Characterization of heparin binding by a peptide from amyloid P component using capillary electrophoresis, surface plasmon resonance and isothermal titration calorimetry

Abstract

Synthetic peptides based on amino-acid residues 27-38 of human serum amyloid P component represent a novel type of heparin binders as they do not contain clusters of basic amino acids or other known features associated with protein or peptide heparin binding. Here, we characterize the binding using capillary electrophoresis (CE), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). By CE, heparin-binding activity was readily apparent for both a regular peptide and a slightly N-terminally modified form, while a sequence-scrambled peptide had no measurable binding. Dissociation constants in the 1-15 lM range were estimated, but only a minor part of the binding isotherm was covered by the experiments. SPR measurements using immobilized peptides verified heparin binding, the range of the binding constants, and the reduced binding of the sequence-scrambled peptide. Structurally defined heparin oligosaccharides were used to establish that while the tetrasaccharide is too small to exhibit strong binding, little difference in binding strength is observed between hexa-and tetradeca-saccharides. These experiments also confirmed the almost complete lack of activity of the sequence-scrambled peptide. The amino-acid sequencedependent binding and the importance of a disulfide bond in the peptide were verified by ITC, but the experimental conditions had to be modified because of peptide precipitation and ITC yielded significantly weaker binding constants than the other methods. While the precise function of the peptide in the intact protein remains unclear, the results confirm the specificity of the glycosaminoglycan interaction with regard to peptide sequence by applying two additional biophysical techniques and showing that the N-terminal part of the peptide may be modified without changing the heparin binding capabilities.Keywords: amyloid P component; heparin binding; surface plasmon resonance; capillary electrophoresis; isothermal titration calorimetry.Amyloid P component (AP) is a heparin-binding serum protein, which is also found outside the circulation in basement membranes, connective tissue, and in all types of amyloid deposits [1][2][3][4][5]. Because of its largely unknown physiological function [6], its affinity for amyloid, and possible involvement in the pathogenesis of systemic lupus erythematosus [7], the structure and activity of AP are intensely studied [8][9][10][11][12]. In a search for functional peptides in the AP structure with homology to known cell attachment peptides, a control peptide representing residues 27-38 of AP (AP-1) was surprisingly found to support cell attachment [13]. The peptide was subsequently found to bind heparin and other anionic carbohydrates including DNA, with apparent dissociation constants in the 0.5-10 lM range based on capillary electrophoresis (CE) and solid-phase inhibition assays [14][15][16]. We found that the tryptic glycopeptide corresponding to amino acids 14-38 of AP glycopeptide binds heparin [16] and that the attached carbohydrate protects a...

Search of ligands for the amyloidogenic protein β2-microglobulin by capillary electrophoresis and other techniques

“…A few molecules are known to bind the native form of b 2 -m, namely Congo Red [13][14][15], 8-anilino-1-naphthalene sulfonic acid [14], heparin [15][16][17], and the drug suramin [11], although none of these compounds has so far been demonstrated to stabilize the native form of b 2 -m.…”

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

“…Amyloid P component is a heparin-binding serum protein of largely unknown physiological function [95] with affinity for amyloid and presumably related to the pathogenesis of systemic lupus erythematosus [96]. Pre-eq CZE has been utilized in the search for structure-function relationships by investigation of the affinity of various amyloid P component-derived peptide sequences for heparin [44,61,97]. Structure dependent binding was found and affinity constants determined with pre-eq CZE.…”

Recent advances in capillary electrophoresis and capillary electrochromatography of peptides

“…Synthetic peptides derived from the amino acid residues 27-38 of human serum amyloid P component have been found as a novel type of heparin binders by CE of the mixture of these peptides with heparin [284]. The heparinbinding activity was readily apparent for both a regular peptide and a slightly N-terminally modified form with dissociation constants in the 1-15 mM range, while a sequencescrambled peptide did not exhibit any measureable binding.…”