Biochemical and Biophysical Research Communications
Structural determinants of heparan sulfate interactions with Slit proteins
Section snippets
Materials and methods
Materials. Heparin (MW: 14,000) from porcine intestinal mucosa, sodium salts, was prepared by Celsus Laboratories (Cincinnati, OH). LMW heparin from porcine intestinal mucosa, sodium salt (MW: 5000) prepared using nitrous acid was purchased from Calbiochem–Novabiochem (La Jolla, CA). Heparin lyase I (heparinase, EC 4.2.2.7) was purchased from Sigma Chemical (St. Louis, MO). Gel permeation chromatography was performed on Bio-Gel P2, P6 (superfine) from Bio-Rad (Richmond, CA) or Sephadex G10 from
ELISAs of the interactions of glypican-1 with Slit
Using ELISAs in which we quantitated the inhibition of glypican-1 binding to Slit-2 by heparin oligosaccharides of defined structure [20], it was found that maximum inhibition of binding begins to be seen with a heparin decasaccharide, and is somewhat more extensive with dodecasaccharides and tetradecasaccharides (i.e., 6–7 disaccharide units; Fig. 1). Using the same test system, it was found that the small hexasulfonated molecule suramin (MW 1429 for the sodium salt) at a concentration of 5 μM
Conclusions
The studies described here have provided additional information on the fine structural features required for the heparan sulfate-mediated binding of glypican-1 to Slit proteins, and also demonstrated that significant inhibition of these interactions can be obtained by small sulfated molecules whose structures are entirely unrelated to those of heparin and heparan sulfate. In addition to earlier evidence for the role of cell surface heparan sulfate in the repulsive guidance activities of Slit-2
Acknowledgements
This research was supported by Grants HL52662 (R.J.L.) and NS13876 (R.U.M.) from the National Institutes of Health and by the Ron Shapiro Charitable Foundation.
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2013, BiochimieCitation Excerpt :These observations suggest that HS may interact with Slit and Robo simultaneously, resulting in the formation of ternary Slit/Robo/HS signaling complexes on cell surface thereby facilitating the Slit–Robo signaling, reminiscent of the well-accepted FGF/FGFR/HS and VEGF/VEGFR/HS models [38–42]. In previous studies we used heparin, a highly sulfated form of HS that is abundantly available, as a model of HS to determine the structural features required for HS to interact with Slit2 and Slit3, and demonstrated Slit shows a heparin/HS-specific interaction with high affinity and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfo groups and carboxyl moiety of the polysaccharide [43,44]. In the present study, we carried out surface plasmon resonance (SPR) analysis to determine the HS structure that is important for HS interaction with Robo1.
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2012, Advances in Cancer ResearchCitation Excerpt :However, it is unclear whether or not these GAGs constitute membrane bound proteoglycans. Genetic studies in Drosophila suggest that this may be the case, as the heparan sulfate proteoglycan, glypican, which is associated with the cell surface via a glycosylphosphatidylinositol (GPI) linkage, interacts with Slit and regulates its distribution (Liang et al., 1999; Ronca et al., 2001; Smart et al., 2011; Zhang et al., 2004). Thus, heparan sulfate GAGs, either cell associated or present as free sugars in the extracellular matrix, concentrate, and localize Slits, shaping the signaling environment by regulating their concentration and accessibility.
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2007, Chemistry and BiologyCitation Excerpt :We also sought to investigate whether HS might interact with a variety of chemotactic proteins and thus participate more broadly in axon guidance. We found that slit2 bound to heparin on the microarray, consistent with cellular studies in which 125I-labeled slit interacted with the surface of HS-expressing cells and recent SPR studies with HS oligosaccharides (Figure 5A) [16, 54]. More importantly, we observed a strong preference of slit2 for 6-O-sulfated heparin, as indicated by a significant decrease in slit2 binding upon desulfation at the 6-position.