Ari-Pekka Kvist

A short sequence in the N‐terminal region is required for the trimerization of type XIII collagen and is conserved in other collagenous transmembrane proteins

The recombinant transmembrane protein type XIII collagen is shown to reside on the plasma membrane of insect cells in a ‘type II’ orientation. Expressions of deletion constructs showed that sequences important for the association of three α1(XIII) chains reside in their N‐ rather than C‐terminal portion. In particular, a deletion of residues 63–83 immediately adjacent to the transmembrane domain abolished the formation of disulfide‐bonded trimers. The results imply that nucleation of the type XIII collagen triple helix occurs at the N‐terminal region and that triple helix formation proceeds from the N‐ to the C‐terminus, in opposite orientation to that of the fibrillar collagens. Interestingly, a sequence homologous to the deleted residues was found at the same plasma membrane‐adjacent location in other collagenous transmembrane proteins, suggesting that it may be a conserved association domain. The type XIII collagen was secreted into insect cell medium in low amounts, but this secretion was markedly enhanced when the cytosolic portion was lacking. The cleavage occurred in the non‐collagenous NC1 domain after four arginines and was inhibited by a furin protease inhibitor.

Lack of Cytosolic and Transmembrane Domains of Type XIII Collagen Results in Progressive Myopathy

Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1(N/N)) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1(N/N) mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal alpha1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains.

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

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.

Metabolic adaptation allows Amacr-deficient mice to remain symptom-free despite low levels of mature bile acids

Bile acids play multiple roles in the physiology of vertebrates; they facilitate lipid absorption, serve as signaling molecules to control carbohydrate and lipid metabolism, and provide a disposal route for cholesterol. Unexpectedly, the α-methylacyl-CoA racemase (Amacr) deficient mice, which are unable to complete the peroxisomal cleavage of C27-precursors to the mature C24-bile acids, are physiologically asymptomatic when maintained on a standard laboratory diet. The aim of this study was to uncover the underlying adaptive mechanism with special reference to cholesterol and bile acid metabolism that allows these mice to have a normal life span. Intestinal cholesterol absorption in Amacr-/- mice is decreased resulting in a 2-fold increase in daily cholesterol excretion. Also fecal excretion of bile acids (mainly C27-sterols) is enhanced 3-fold. However, the body cholesterol pool remains unchanged, although Amacr-deficiency accelerates hepatic sterol synthesis 5-fold. Changes in lipoprotein profiles are mainly due to decreased phospholipid transfer protein activity. Thus Amacr-deficient mice provide a unique example of metabolic regulation, which allows them to have a normal lifespan in spite of the disruption of a major metabolic pathway. This metabolic adjustment can be mainly explained by setting cholesterol and bile acid metabolism to a new balanced level in the Amacr-deficient mouse.

Physical mapping of mouse collagen genes on Chromosome 10 by high-resolution FISH

Abstract. Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere–Col10a1–Col13a1–Col6a2–Col6a1–Col18a1–telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere–5′→3′–centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.

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.