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

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 × 10(6) M(-1)s(-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.

Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

Following protein-glycosaminoglycan polysaccharide interactions with differential scanning fluorimetry.

Studies of the structural changes invoked in proteins by the binding of the glycosaminoglycan (GAG) polysaccharide portion of proteoglycans are of increasing importance to research in a wide range of fields, from biochemistry and molecular biology to biotechnology and medicine. One important aspect is the degree of stabilisation or destabilisation induced in a protein by the binding of these anionic materials, and this can affect enzyme activity, the stability of complexes, folding and the formation of aggregates, including those in neurodegenerative processes. A simple method, able to determine the effect of interactions with GAG polysaccharides on protein stability is described, based on the propensity of a fluorescent dye-Sypro™ Orange-to present differentiable fluorescence emission spectra following contact with exposed core amino acid residues. The method requires only commonly available and inexpensive equipment and is suitable for a multi-well format, allowing multiple readings to be made simultaneously.

Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis.

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.

Comparable stabilisation, structural changes and activities can be induced in FGF by a variety of HS and non-GAG analogues: implications for sequence-activity relationships.

The activities of heparan sulfate (HS) and heparin do not correlate simply with sulfation levels or sequence. The alternative hypothesis, that appropriate charge and conformational characteristics for protein binding and activity can be provided by other sequences in heparan sulfate and, possibly, also in unrelated sulfated polysaccharides, is explored. Differential scanning fluorimetry was used to measure the thermostabilisation bestowed by modified heparin polysaccharides (proxies for heparan sulfate) on fibroblast growth factor-1 (FGF-1) and fibroblast growth factor-2 (FGF-2), prototypical heparan sulfate-binding proteins, revealing varied abilities and primary sequence-activity redundancy. The effect of substitution pattern on the heparin/heparan sulfate backbone was explored using principal component analysis of (13)C NMR chemical shift data for homogeneously modified heparin polysaccharides revealing complex conformational effects. No simple relationship emerged between these polysaccharides, with their distinct charge distributions and geometries, and their ability to signal. Other, structurally unrelated sulfated polysaccharides were also able to support signalling. These influenced FGF stabilisation in a similar manner to the HS analogues and provided analogous cell signalling activity. For FGF-1, but not FGF-2, signaling correlated strongly with protein stabilisation and circular dichroism spectroscopy demonstrated that some non-HS polysaccharides invoked comparable secondary structural changes to those induced by heparin. Active conformations can readily be found in several heparin derivatives, as well as among non-HS polysaccharides, which comprise unrelated primary sequences, confirming the hypothesis and implying that the level of unique information contained in HS sequences may be much lower than previously thought.

Differential scanning fluorimetry measurement of protein stability changes upon binding to glycosaminoglycans: a screening test for binding specificity.

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.

Ciz1, a p21 cip1/Waf1-interacting zinc finger protein and DNA replication factor, is a novel molecular partner for human enhancer of rudimentary homolog.

Enhancer of rudimentary homolog (Drosophila) (ERH) is a small, highly conserved, nuclear protein with a unique three-dimensional structure, whose gene has been identified in animals, plants and protists, but not in fungi. Involvement of ERH in fundamental processes such as regulation of pyrimidine metabolism, cell cycle progression, transcription and cell growth control has been suggested. Here, employing a yeast two-hybrid system, a glutathione S-transferase pull-down assay and tandem MS, we demonstrate that Ciz1 is a bona fide interactor of human ERH. Ciz1 is a nuclear zinc finger protein interacting with p21(Cip1/Waf1), a universal inhibitor of cyclin-dependent kinases, and is a DNA replication factor. The region of Ciz1 necessary for the interaction with ERH spans residues 531-644, encompassing its first zinc finger motif. This region overlaps the p21(Cip1/Waf1)-binding site, suggesting that the interaction with ERH could block the binding of p21(Cip1/Waf1) by Ciz1 in the cell. When ERH and Ciz1 are coexpressed in HeLa cells, Ciz1 recruits ERH to DNA replication foci.

Human enhancer of rudimentary is a molecular partner of PDIP46/SKAR, a protein interacting with DNA polymerase delta and S6K1 and regulating cell growth.

Enhancer of rudimentary (ER) is a small protein that has a unique amino acid sequence and structure. Its highly conserved gene has been found in all eukaryotic kingdoms with the exception of fungi. ER was proposed to be involved in the metabolism of pyrimidines and was reported to act as a transcriptional repressor in a cell type-specific manner. To further elucidate ER functions, we performed the yeast two-hybrid screen of the human lung cDNA library for clones encoding proteins interacting with the human ER protein. The screen yielded polymerase delta interacting protein 46 or S6K1 Aly/REF-like target (PDIP46/SKAR), a protein possessing one RNA recognition motif (RRM) and being a protein partner of both the p50 subunit of DNA polymerase delta and p70 ribosomal protein S6 kinase 1 (S6K1). This interaction was further confirmed in vitro by the glutathione S-transferase-ER pull-down of a protein of 46 kDa from a nuclear extract from human cells which was identified as PDIP46/SKAR by tandem mass spectrometry. The bipartite region of PDIP46/SKAR interacting with ER comprising residues 274-421 encompasses the docking site for S6K1 within the RRM and two serines phosphorylated by S6K1. ER and both isoforms of PDIP46/SKAR share the same nuclear localization in the mammalian cells and their genes display a ubiquitous pattern of expression in a variety of human tissues, so the interaction between ER and PDIP46/SKAR has an opportunity to occur universally in mammalian cells. Because PDIP46/SKAR is involved in the regulation of cell growth its interaction with ER may suggest some function for ER in that control.