## 3 Discussion  
Chemokines are small proteins that have two main interaction partners: chemokine receptors, which are G protein-coupled receptors (GPCRs), and GAGs [24,25]. The binding of chemokines to GAGs enables the formation of a chemokine concentration gradient, which is indispensable for leukocyte migration [26]. Interfering with the interactions between GAGs and chemokines can be a useful therapeutic strategy to inhibit inflammation [27]. The chemokine CXCL9 binds to CXCR3 and is characterized by a highly positively charged COOH-terminal tail. A CXCL9-derived peptide, CXCL9(74-103), consisting of the COOH-terminal region of CXCL9 has been synthesized previously in our laboratory. This peptide lacks the capacity to bind to, and signal through, CXCR3 [15]. Interestingly, high affinity of CXCL9(74-103) for GAGs has already been shown in previous studies [15, 16, 17, 18, 19]. Since we aimed to elucidate the structural requirements of proteins for binding to GAGs, we started our research from this known GAG-binding peptide. As CXCL9(74-103) contains the two different GAG-binding motifs described by [PERSON] and [PERSON]: BBXB and BBBXXB, we selected these two motifs to be part of the amino acid sequences of seven new peptides (Table 1) [7]. [PERSON] et al. investigated the interactions of Arg and Lys with heparin [28]. A higher affinity of Arg-containing peptides for heparin was observed in comparison with Lys-containing peptides. In addition, acetyl-Arg-amide bound approximately 2.5 times more tightly to heparin than the corresponding acetyl-Lys-amide. This study suggests that the difference between these amino acids is due to the intrinsic properties of their side chains. To be specific, the tighter interaction between the guanidino group of the Arg side chain and the sulfates in heparin may be due to stronger hydrogen bonding and a more exothermic electrostatic interaction [28]. Despite this knowledge, we designed peptides with Lys and not Arg residues as basic amino acids because CXCL9(74-103) contains mainly Lys residues (13 Lys versus 3 Arg residues). Moreover, the GAG-binding motifs that are present in this CXCL9-derived peptide only contain Lys residues as basic amino acids. In order to study the interactions between several GAGs ((ds)LMWH, HS and HA) and peptides, IFT experiments were performed, and the resulting binding isotherms were used to calculate the K\({}_{\text{d}}\) values. In general, LMWH is more sulfated than HS, which consists of sulfated, non-sulfated and mixed domains [1; 29]. The dsLMWH is partially (2-O, 3-O) desulfated LMWH, but is expected to be still more sulfated than HS, although the sulfated domains of HS could have a higher density in sulfate groups. Thus, we estimate that the order of increasing sulfation of the GAGs used for IFT experiments is: HS < dsLMWH < LMWH. No significant difference in affinity for HS was detected between the long (KKKQQK) and short (KKQK) GAG-binding motifs (Figure 2). Trimer peptides (KKAK-, KKQKASAS- and KKQK-3 mers) showed higher affinity for HS in comparison with dimer peptides (KKAK-, KKQKASAS-, KKQK- and KKKQK-2 mers), indicating the contribution of the additional GAG-binding motif to the HS-binding affinity. This is consistent with the report that peptides containing multiple copies of the GAG-binding motifs XBBXXBX and XBBBXXB had higher affinity for heparin [30]. It is important to point out that CXCL9(74-103) had the highest affinity for HS, whereas it had only two GAG-binding motifs. Hence, not only the number, but also the type of GAG-binding motifs seems to determine the affinity for GAGs, as CXCL9(74-103) contains two different GAG-binding motifs, unlike the other peptides that were synthesized. In contrast to the affinities for HS, we observed higher affinities of the KKKQK-2 mer for binding to the two highest sulfated GAGs: LMWH and dsLMWH, in comparison with the KKQK-2 mer (Figure 4). Consequently, the BBBXXB GAG-binding motif appears to be an important element for binding to these GAGs.