Identification of novel binding sites for heparin in receptor protein-tyrosine phosphatase (RPTPσ): Implications for proteoglycan signaling

Abstract

Receptor protein-tyrosine phosphatase RPTPσ has important functions in modulating neural development and regeneration. Compelling evidence suggests that both heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs) bind to a series of Lys residues located in the first Ig domain of RPTPσ. However, HS promotes and CS inhibits axonal growth. Mutation of these Lys residues abolished binding and signal transduction of RPTPσ to CS, whereas HS binding was reduced, and signaling persisted. This activity was mediated through novel heparin-binding sites identified in the juxtamembrane region. Although different functional outcomes of HS and CS have been previously attributed to the differential oligomeric state of RPTPσ upon GAG binding, we found that RPTPσ was clustered by both heparin and CS GAG rich in 4,6--disulfated disaccharide units. We propose an additional mechanism by which RPTPσ distinguishes between HS and CS through these novel binding sites.

Crystal and solution structures of fragments of the human leucocyte common antigen-related protein

“…In agreement with these results, we found that LAR Ig1-3 was retained on a heparin column and that LAR Ig1-3 bound Hep-FITC with a nanomolar affinity. This is compatible with the values obtained for the affinity of PTPR for CS (K d = 11 nM; Shen et al, 2009) and for heparin (K d = 0.3 nM; Katagiri et al, 2018) considering that these values were obtained using immobilized ligands, whereas the K d measured here was for the components in solution. Furthermore, we found that Innohep induced a shift in hydrodynamic size for both LAR Ig1-3 and LAR FN5-8 while the chemically well defined pentameric LMWH drug Arixtra did not.…”

“…This was confirmed by the MST measurements of FITC-Hep, which showed affinity in the low micromolar range. This is in contrast to the heparin affinity of the PTPR juxtamembrane domain, which was 0.6 nM (Katagiri et al, 2018). As for LAR Ig1-3 , analysis by SEC showed that Innohep was likely to induce oligomerization of LAR FN5-8 while Arixtra did not, indicating that the minimal sugar length for oligomerization is larger than five.…”

“…These soluble protein fragments were heterogeneously expressed in bacterial or insect cells and purified to homogeneity for subsequent biochemical and structural studies. Binding to sulfated proteoglycans has been shown to induce oligomerization for PTPR, thus modulating its biological activity, and a recent study identified an additional heparin-binding site in PTPR (Coles et al, 2011;Katagiri et al, 2018). To test whether human LAR contains additional heparin-binding sites, we performed heparin-affinity chromatography.…”

“…A recent study has identified an additional binding site for heparin on PTPR (Katagiri et al, 2018) located in or near the juxtamembrane FNIII domain. We found that LAR FN5-8 was retained on the heparin column, although it eluted at a lower NaCl concentration than LAR Ig1-3 , suggesting weaker affinity for heparin.…”

“…The binding site for both HSPG and CSPG has been mapped to a cluster of alkaline residues on the first Ig domain of all three PTPRs (Coles et al, 2011;Gao et al, 2018). However, a recent study has identified a second binding site specific for HSPG near the fourth FNIII domain of PTPR (Katagiri et al, 2018). The biological significance of this second binding site remains to be fully understood.…”