Structural determinants of heparan sulfate interactions with Slit proteins

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Abstract

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican–Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin–Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin–heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (∼60% at 2 μM concentration) was attained with a dodecasaccharide.

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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