Structural determinants of heparan sulfate interactions with Slit proteins.
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 (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.
Zhang
Fuming
F
Departments of Chemistry, Biology, and Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Ronca
Francesca
F
Linhardt
Robert J
RJ
Margolis
Richard U
RU
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029721:Drosophila Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013552:Swine
D029721:Drosophila Proteins; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D009419:Nerve Tissue Proteins; C066819:sli protein, Drosophila