Type XIII collagen:organization and chromosomal localization of the mouse gene, distance between human COL13A1 and prolyl 4-hydroxylase α-subunit genes, and generation of mice expressing an N-terminally altered type XIII collagen

Abstract The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15. The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp. To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein

Phytol is lethal for Amacr-deficient mice

α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr-/- mice died within 36weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr-/- mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr-/- mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr-/- mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.publisher: Elsevier articletitle: Phytol is lethal for Amacr-deficient mice journaltitle: Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids articlelink: http://dx.doi.org/10.1016/j.bbalip.2015.07.008 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe

Abnormal adherence junctions in the heart and reduced angiogenesis in transgenic mice overexpressing mutant type XIII collagen

Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated α1(XIII) chains. Shortened α1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development

The Activities of Lysyl Hydroxylase 3 (LH3) Regulate the Amount and Oligomerization Status of Adiponectin

<div><p>Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.</p> </div

Distribution of recombinant adiponectin in MEF cell lysate and cell culture media.

A<p>Average of three experiments in which equal number of cells were used.</p>B<p>The results are calculated as a percentage of adiponectin from the total adiponectin on a cell culture plate (ng in lysate+ng in media measured by ELISA) and presented as average values ± S.D. from 3 different transfections.</p

Immunoblot and mass analyses of adiponectin produced in LH3 manipulated cell lines.

<p>(A) Recombinant adiponectin was produced in MEF cells and the medium containing the secreted adiponectin was collected for the analysis. The immunoblot analysis of recombinant adiponectin monomers from the concentrated medium of wild type, LH3^−/− knockout and LH mutant MEF cells indicates a clear size difference. (B) Schematic picture of the collagenous domain of mouse adiponectin shows the positions of posttranslational modifications of lysine and proline residues reported earlier. (C) Mass spectrometry identification of peptides and modifications from tryptic digests of mouse adiponectin produced in wild type, LH3^−/− knockout and LH mutant MEFs. Abbreviations: WT = wild type; KO = LH3^−/− knockout; MUT = LH mutant; Hyl = hydroxylysine; Gal = galactosyl; Glc = glucosyl.</p

The distribution of adiponectin oligomers is altered in the serum of LH mutant mice.

<p>(A) The ratio of HMW/total adiponectin was significantly increased and MMW/total decreased in serum of 2 months old male mice, even though the total amount of adiponectin was lowered as seen in the elution profile of adiponectin (B). The oligomeric forms were separated with gel filtration chromatography and quantified either from adiponectin immunoblots (A) or by ELISA (B). The level of oligomeric forms (A) was calculated as a proportion of total adiponectin. The values represent the average ± SD of the experiments. P values were calculated using unpaired homoscedastic student t-tests with two-tailed distribution.* p<0.05, ** p<0.01, *** p<0.001.</p

The expression of genes involved in β-oxidation, fatty acid synthesis and gluconeogenesis in LH mutant mice.

<p>Quantitative RT-PCR was used to determine changes in the gene expressions in LH mutant mice after 4 months of high fat diet. The columns represent the relative expression levels of muscular genes involved in the mitochondrial β-oxidation; CPT-1 and VLCAD and hepatic genes involved in the mitochondrial β-oxidation, fatty acid synthesis and gluconeogenesis; VLCAD, ACACA and PEPCK-C, respectively. For relative quantification of gene expression, the results were normalized using GADPH and β-actin as endogenous controls, and the expression of the wild type samples were set to 1. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050045#s3" target="_blank">Results</a> represent means with 95% confidence interval of 10 independently analyzed mice for each genotype. P values were calculated using unpaired heteroscedastic student t-test with one-tailed distribution. * p<0.05, ** p<0.01, *** p<0.001. Abbreviations: CPT-1 = carnitine palmitoyltransferase, VLCAD = very long chain acyl-CoA dehydrogenase, Acaca = acetyl-CoA carboxylase, PEPCK-C = phosphoenoylpyruvate carboxykinase.</p