have small eyes. Apoptosis and leakage of cellular material through the lens capsule are observed in neonatal lenses, and lenses degenerate within 3 weeks of birth. Electron microscopy revealed altered structure of the lens capsule through which cells had formed extensions. No kidney malfunction, such as proteinuria, was detected in Hspg2 D3/D3 mutant mice, nor were ultrastructural changes observed in the glomerular basement membranes (BMs). To achieve further depletion in the HS content of the BMs, Hspg2 D3/D3 mice were bred with collagen XVIII null mice. Lens defects were more severe in the newborn Col18a1 ±/± 3 Hspg2 D3/D3 mice and degeneration proceeded faster than in Hspg2 D3/D3 mice. The results suggest that in the lens capsule, HS chains have a structural function and are essential in the insulation of the lens from its environment and in regulation of incoming signals.

Lysyl hydroxylase (EC 1.14.11.4) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.Collagens are extracellular proteins found essentially in all tissues. They play a crucial role in maintenance of the structural integrity of tissues and in regulation of cellular behavior. The collagens, like other extracellular proteins, bind to growth factors and other regulatory components of cells and modulate cellular metabolism. To date, 19 genetically distinct collagen types have been identified (1-3). The collagen molecule is composed of three polypeptide chains, which coil around each other into a triple helical structure. Some of the collagen types, such as type I, II, and III collagens, have a rod-like structure without any interruptions in the helical region, whereas in the other types the triple-helical regions are interrupted with multiple short nonhelical sequences. Collagen molecules are aggregated in tissues into supramolecular structures such as fibrils, beaded filaments, or net-like or other kinds of structures depending on the type of collagen (3).The biosynthesis of collagen involves several post-translational modifications, which include hydroxylation of lysyl residues, galactosylation of hydroxylysyl residues, and glucosylation of galactosylhydroxylysyl residues. These reactions occur in the endoplasmic reticulum before triple helix formation. Hydroxylysine occurs in the Y position of the repeating XaaYaa-Gly triplet within the helical region of collagen molecules and also in the sequence of the nonhelical telopeptide regions of some collagen types when glycine is replaced either by serine or alanine (2, 4). The hydroxy groups of hydroxylysyl residues provide attachment sites for glycosyl residues, either the monosaccharide galactose or the disaccharide glucosylgalactose.The hydroxy groups also play a crucial role in the formation of inter-and intramolecular collagen cross-links. The biological role of the hydroxylysyl-linked carbohydrates, which are collagen-specific structures, is not clear. These carbohydrates point outward from the collagen helix and, thus, are located at the surface of the protein where they probably play an important role in lateral interactions between collagen triple helices and between collagen molecules and other extracellular ...

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the ␣ subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm Ϫ/Ϫ mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1␣ and Hif-2␣ protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm Ϫ/Ϫ mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2 gt/gt ) and Hif-p4h-3 Ϫ/Ϫ mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm Ϫ/Ϫ and wild-type mice, but caused higher increases in both values in the Hif-p4h-2 gt/gt mice and in hematocrit value in the Hif-p4h-3 Ϫ/Ϫ mice than in the wild-type. Hif-p4h-2 gt/gt /P4h-tm Ϫ/Ϫ double genemodified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2 gt/gt or P4h-tm Ϫ/Ϫ mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis. (Blood. 2012;120(16): 3336-3344) IntroductionErythropoiesis is a tightly controlled process, its key regulator being erythropoietin (EPO). During embryonic development most of the EPO production occurs in the liver, whereas the major EPO source in adults is the kidney, although the liver maintains a capacity for its expression. 1,2 Hypoxia-inducible transcription factor (HIF) plays a pivotal role in the regulation of the transcription of the EPO gene and numerous other hypoxia-regulated genes, including many additional genes influencing erythropoiesis. [1][2][3][4] The HIF-␣ subunit isoforms HIF-1␣ and HIF-2␣ are synthesized constitutively, and hydroxylation of 2 critical prolines generates 4-hydroxyproline residues that target HIF-␣ for rapid degradation in normoxia. [5][6][7] In hypoxia, this hydroxylation is inhibited, so that HIF-␣ escapes degradation, translocates into the nucleus, and dimerizes with HIF-␤. 5-7 HIF-1␣ is expressed in all nucleated cells, whereas HIF-2␣ expression is restricted to specific cell types, including renal interstitial cells and hepatocytes. 1,2 Renal and hepatic EPO production in adults is primarily regulated by HIF-2␣, but HIF-1␣ also plays a role in several situations. 1,2 Erythropoiesis requires iron. Hepcidin, a 25-amino-acid peptide secreted predominantly from hepatocytes, is the central regulator of iron metabolism. It down-regulates ferroportin and thus inhibits the absorption of dietary iron and the release of iron form erythrocytes and macrophages. 8,9 Hepcidin expression is lowered by hypoxia and agents that stabilize HIF-2␣ leading to increased serum EPO concentration and erythropoiesis, its regulation also involving the hemojuvelin/...

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.

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