Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis.

Roundabout 4 (Robo4) is a transmembrane receptor that expresses specifically in endothelial cells. Soluble Robo4 was reported in the human plasma and mouse serum and is inhibitory towards FGF- and VEGF-induced angiogenesis. It remains unknown how soluble Robo4 is generated and if soluble Robo4 regulates additional angiogenic signaling. Here, we report soluble Robo4 is the product of constitutive ectodomain shedding of endothelial cell surface Robo4 by disintegrin metalloproteinases ADAM10 and ADAM17 and acts to inhibit angiogenic Slit3 signaling. Meanwhile, the ligand Slit3 induces cell surface receptor Robo4 endocytosis to shield Robo4 from shedding, showing Slit3 inhibits Robo4 shedding to enhance Robo4 signaling. Our study delineated ADAM10 and ADAM17 are Robo4 sheddases, and ectodomain shedding, including negative regulation by its ligand Slit3, represents a novel control mechanism of Robo4 signaling in angiogenesis.

A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia.

Mitochondrial genome content and structure vary widely across the eukaryotic tree of life, with protists displaying extreme examples. Apicomplexan and dinoflagellate protists have evolved highly reduced mitochondrial genome sequences, mtDNA, consisting of only three cytochrome genes and fragmented rRNA genes. Here, we report the independent evolution of fragmented cytochrome genes in Toxoplasma and related tissue coccidia and evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) totaling 5.9 kb that exist as nonrandom concatemers. Single-molecule Nanopore reads consisting entirely of SBs ranging from 0.1 to 23.6 kb reveal both whole and fragmented cytochrome genes. Full-length cytochrome transcripts including a divergent coxIII are detected. The topology of the mitochondrial genome remains an enigma. Analysis of a cob point mutation reveals that homoplasmy of SBs is maintained. Tissue coccidia are important pathogens of man and animals, and the mitochondrion represents an important therapeutic target. The mtDNA sequence has been elucidated, but a definitive genome architecture remains elusive.

Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements.

Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.

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.

High-resolution probing heparan sulfate-antithrombin interaction on a single endothelial cell surface: single-molecule AFM studies.

Heparan sulfate (HS) plays diverse functions in multiple biological processes by interacting with a wide range of important protein ligands, such as the key anticoagulant factor, antithrombin (AT). The specific interaction of HS with a protein ligand is determined mainly by the sulfation patterns on the HS chain. Here, we reported the probing single-molecule interaction of AT and HS (both wild type and mutated) expressed on the endothelial cell surface under near-physiological conditions by atomic force microscopy (AFM). Functional AFM imaging revealed the uneven distribution of HS on the endothelial cell surface though they are highly expressed. Force spectroscopy measurements using an AT-functionalized AFM tip revealed that AT interacts with endothelial HS on the cell surface through multiple binding sites. The interaction essentially requires HS to be N-, 2-O- and/or 6-O-sulfated. This work provides a new tool to probe the HS-protein ligand interaction at a single-molecular level on the cell surface to elucidate the functional roles of HS.

Heparan sulfate modulates Slit3-induced endothelial cell migration.

Heparan sulfate is a long, linear polysaccharide with sulfation modifications and belongs to the glycosaminoglycan family. Our recent studies elucidated that the axon guidance molecule Slit3 is a new heparan sulfate-binding protein and a novel angiogenic factor by interacting with its cognate receptor Robo4, which is specifically expressed in endothelial cells. Here we describe using heparan sulfate-deficient mouse endothelial cells to determine the co-reception function of heparan sulfate in Slit3-induced endothelial cell migration in a Boyden chamber trans-well migration assay.

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.