PATTERN AND SPACING OF BASIC-AMINO-ACIDS IN HEPARIN-BINDING SITES

Glycosaminoglycan (GAG)-protein interactions regulate a myriad of phys iologic and pathologic processes, yet an understanding of how these mo lecules interact is lacking, The role of the pattern and spacing of ba sic amino acids (arginine (R) and lysine (K)) in heparin binding sites was investigated using peptide analogs as well as by examining known heparin binding sites, Peptides having the general structure RnW (n = 3-9, where tyrosine (W) was added for peptide detection) were synthesi zed and their interaction with heparin was determined by isothermal ti tration calorimetry, Binding affinity increased with increasing number of R residues, A 9-mer of R (R9W) bound as tightly to heparin as acid ic fibroblast growth factor under physiologic conditions, Despite thei r high affinity for heparin, long stretches of basic amino acids are u ncommon in heparin binding proteins, Known heparin binding sites most commonly contain single isolated basic amino acids separated by one no nbasic amino acid, Peptides having the structure, H3CCONH-GRRG(m)RRG(5 -m)-CONH2 (denoted as the RRG(m)RR peptide series) and H3CCONH-GRRRG(m )RG(5-m)-CONH2 (denoted as the RRRG(m)R peptide series), where m = 0-5 , were synthesized to test the hypothesis that the spacing of basic am ino acids in heparin binding sites is optimally arranged to interact w ith different GAGs, The peptides in both the -RRG(m)RR- and -RRRG(m)R- peptide series, when m = 0, bound most tightly with heparin, as measu red by affinity chromatography. In contrast, the -RRG(m)RR-peptide ser ies interacted most tightly with heparan sulfate when m = 0 or 1, wher eas the -RRRG(m)R- peptide series bound tightest when m = 3, These res ults are consistent with our understanding of heparin and heparan sulf ate structure, A highly sulfated GAG, such as heparin, interacts most tightly with peptides (or peptide sequences within proteins) containin g a complementary binding site of high positive charge density, Hepara n sulfate, having fewer and more highly spaced negatively charged grou ps, interacts most tightly with a complementary site on a peptide (or peptide sequences with proteins) that has more widely spaced cationic residues. (C) 1997 Academic Press