8 research outputs found
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
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Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia.
Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development
Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia
Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1(ECKO) mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1(ECKO) mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development
Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1
An
integrated methodology is described to establish ligand requirements
for heparan sulfate (HS) binding proteins based on a workflow in which
HS octasaccharides are produced by partial enzymatic degradation of
natural HS followed by size exclusion purification, affinity enrichment
using an immobilized HS-binding protein of interest, putative structure
determination of isolated compounds by a hydrophilic interaction chromatography–high-resolution
mass spectrometry platform, and chemical synthesis of well-defined
HS oligosaccharides for structure–activity relationship studies.
The methodology was used to establish the ligand requirements of human
Roundabout receptor 1 (Robo1), which is involved in a number of developmental
processes. Mass spectrometric analysis of the starting octasaccharide
mixture and the Robo1-bound fraction indicated that Robo1 has a preference
for a specific set of structures. Further analysis was performed by
sequential permethylation, desulfation, and pertrideuteroacetylation
followed by online separation and structural analysis by MS/MS. Sequences
of tetrasaccharides could be deduced from the data, and by combining
the compositional and sequence data, a putative octasaccharide ligand
could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S).
A modular synthetic approach was employed to prepare the target compound,
and binding studies by surface plasmon resonance (SPR) confirmed it
to be a high affinity ligand for Robo1. Further studies with a number
of tetrasaccharides confirmed that sulfate esters at C-6 are critical
for binding, whereas such functionalities at C-2 substantially reduce
binding. High affinity ligands were able to reverse a reduction in
endothelial cell migration induced by Slit2-Robo1 signaling