The Heparin-Binding Sites in the Collagenic Tail of Acetylcholinesterase

Abstract

The collagenic subunit (ColQ) allows the anchorage and localization of the asymmetric (A) forms of acetylcholinesterase (AChE) to the basal lamina at the neuromuscular junction. The anchoring of the enzyme involves an interaction with heparan sulfate proteoglycans (HSPG). Analysis of the primary structure of Torpedo marmorata ColQ, as well as competition studies using synthetic peptides, led us to describe the presence of two putative heparin-binding domains (HBDs) characterized by a core of basic residues (BBXB), that could account for the interaction with HSPG (1 and the preceding abstract). We now present an analysis of these sites using site-directed mutagenesis of the recently cloned mammalian ColQ, in which the HBDs are highly conserved with respect to the Torpedo collagenic tail (2). Wild type (wt) and mutated A forms were obtained by co-expression of AChET and rat ColQ mRNA in Xenopus oocytes and then where isolated by sucrose gradient velocity sedimentation. The relative heparin affinities were evaluated in terms of the concentration of NaCl required for elution of the bound enzyme from heparin-agarose columns using NaCl steps. The first set of mutations was designed to alter the core of the HBDs by replacing a doublet of basic residues with Asp-Pro (DP). We mutated either the N-terminal HBD domain (RKGR → DPGR), the C-terminal one (KRGK → DPGK) or both. The double mutant bound heparin poorly, demonstrating that the two identified HBDs are the main heparin-binding sites in ColQ. Interestingly, we found that the C-terminal HBD interacted more strongly with heparin than the N-terminal one, a fact consistent with the results obtained with the triple-helical peptides derived from Torpedo ColQ (see the preceding abstract).