AffiliationsKeywords: arginine, phenylglyoxal, p-hydroxyl phenylglyoxal, fibroblast growth factor, heparansulfate, heparin binding site.
I-AbstractThe activities of hundreds of proteins in the extracellular space are regulated by binding to the glycosaminoglycan heparan sulfate (HS). These interactions are driven by ionic bonds between sulfate and carboxylate groups on the polysaccharide and the side chains of basic residues in the protein. Here we develop a method to selectively label the guanidino side chains of arginine residues in proteins that engage the anionic groups in the sugar. The protein is bound to heparin (a common experimental proxy for HS) on an affinity column.Arginine side chains that are exposed to solvent, and thus involved in binding, are protected by reaction with the dicarbonyl phenylgyoxal (PGO). Elution of the bound proteins then exposes arginine side chains that had directly engaged with anionic groups on the polysaccharide. These are reacted with hydroxyl-phenylglyoxal (HPG). PGO was found to generate three products: a 1:1 product, the 1:1 water condensed product and a 2:1 PGO:arginine product. These three reaction products and that of HPG had distinct masses.Scripts were written to analyse the mass spectra and so identify HPG labelled arginine residues. Proof of principle was acquired on model peptides. The method was then applied to the identification of heparin binding arginine residues in fibroblast growth factors (FGF) 1 and 2. The data demonstrate that four out of eleven arginine residues on FGF2 and five out of six arginine residues of FGF1 engage heparin. Our approach provides a rapid and reliable means to identify arginines involved in functional complexes such as those of proteins with heparin.
III-Materials and Methods
Heparin-binding proteinsRecombinant human FGF1 (UniProt accession P05230 -residues 1-155) and FGF2 (UniProt accession P09038 -residues 134-288) were expressed in C41 Escherichia coli cells using the pET-14b system (Novagen, Merck, Nottingham, UK) and purified, as described previously [29].
SDS-PAGE and Silver stainSamples were separated on 15% (w/v) polyacrylamide-SDS gels. Silver staining was done according to Heukeshoven and Dernick [30].
Selective protection and labelling of arginine side chains in HBSs of proteins using PGOand HPG (Fig 1A) A step by step guide is available at protocol.io at the following link: https://www.protocols.io/view/selective-protection-and-labelling-of-arginine-lys-qqmdvu6Step 1: Binding AF-heparin beads (Tosoh Biosciences GmbH, Stuttgart, Germany; binding capacity of 4 mg antithrombin III/mL) previously used in the lysine selective labelling protocol [11] were employed. A mini affinity column was made by placing a plastic air filter as a frit at the end of a P10 pipette tip (Star Lab Ltd., Milton Keynes, UK) and then packed with 20 µL AFheparin beads. The mini-column was equilibrated with 4 × 50 µL 200 mMNaCl, 0.2 M NaHCO 3, pH 9.5 (Na-1 buffer). The buffer was dispensed slowly into the column using a 2 mL sterile syringe. A minimu...