Identification of Heparin-binding Sites in Proteins by Selective Labeling

Abstract

Heparan sulfate proteoglycans are a ubiquitous component of the extracellular space of complex organisms (1). They are characterized by their size and their plasticity that derives mainly from the complex and dynamically regulated structure of the glycosaminoglycan (GAG) 1 moiety.They participate in the structural organization of the extracellular space (2, 3) and play an active role in molecular networks driving complex biological phenomena such as development (4 -6), inflammation and immune response (7,8), and disease (9). Heparan sulfate proteoglycans exert their functions by interacting with a vast number of protein partners and so regulating their activity (8, 10).In the last decade, a number of techniques have been used to investigate the interaction between proteins and GAGs (for reviews, see . However, despite the identification of more than 200 human heparin-interacting proteins (10, 16), our understanding of the structural features mediating the interaction remains quite poor (10, 13). Some models have been proposed, but they are based on the structural features of only restricted groups or families of heparin-binding proteins (HBPs) (11,17,18). Important limitations for modeling of heparin-binding sites (HBSs) are that many interactions are described solely at a qualitative level and that a three-dimensional structure of the sugar-protein complex is available for less than 10% of the annotated interactions (10). The available data derive mainly from x-ray crystallography (e.g. Refs. 19 and 20), NMR spectroscopy (e.g. Ref. 21), and quantitative biophysics with sitedirected mutagenesis (e.g. Ref. 22) or HBP-derived synthetic peptides (e.g. Ref. 23). Although providing detailed structural and kinetic information about the interaction, all these methods are limited to the study of single protein-sugar interactions, and they cannot be translated into a high throughput format.In 1989 Chang (24) used a lysine-reactive chromophoric reagent to investigate the HBS of antithrombin III (ATIII). ATIII was modified in the presence or absence of heparin, and the colored peptides generated by tryptic digestion were analyzed by RP-HPLC to obtain important insights into the heparin-ATIII interaction. N-Hydroxysuccinimide (NHS) esters have a strong and selective reactivity toward primary amines, in particular -amines on lysines, and their relative stability in neutral/weakly basic aqueous solution makes them compounds of choice for the investigation of HBSs, which are characterized by a high content of basic residues.We developed a rapid and reliable method for the localization of HBSs that can represent an important complement to any structural investigation of heparin-protein interactions because of its low requirement in terms of sample quantity and handling time. The new method, called the "protect and label" strategy, is based on the protection against chemical modification given by heparin/HS to residues located in the HBSs. Thus, an acetyl NHS ester was used to protect residues From the ‡School of Biological Sci...

Landscape of nuclear transport receptor cargo specificity

“…Thereby, primarily non‐biotinylated peptides elute after tryptic digestion and the labeled proteins are identified indirectly. Here, we introduced a subsequent elution step under harsh conditions using organic solvents (see Materials and Methods and Ori et al , 2009) to effectively elute the biotinylated peptides that are then identified directly. The combined approach is not only very effective to identify even transient interactors of NTRs, but it also provides the exact biotinylation sites as an additional layer of information.…”

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions

“…Taking the present Protect and Label data (Figs. 1 and 3-5) together with those published previously (26), how some aspects of heparin binding may have evolved across the FGFs can be proposed. As expected from sequence alignments, Protect and Label identifies the consensus heparin-binding site, HBS-1, in all the FGFs (Figs.…”

“…However, other heparin-binding sites have been demonstrated in FGF-1 and FGF-2 (36,37). Moreover, these sites have been successfully confirmed in FGF-2 by a Protect and Label analysis (26). In this approach, lysine side chains that remain exposed to solvent when the proteins bound to heparin are blocked with N-hydroxysuccinimide acetate.…”

“…Binding parameters of the association and dissociation phases were calculated using the nonlinear curvefitting software provided with the instrument (Fastfit, Farfield Protect and Label-Heparin-binding sites in FGF-1, FGF-7, FGF-9, and FGF-18 were identified by the "Protect and Label" approach, as described for FGF-2 (26), except that 1.5 nM FGF protein was used instead of 1.2 nM. Digested and biotinylated peptides were purified on a C18 ZipTip (Millipore Ltd., Watford, UK) (26) and then analyzed by tandem mass spectrometry. Up to 1 g of biotinylated peptides was injected into a LTQOrbitrap Velos instrument (Thermo) using a nanoAcquity UPLC system (Waters Associates).…”

HPLC-MS/MS of Highly Polar Compounds

“…The introduction of soft ionization methods (ESI), permitting an easy interfacing to HPLC has first provided an online separation and structure elucidation of complex oligosaccharide mixtures (Henriksen et al, 2006;Barosso et al, 2005;Thanawiroon et al, 2004). As a complement, matrix-assisted laser desorption ionization (MALDI) has proved to be a valuable tool for the analysis of protein/peptide-heparin/HS oligosaccharide complexes (Ori et al, 2009;Venkataram et al, 1999). The first important point to consider is the kind of oligosaccharides and disaccharides to be analyzed in particular if they are sulphated or not sulphated.…”