## 3 Discussion
The additional parameters that contribute to the GAG-specificity of peptides include non-basic amino acids in the GAG-binding motifs and linker sequences (Figures 3 and 5). To be specific, Gln residues increased the binding affinity of trimer peptides for LMWH, dsLMWH and HS, whereas Ala residues increased the binding affinity for HA. The importance of the non-basic amino acid residues for high affinity binding to GAGs was also demonstrated for mouse serglycin-derived peptides [30]. A fragment (YPARRARYQWVRCKP) from the native mouse serglycin core protein that contained a BBXB GAG-binding motif had significant heparin-binding affinity. A comparison of this native peptide with a modified peptide (AAARRARAAAAARAKA), in which Ala residues replaced all non-basic amino acids, revealed a 350-fold decrease in heparin-binding affinity [30]. As expected, the presence of sulfate groups on the GAGs also appears to be important for the binding of peptides to GAGs, since a lower affinity for dsLMWH in comparison with LMWH was observed for all peptides (Figure 5). Surprisingly, the KAK-3 mer, and not CXCL9(74-103), showed the highest affinity for the non-sulfated HA. In order to study the binding of peptides to cellular GAGs, a cell line that could function as an appropriate negative control was needed. Therefore, we chose to use CHO-K1 cells and pgsB-618 cells, which do not produce GAGs. CHO cells are known to produce HS, CS and DS [31; 32; 33]. The pgsB-618 cell line was developed by [PERSON] et al., who treated CHO-K1 cells with ethyl methanesulfonate, and subsequently screened for mutants that were defective in proteoglycan synthesis [22]. Five CHO cell mutants, showing less than 2% of the \(\beta\)4 Gal-T7 activity that was observed in wildtype CHO-K1 cells, were isolated. The synthesis of both HS and CS was abolished in these \(\beta\)4 Gal-T7-deficient cells [22]. The enzyme \(\beta\)4 Gal-T7 is responsible for the attachment of the first galactose in the biosynthesis of the tetrasaccharide linkage region (GlcA\(\beta\)1-3 Gal\(\beta\)1-3 Gal\(\beta\)1- 4 Xyl\(\beta\)1-O-Ser) that is common to HS, heparin, CS and DS [23; 34]. Surprisingly, the pgsB-618 cells appeared to be a mixed population of cells as both the HS\({}^{+}\) and HS\({}^{-}\) subpopulations were detected using flow cytometry (Figure S1). Therefore, either this cell line was not derived from a single cell, the cell line had been contaminated with an HS\({}^{+}\) CHO cell line or a post-cloning genetic event had occurred, resulting in the mixed population of cells [35]. [PERSON] et al. mentioned the possible presence of revertant cells, and therefore only subcultured the cells less than 15 times. However, the cells we received had a low passage number (#8), so the presence of revertant cells at the time the expression of HSwas tested initially was very unlikely. Furthermore, [PERSON] et al. reported the \(\beta\)4 Gal-T7 activity of the pgsB-618 cells to be 1.6% in comparison with the wildtype CHO cells. After subcloning the pgsB-618 cells, several HS\({}^{-}\) and HS\({}^{+}\) clones were obtained. The binding of TAMRA-labeled peptides (KKAK-2 mer, KKQK-3 mer, KKKQQK-2 mer and CXCL9(74-103)) to cellular GAGs was assessed using HS\({}^{+}\) and HS\({}^{-}\) CHO cells in parallel. The four peptides bound significantly better to HS\({}^{+}\) than to HS\({}^{-}\) CHO cells (Figure 7). A trend towards an increased binding capacity of the KKKQQK-2 mer peptide in comparison with the KKQK-3 mer for cellular GAGs could be noticed. This observation is not in accordance with the presence of less GAG-binding motifs in the KKKQQK-2 mer, suggesting a possible contribution of the BBBXXB GAG-binding motif to the binding capacity for cellular GAGs. Furthermore, the KKAK-2 mer and CXCL9(74-103) showed the lowest and highest binding capacity for cellular GAGs, respectively, which is in line with the IFT data. Remarkably, the binding of these four peptides to HS\({}^{-}\) CHO cells was not entirely abrogated (Figure 7B).