###### Abstract  
Although glycosaminoglycan (GAG)-protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs. 10 December 2021 2  
processes; for instance, embryonic development, assembly of the ECM, cell signaling, cell proliferation, angiogenesis and anti-coagulation. In addition, GAG-protein interactions are involved in many pathological contexts and human diseases, such as tumor progression, metastasis, inflammation, infectious diseases, mucopolysaccharides, and cardiovascular and neurodegenerative diseases. Hence, targeting specific GAG-protein interactions may be a valuable strategy for use in therapeutic drug development [1; 3; 4]. In general, the binding of GAGs with proteins is considered to be based mainly on electrostatic interactions between negatively charged sulfate or carboxylate groups of GAGs and positively charged amino acids in proteins. These amino acids include Arg, Lys and, to a lesser extent, His. Besides electrostatic interactions, van der Waals forces, hydrogen bonds and hydrophobic interactions with the carbohydrate backbone are also involved in the binding of proteins to GAGs [1; 5; 6]. Several amino acid consensus sequences have been demonstrated or have been predicted to be the sequences in proteins that interact with GAGs. As positively charged amino acids are clearly of major importance in GAG binding, these consensus sequences are characterized by specific arrangements of basic amino acids. [PERSON] and [PERSON] investigated the amino acid sequences of 21 heparin-binding proteins and determined two consensus sequences for the recognition of GAGs: XBBXBX and XBBBXXBX [7]. B and X represent basic and hydrophilic amino acid residues, respectively. The basic residues were most commonly Lys or Arg, as opposed to His, which was infrequently found. The amino acid residues Asn, Ser, Ala, Gly, Ile, Leu and Tyr were relatively abundant at the X positions in the XBBBXXBBX consensus sequence, whereas Cys, Glu, Asp, Met, Phe and Trp rarely occurred at these positions [7]. [PERSON] et al. assessed the amino acid sequences of the established heparin-binding domains of several proteins and observed a clear pattern of clustered cationic residues that are separated by predominantly non-polar, neutral amino acid residues. As a result, they proposed an optimal, palindromic consensus sequence: XBBXXBBBX, where cationic residues are represented by B. Moreover, they identified a 23-residue heparin-binding domain in human von Willebrand factor (Tyr565-Ala587), which closely approximates the proposed consensus sequence [8].