###### Abstract
Furthermore, a structural analysis of the acidic fibroblast growth factor (FGF), basic FGF and transforming growth factor-\(\beta\)1 (TGF-\(\beta\)1) by [PERSON] et al. led to the description of another type of consensus sequence: TXXBXTBXXTBB. In this consensus sequence, turns, basic amino acid residues (Arg, Lys or occasionally Gln) and hydrophilic amino acid residues are denoted by T, B and X, respectively. Basic amino acid residues are brought into proximity by the turns, which emphasizes the importance of the secondary structure in GAG-binding motifs [9]. In addition, [PERSON] et al. searched for a conserved structural pattern in 20 heparin-binding proteins and reported a cation-polar-cation (CPC) clip motif in heparin-binding proteins. This structural motif consists of one polar (preferentially Asn, Gln, Thr, Tyr or Ser) and two positively charged amino acid residues (Arg or Lys). Moreover, the spatial arrangement of this CPC clip motif is characterized by regular distances between the \(\alpha\) carbons as well as the center of gravity of the side chains of the amino acid residues. This structural motif acts as a conserved structural signature for heparin-binding proteins [10]. [PERSON] et al. sought potentially conserved HS/heparin-binding motifs in 437 HS/heparin-binding proteins and concluded that these motifs may be formed on protein surfaces by short, spaced, basic amino acid sequences in a three-dimensional arrangement [11]. This study, as well as examples such as antithrombin and XCL1 (lymphotactin), indicates that not only the primary sequence, but also the tertiary structure of proteins determines GAG-protein interactions [12; 13; 14]. The spatial proximity of the basic residues in the primary amino acid sequence is therefore not essential. Nevertheless, despite research concerning the molecular mechanisms underlying GAG-protein interactions, the structural requirements of proteins for binding to GAGs are still not well defined. In view of this, detailed studies of the elements contributing to GAG binding are valuable. Therefore, in this study, we aimed to elucidate the structural requirements for the binding of proteins to GAGs. Considering this, we investigatedthe role of consensus sequences, hereafter referred to as \"GAG-binding motifs\", in the interactions between GAGs and peptides. The chemokine-derived peptide CXCL9(74-103) was previously synthesized in our laboratory and consists of the 30 COOH-terminal amino acids of the chemokine CXCL9. Remarkably, more than 50% of the amino acids of this peptide are positively charged and it contains two GAG-binding motifs: \({}^{75}\)KKQK\({}^{78}\) (BBXB) and \({}^{85}\)KKKVLK\({}^{90}\) (BBBXXB). Several studies have demonstrated the binding of this peptide to GAGs with high affinity. Moreover, this CXCL9-derived peptide could compete with several chemokines and vascular endothelial growth factor (VEGF) for binding to GAGs [15; 16; 17; 18; 19; 20]. Therefore, CXCL9(74-103) served as a starting point for our research concerning the elements contributing to GAG binding. In essence, different peptides with varying lengths and varying numbers and types of GAG-binding motifs were designed. In order to evaluate the contribution of specific amino acids to GAG binding, we altered the amino acid sequences of the GAG-binding motifs and the sequences in between the GAG-binding motifs, hereafter referred to as \"linker sequences\". The peptides were chemically synthesized and their binding affinity for various GAGs (low-molecular-weight heparin (LMWH), partially desulfated LMWH (dsLMWH), HS and HA) was determined using isothermal fluorescence titration (IFT). In addition, we assessed the binding of several of these peptides to cellular GAGs on Chinese hamster ovary (CHO) cells with flow cytometry. Moreover, the binding of the peptides to cellular GAGs in the presence of soluble GAGs was investigated.