Interaction of the collagen-like tail of asymmetric acetylcholinesterase with heparin depends on triple-helical conformation, sequence and stability

Abstract

The collagen-like tail of asymmetric acetylcholinesterase (AChE) contains two heparin-binding domains (HBDs) that interact with heparan sulphate proteoglycans, determining the anchoring of the enzyme at the basal lamina and its specific localization at the neuromuscular junction. Both HBDs are characterized by a cluster of basic residues containing a core with the BBXB consensus sequence (where B represents a basic residue and X a non-basic residue). To study the interaction of such HBDs with heparin we have used synthetic peptides to model the N-terminal and C-terminal sites. CD spectroscopy showed that all peptides are triple-helical at low temperatures, and undergo trimer-to-monomer transitions. Displacement assays of asymmetric AChE bound to heparin were performed using the peptides in both monomeric and triple-helical states. In the monomeric conformation, all the peptides were able to displace low levels of AChE depending on the basic charge content. In the triple-helical conformation, peptides containing the consensus sequence showed a large increase in the ability to displace bound AChE. Results suggest that the specific binding of the collagen-like-tail peptides to heparin depends both on the presence of the core sequence and on the triple-helical conformation. Moreover, BBXB-containing peptides that are less stable are more effective in displacing AChE, suggesting that the interaction region needs a significant amount of structural flexibility to better accommodate the ligand.

Two Different Heparin-binding Domains in the Triple-helical Domain of ColQ, the Collagen Tail Subunit of Synaptic Acetylcholinesterase

“…1A). Consistent with this, a decrease in stability was observed when these arginines were replaced by alanines in triple-helical peptides modeling the Torpedo N-terminal HBD (13). Thus, depending on the contacts they establish, arginines could alternatively participate directly in heparin interactions or play a structural role, which in turn can affect the accessibility of the neighboring residues to the ligand.…”

“…Triple-helix Structural Properties as Modulators of Heparin Affinity-Previous studies using synthetic peptides have shown a negative correlation between affinity and triple-helical stability (13). Here, ColQ mutants in which a same residue was substituted by alanine or proline, generating an identical array of basic residues but likely to be subjected to different local stability, showed different affinities for heparin.…”

“…Equilibrium Binding Assays-Binding assays were performed in MaxiSorp 96-well plates (Nunc, Roskilde, Denmark) coated with heparin coupled to albumin as described previously (13). After coating and blocking steps, the plates were incubated with 0.25 milliunits of recombinant A 12 AChE (non-saturating amount of enzyme) in 50 l of 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl for 18 h at 4°C with constant agitation.…”

“…Purification of Torpedo Asymmetric AChE-Asymmetric AChE was purified from Torpedo californica electric organ (Pacific Bio-Marine Laboratory, Venice, CA) using sequential extraction and affinity chromatography, as described previously (13).…”

“…We have characterized the structural properties of those putative heparin-binding domains (HBDs) by using molecular modeling and synthetic peptides (12)(13)(14). Here, we analyzed ColQ-heparin interactions by heparin affinity chromatography of the entire A 12 AChE, carrying different mutations in ColQ.…”