Characterisation of the interaction of neuropilin-1 with heparin and a heparan sulfate mimetic library of heparin-derived sugars

Abstract

Background. Neuropilin-1 (NRP-1) is a multidomain membrane protein with soluble isoforms interacting with a complex network of other membrane receptors, their respective ligands and heparan sulfate (HS). It is involved in the development of vasculature, neural patterning, immunological responses and pathological angiogenesis.Methods. We have characterised the binding of a Fc fusion of rat NRP-1 (Fc rNRP-1) and of a soluble isoform, corresponding to the first four extracellular domains of human NRP-1, shNRP-1, using optical biosensor-based binding assays with a library of heparin derivatives. Selective labelling of lysines protected upon heparin binding allowed their identification by mass spectrometry.Results. Fc rNRP-1 bound to heparin with high affinity (2.5 nM) and fast ka (9.8 × 106 M−1s−1). Unusually, NRP-1 bound both highly sulfated and completely desulfated stretches of heparin and exhibited a complex pattern of preferences for chemically modified heparins possessing one or two sulfate groups, e.g., it bound heparin with just a 6-O sulfate group better than heparin with any two of N-sulfate, 6-O sulfate and 2-O sulfate. Mass-spectrometry based mapping identified that, in addition to the expected the b1 domain, the a1, and c domains and the L2 linker were also involved in the interaction. In contrast, shNRP-1 bound heparin far more weakly. This could only be shown by affinity chromatography and by differential scanning fluorimetry.Discussion. The results suggest that the interaction of NRP-1 with HS is more complex than anticipated and involving a far greater extent of the protein than just the b1–b2 domains. NRP-1’s preference for binding long saccharide structures suggests it has the potential to bind large segments of HS chains and so organise their local structure. In contrast, the four domain soluble isoform, shNRP-1 binds heparin weakly and so would be expected to diffuse away rapidly from the source cell.

Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

“…Three lysines of the peptide ' 131 YLAMNKKGKLY 141 ' (Lys-125, Lys-126 and Lys-128) of FGF10 were found to be both acetylated and biotinylated (figure 9). This has been observed previously in other proteins [34,44], and is considered to be due to the local dissociation of a lysine side chain from its interaction with the polysaccharide. In the presence of the NHSacetate used in the protection step, the transiently dissociated lysine side chain becomes acetylated, and this would likely preclude its re-binding to the polysaccharide.…”

Heparin/Heparan Sulfate Proteoglycans Glycomic Interactome in Angiogenesis: Biological Implications and Therapeutical Use

“…NPR-1 can be shed as a short form composed of its extracellular domain. Both membrane-associated and free forms of NRP-1 bind heparin, with possible, still unexplored, biological consequences [ 129 ]. Robo binds heparin with low affinity (Kd equal to 650 nM) [ 86 ], the consequences of such interaction have been already described above for its ligand Slit.…”

Neuropilin-1 is a receptor for extracellular miRNA and AGO2/miRNA complexes and mediates the internalization of miRNAs that modulate cell function

“…The molecular basis for these numerous interactions is not well understood, but crystal structure studies and other investigations have identified the binding sites of VEGF. Importantly, it binds negatively charged heparin [ 15 , 16 ], which raised the possibility that it might also bind nucleic acids. Here, we report that NRP1 binds miRNAs with high affinity and promotes their entry into the cell.…”