Xylosyltransferase 2 deficiency and organ homeostasis.

In this paper we characterize the function of Xylosyltransferase 2 (XylT2) in different tissues to investigate the role XylT2 has in the proteoglycan (PG) biochemistry of multiple organs. The results show that in all organs examined there is a widespread and significant decrease in total XylT activity in Xylt2 knock out mice (Xylt2-/-). This decrease results in increased organ weight differences in lung, heart, and spleen. These findings, in addition to our previous findings of increased liver and kidney weight with loss of serum XylT activity, suggest systemic changes in organ function due to loss of XylT2 activity. The Xylt2-/- mice have splenomegaly due to enlargement of the red pulp area and enhanced pulmonary response to bacterial liposaccharide. Tissue glycosaminoglycan composition changes are also found. These results demonstrate a role of XylT2 activity in multiple organs and their PG content. Because the residual XylT activity in the Xylt2-/- is due to xylosyltransferase 1 (XylT1), these studies indicate that both XylT1 and XylT2 have important roles in PG biosynthesis and organ homeostasis.

Adipose tissue loss and lipodystrophy in xylosyltransferase II deficient mice.

BACKGROUND/OBJECTIVES: The cellular and extracellular matrix (ECM) interactions that regulate adipose tissue homeostasis are incompletely understood. Proteoglycans (PGs) and their sulfated glycosaminoglycans (GAGs) provide spatial and temporal signals for ECM organization and interactions with resident cells by impacting growth factor and cytokine activity. Therefore, PGs and their GAGs could be significant to adipose tissue homeostasis. The purpose of this study was to determine the role of ECM sulfated GAGs in adipose tissue homeostasis. METHODS: Adipose tissue and metabolic homeostasis in mice deficient in xylosyltransferase 2 (Xylt2-/-) were examined by histologic analyses, gene expression analyses, whole body fat composition measurements, and glucose tolerance test. Adipose tissue inflammation and adipocyte precursors were characterized by flow cytometry and in vitro culture of mesenchymal stem cells. RESULTS: Xylt2-/- mice have low body weight due to overall reductions in abdominal fat deposition. Histologically, the adipocytes are reduced in size and number in both gonadal and mesenteric fat depots of Xylt2-/- mice. In addition, these mice are glucose intolerant, insulin resistant, and have increased serum triglycerides as compared to Xylt2 + / + control mice. Furthermore, the adipose tissue niche has increased inflammatory cells and enrichment of proinflammatory factors IL6 and IL1ß, and these mice also have a loss of adipose tissue vascular endothelial cells. Lastly, xylosyltransferease-2 (XylT2) deficient mesenchymal stem cells from gonadal adipose tissue and bone marrow exhibit impaired adipogenic differentiation in vitro. CONCLUSIONS: Decreased GAGs due to the loss of the key GAG assembly enzyme XylT2 causes reduced steady state adipose tissue stores leading to a unique lipodystrophic model. Accumulation of an adipocytic precursor pool of cells is discovered indicating an interruption in differentiation. Therefore, adipose tissue GAGs are important in the homeostasis of adipose tissue by mediating control of adipose precursor development, tissue inflammation, and vascular development.

PDGFRß signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (VSMCs). However, the direct effects of PDGF receptor ß (PDGFRß) activation on VSMCs have not been studied in the context of atherosclerosis. Here we present a new mouse model of atherosclerosis with an activating mutation in PDGFRß. Increased PDGFRß signalling induces chemokine secretion and leads to leukocyte accumulation in the adventitia and media of the aorta. Furthermore, PDGFRß(D849V) amplifies and accelerates atherosclerosis in hypercholesterolemic ApoE(-/-) or Ldlr(-/-) mice. Intriguingly, increased PDGFRß signalling promotes advanced plaque formation at novel sites in the thoracic aorta and coronary arteries. However, deletion of the PDGFRß-activated transcription factor STAT1 in VSMCs alleviates inflammation of the arterial wall and reduces plaque burden. These results demonstrate that PDGFRß pathway activation has a profound effect on vascular disease and support the conclusion that inflammation in the outer arterial layers is a driving process for atherosclerosis.

Homozygosity for frameshift mutations in XYLT2 result in a spondylo-ocular syndrome with bone fragility, cataracts, and hearing defects.

Heparan and chondroitin/dermatan sulfated proteoglycans have a wide range of roles in cellular and tissue homeostasis including growth factor function, morphogen gradient formation, and co-receptor activity. Proteoglycan assembly initiates with a xylose monosaccharide covalently attached by either xylosyltransferase I or II. Three individuals from two families were found that exhibited similar phenotypes. The index case subjects were two brothers, individuals 1 and 2, who presented with osteoporosis, cataracts, sensorineural hearing loss, and mild learning defects. Whole exome sequence analyses showed that both individuals had a homozygous c.692dup mutation (GenBank: NM_022167.3) in the xylosyltransferase II locus (XYLT2) (MIM: 608125), causing reduced XYLT2 mRNA and low circulating xylosyltransferase (XylT) activity. In an unrelated boy (individual 3) from the second family, we noted low serum XylT activity. Sanger sequencing of XYLT2 in this individual revealed a c.520del mutation in exon 2 that resulted in a frameshift and premature stop codon (p.Ala174Profs(∗)35). Fibroblasts from individuals 1 and 2 showed a range of defects including reduced XylT activity, GAG incorporation of (35)SO4, and heparan sulfate proteoglycan assembly. These studies demonstrate that human XylT2 deficiency results in vertebral compression fractures, sensorineural hearing loss, eye defects, and heart defects, a phenotype that is similar to the autosomal-recessive disorder spondylo-ocular syndrome of unknown cause. This phenotype is different from what has been reported in individuals with other linker enzyme deficiencies. These studies illustrate that the cells of the lens, retina, heart muscle, inner ear, and bone are dependent on XylT2 for proteoglycan assembly in humans.

Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum.

Circulating glycosyltransferases including xylosyltransferases I (XylT1) and II (XylT2) are potential serum biomarkers for various diseases. Understanding what influences the serum activity of these enzymes as well as the sources of these enzymes is important to interpreting the significance of alterations in enzyme activity during disease. This article demonstrates that in the mouse and human the predominant XylT in serum is XylT2. Furthermore, that total XylT levels in human serum are approximately 200% higher than those in plasma due in part to XylT released by platelets during blood clotting in vitro. In addition, the data from Xylt2 knock-out mice and mice with liver neoplasia show that liver is a significant source of serum XylT2 activity. The data presented suggest that serum XylT levels may be an informative biomarker in patients who suffer from diseases affecting platelet and/or liver homeostasis.

Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis.

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects approximately 500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.

Biosynthesis of chondroitin and heparan sulfate in chinese hamster ovary cells depends on xylosyltransferase II.

Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.

Complex control of mouse apolipoprotein B gene expression revealed by targeted duplication.

An elevated plasma level of apolipoprotein B-containing lipoproteins is a risk factor for atherosclerotic cardiovascular disease. Subtle genetic abnormalities in gene expression including an increased expression of the APOB gene may play an important role in determining overall risk. In an attempt to increase mouse Apob expression, we used gene targeting and duplicated approximately 65 kb of genomic DNA containing the Apob locus in its natural genomic position in mice. While we successfully generated mice carrying the Apob gene duplication, the amount of the total Apob mRNA was not increased in their liver. In the intestine, total Apob mRNA was reduced to half of the wild-type mice. Plasma lipids in the Apob duplication mice were not altered. Expression analyses showed that the proximal Apob gene in the duplicated locus was preferentially expressed in both tissues suggesting a limitation of tissue-specific enhancer function. The previously characterized distant intestinal control element was not duplicated, explaining the unequal ratio of intestinal Apob expression. While the existence of an additional liver-specific enhancer element is unknown, our findings suggest the presence of an additional enhancer outside the duplicated region, and that Apob gene expression is more complicated than previously thought.

ApoB-48 and apoB-100 differentially influence the expression of type-III hyperlipoproteinemia in APOE*2 mice.

Apolipoprotein E (apoE) is essential for the clearance of plasma chylomicron and VLDL remnants. The human APOE locus is polymorphic and 5-10% of APOE*2 homozygotes exhibit type-III hyperlipoproteinemia (THL), while the remaining homozygotes have less than normal plasma cholesterol. In contrast, mice expressing APOE*2 in place of the mouse Apoe (Apoe(2/2) mice) are markedly hyperlipoproteinemic, suggesting a species difference in lipid metabolism (e.g., editing of apolipoprotein B) enhances THL development. Since apoB-100 has an LDLR binding site absent in apoB-48, we hypothesized that the Apoe(2/2) THL phenotype would improve if all Apoe(2/2) VLDL contained apoB-100. To test this, we crossed Apoe(2/2) mice with mice lacking the editing enzyme for apoB (Apobec(-/-)). Consistent with an increase in remnant clearance, Apoe(2/2). Apobec(-/-) mice have a significant reduction in IDL/LDL cholesterol (IDL/LDL-C) compared with Apoe(2/2) mice. However, Apoe(2/2).Apobec(-/-) mice have twice as much VLDL triglyceride as Apoe(2/2) mice. In vitro tests show the apoB-100-containing VLDL are poorer substrates for lipoprotein lipase than apoB-48-containing VLDL. Thus, despite a lowering in IDL/LDL-C, substituting apoB-48 lipoproteins with apoB-100 lipoproteins did not improve the THL phenotype in the Apoe(2/2).Apobec(-/-) mice, because apoB-48 and apoB-100 differentially influence the catabolism of lipoproteins.