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.
Highlights
Neuropilin-1 (NRP-1, reviewed Uniewicz & Fernig, 2008), was first described in 1987 as antigen A5 expressed by neural cells of Xenopus tadpoles, with a role as a neuronal recognition molecule regulating neural path finding and cell differentiation
The commercially available NRP-1 used in this study, Fc rNRP-1, encompasses most of the extracellular part of the native protein with the omission of amino acid residues 811–828
The interaction of shNRP-1 with heparin Given the differences found in the interaction of Fc rNRP-1 with heparin compared to that of the b1–b2 domain, we investigated the properties of shNRP-1 (Fig. S1). shNRP-1 covers the sequence of a naturally occurring alternatively spliced soluble human isoform of NRP-1 and corresponds to domains a1, a2, b1, b2, but lacks the L2 linker and the c domain (Fig. S1)
Summary
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. This could only be shown by affinity chromatography and by differential scanning fluorimetry. 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. The four domain soluble isoform, shNRP-1 binds heparin weakly and so would be expected to diffuse away rapidly from the source cell.
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