title: Selective labelling of arginine residues in protein sulfated glycosaminoglycan binding sites
Creators: name: Bui, TP
Publisher: name: University of Liverpool Repository
Year: 2019

description: In animals, the interaction of numerous proteins with glycosaminoglycans (GAGs) regulates many aspects of their functions, and so organism development and homeostasis, as well many related pathologies. The interactions between the GAG heparan sulfate (HS) and the fibroblast growth factors (FGFs) have become a major paradigm for how such interactions regulate biological outcomes. Thus, binding HS controls FGF stability, its diffusion between cells and the generation of intracellular signals via the cognate FGF receptors (FGFRs). The basic arginine and lysine amino acids on the surface of FGFs have an important role in binding the polysaccharide via its numerous sulfate and carboxyl groups. Indeed electrostatic bonds between FGF and GAG dominate the interaction, at least kinetically. However, the contribution of arginine residues on the protein surface to GAG binding has generally not been established. This has been addressed directly in this thesis. A means to selectively label and so identify arginine side chains involved in binding heparin as an approximation for cellular GAGs was developed and evaluated, using the two most studied FGFs, FGF1 and FGF2. Two chemicals which selectively and specifically react with the guanidino group of arginine, phenylglyoxal (PGO) and hydroxyphenylglyoxal (HPG) were chosen for the purpose of this work. By combining mass spectrometry and an automatic programming language, the multiple products of PGO’s reaction with arginine were readily deconvoluted. The method was then applied to the other thirteen paracrine FGFs, which were produced as recombinant proteins. In addition, lysine selective labelling data were acquired for those FGFs where this was lacking. Thus, a complete description of the surface electrostatic map on fifteen paracrine FGFs guiding heparin engagement was obtained. The addition of arginine residues into the map of binding makes adjustments to the definition of heparin binding sites (HBSs) in some FGFs, for instance, FGF4. The refinement of the assignment of residues to secondary HBSs is consistent with data obtained by others on the ability of particular FGFs (FGF4, FGF6, FGF17, FGF18 and FGF10) to cross-link HS chains. In addition, the data raise some interesting properties of GAG binding, for example, the dual specificity of the secondary heparin binding site, HBS-3, in some FGFs, which is also involved in binding the FGFR. While the results generally support the idea that the conservation of structures for GAG binding in FGFs is related to their evolutionary divergence, it also suggests that changes in the structures in GAG binding may in some instances have diverged more rapidly since an amino acid substitution between members of a subfamily enables a significant alteration in heparin engagement. This suggests that in some instances, changes to the heparin binding properties of an FGF may contribute to a diversification of function within a subfamily. A further method was developed, whereby the entire selective labelling was done in solution, without the need for a heparin affinity column. The method was applied to the interactions of members of the FGF7 subfamily (FGF3, FGF7, and FGF10) with heparin, followed by chondroitin sulfate (CS), dermatan sulfate (DS). This allowed the analysis of lysine residues involved in binding physiologically relevant GAGs rather than heparin. Finally, the wider applicability of the method was acquired by determining the lysine and arginine residues that bind heparin in a ‘phage display antibody’, HS4C3. Due to the selectivity of HS4C3 for anticoagulant structures in heparin through its CDR3 loop, it was possible to use the data to propose a means to model protein-GAG binding. This was demonstrated using the knowledge of the binding specificity of HS4C3 for 3-O-sulfate and the positions of residues identified by the selective labelling. Taken together, the work in this thesis provides new insight into the involvement of arginine and lysine residues in the engagement of proteins to GAGs. This may enable future ‘omics’ approaches to identify GAG binding sites in proteins in cells and tissues, and to predict from principles where such sites are on a protein surface.