Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo

Fukuhara, Noémi; Howitt, Jason; Hussain, Sadaf-Ahmahni; Hohenester, Erhard

doi:10.1074/jbc.m800688200

Cited by 83 publications

(106 citation statements)

References 41 publications

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“…However, the large size, heterogeneous structure, and inherent flexibility of GAG chains make the detailed structural interrogation of protein-GAG interactions difficult, particularly for full-length GAG chains. Previous studies have shown that heparin octasaccharides bind to Drosophila Robo directly, and the heparin dp8 binding sites have been identified by mutagenesis and partial direct crystallographic observation of a bound heparin oligosaccharide (21). These observations indicate that HS/heparin plays a direct and essential role in Slit-Robo signaling.…”

mentioning

confidence: 63%

“…Here, this technology is applied to characterizing the interaction of Robo1 Ig1-2 with unfractionated heparin. We have identified basic residues in and around the previously identified site for high affinity heparin binding (21) as well as a previously unidentified second binding site near the N terminus of Robo1 that is evolutionarily conserved and influences heparin binding as determined by site-directed mutagenesis, SPR, and heparin affinity chromatography. We have also identified residues in the hydrophobic core of the Ig2 domain that show altered solvent accessibility upon binding of unfractionated heparin, suggesting a heparin binding-induced conformational change in the Ig2 domain that may be involved in heparin-induced signal transduction.…”

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confidence: 99%

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High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

Moniz

Wang

et al. 2015

Journal of Biological Chemistry

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Background:The molecular basis of full-length heparin activation of Slit-Robo is poorly understood, despite its importance in nervous system development. Results: Two separate binding sites of Robo1-full length heparin interaction were identified. Conclusion: A model for heparin/heparan sulfate binding and activation of the Slit-Robo complex is proposed. Significance: A previously unidentified Robo1 low affinity binding site for heparin may be required for signal transduction.

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confidence: 63%

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confidence: 99%

High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

Moniz

Wang

et al. 2015

Journal of Biological Chemistry

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“…SLIT2 and ROBO1 have previously been shown to bind HS (23)(24)(25)(26), which suggests that SLIT3 and ROBO4 might function in a similar manner to interact with endothelial HS. To test this idea, we generated the N-terminal fragments of SLIT3 (SLIT3-N) and the extracellular domain of ROBO4.…”

Section: -O-sulfation By 20% (mentioning

confidence: 99%

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

Zhang¹,

Xiao²,

Qiu³

et al. 2013

J. Clin. Invest.

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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.

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“…Moreover, as we discuss below, several studies have identified HSPGs as co-receptors for Slits that are necessary for Slit-Robo signalling (Hu, 2001;Steigemann et al, 2004). Recent structural analysis has revealed that Slit/Robo form a ternary complex with a heparin/HS that stabilises and strengthens the Slit-Robo interaction (Fukuhara et al, 2008;Hussain et al, 2006). And, as mentioned above, structural studies have also shown that the Slit D4 region binds HS and promotes Slit homodimerisation (Seiradake et al, 2009).…”

Section: Inferior Olive (Io)mentioning

confidence: 99%

Moving away from the midline: new developments for Slit and Robo

2010

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SummaryIn most tissues, the precise control of cell migration and cellcell interaction is of paramount importance to the development of a functional structure. Several families of secreted molecules have been implicated in regulating these aspects of development, including the Slits and their Robo receptors. These proteins have well described roles in axon guidance but by influencing cell polarity and adhesion, they participate in many developmental processes in diverse cell types. We review recent progress in understanding both the molecular mechanisms that modulate Slit/Robo expression and their functions in neural and non-neural tissue. Key words: Axon guidance, Cell migration, Cell-cell interaction IntroductionIn most organisms, the central nervous system (CNS) develops along a bilateral axis of symmetry located at the midline (Placzek and Briscoe, 2005). During development, the ventral midline or floor plate acts as an organiser through the secretion of diffusible proteins (Placzek and Briscoe, 2005;Gore et al., 2008), which control the growth of axons and dendrites and the migration of neurons across the midline. In the forebrain, glial or neuronal cells delineate the midline (see Glossary, Box 1) and also control axon guidance. For more than two decades, many developmental neurobiologists have tried to understand the mechanisms that control midline crossing in the CNS and to answer some key questions. Firstly, how are commissural axons (see Glossary, Box 1) attracted to the midline and how do their growth cones (see Glossary, Box1) receive and integrate the multiple and contrasting signals released by midline cells? Secondly, what are the molecular and signalling changes that enable commissural axons to leave the midline and often to switch to a longitudinal growth mode? All midline-derived axon guidance factors are expressed at other locations in many developing and mature tissues, where they control a wide range of biological processes.Roundabout receptors (Robo) and their Slit ligands form one of the most crucial ligand-receptor pairings among the axon guidance molecules. Robos were identified in Drosophila in a mutant screen for genes that control the midline crossing of commissural axons (Kidd et al., 1998;Seeger et al., 1993). Similarly, Slit was discovered in Drosophila as a protein secreted by midline glia (Rothberg et al., 1988;Rothberg et al., 1990). Homologues of both proteins have since been discovered in many species (for a review, see . However, the Slit/Robo couple not only functions in axon guidance but also in a variety of developmental Development 137, 1939Development 137, -1952Development 137, (2010 Commissural axons Axons with cell bodies (somata) located on one side of the central nervous system (CNS) that project axons across the midline to contact target cells on the opposite side. Growth cone A specialised bulbous enlargement at the end of growing axons that is characterised by dynamic filamentous extensions, known as filopodia. They sense the environment and respond to adhesi...

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Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo

Cited by 83 publications

References 41 publications

High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

Moving away from the midline: new developments for Slit and Robo

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