The Dependence of Acetylcholinesterase Aggregation at Low Ionic Strength upon a Polyanionic Component

Abstract

The low ionic strength aggregation of Electrophoixs asymmetric forms of acetylcholinesterase has been found to depend upon a distinct aggregating agent, present in large excess in the electric organ extracts. The aggregates appeared, in electron micrographs, as bundles of a few molecules, the tails of which were linked in a side-to-side association, leaving the head groups at both ends. The proportion of aggregated enzyme, but not the sedimentation properties of the aggregates, depended in a complex manner upon ionic conditions and aggregating agent levels. The aggregates were not dissociated by dilution even at very low concentrations. Properties of the aggregating agent suggested that it might be a non-proteic polymer, interacting with positively charged groups of the enzyme tail.Various polyanions were found to promote aggregation of acetylcholinesterase, but induced aggregates of different sedimentation coefficients. Polycarboxylates were effective only at high concentrations while sulfate and phosphate polyanions were very efficient. Aggregates induced by these latter polyanions possessed the same morphological features as those produced by the endogenous preparation.Analysis of the Electrophorus aggregating preparation demonstrated that its chondroitin sulfate content fully accounts for its aggregating capacity, and this was in agreement with its sensitivity to specific mucopolysaccharidases.Aggregates were also obtained in Electrophorusl Torpedo mixed systems but their characteristics were found to depend both on the nature of the enzyme and of the aggregating agent. Aggregation had no effect on catalytic efficiency, and we discuss its relevance to the synaptic localization of the enzyme, through interactions between the tails of acetylcholinesterase and chondroitin-sulfatetype glycosaminoglycan components.It has been known for a long time that certain varieties of acetylcholinesterase from electric fish (Electrophorus and Torpedo) aggregate in low-salt media [l-51. Studies of the pH and ionic strength dependence of aggregate formation [ 1 -31 indicate that this phenomenon is controlled by ionic interactions. We have recently described the structure of the aggregates, as observed by high-resolution electron microscopy. These structures consist of a few, of the order of 6-10, asymmetric molecules, the tails of which are linked side by side. This bundle of rod-like tails constitutes the core of the aggregate, leaving two groups of heads at both extremities [6].In the present paper we examine the nature of the interactions involved in acetylcholinesterase aggregation and demonstrate that it is characteristic of the tailed forms and depends upon the presence of an agAhbipviation. Nbsz, 5,5'-dithio-bis(2 nitrobenzoic acid).gregating polyanionic agent distinct from the acetylcholinesterase molecule. We compare the electron microscopic appearance of aggregates inducted by various polyanions and present some evidence regarding the chemical nature of the endogenous aggregating agent. Finally we discuss the relevan...

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

“…In this context, a possible candidate is chondroitin sulfate. It has been suggested previously that chondroitin sulfate proteoglycans interact with asymmetric AChE (34,35), and we have observed previously a direct interaction between chondroitin sulfate and the peptides modeling the N-and C-terminal HBDs, whose characteristics also differed between the two sites (14).…”

A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down‐Regulation Toward Maturity and After Denervation

“…Various forms of AChE exist in both developing and mature neuromuscular junctions. The predominant form of the enzyme is the asymmetric collagen-tailed AChE that is specifically attached at the synaptic basal lamina (Bon et al, 1978;McMahan et al, 1978), whereas most of the globular forms of AChE are at the membrane of postsynaptic muscle cells (Massoulie et al, 1993). At the neuromuscular junction, the following three cell types can contribute to the synaptic AChE activity: postsynaptic muscle cell, presynaptic motor neuron, and myelinating Schwann cell.…”

“…The globular forms of AChE are monomers (G 1), dimers (G2), and tetramers (G4) of catalytic subunits; they may be attached to the membrane by a hydrophobic domain (Inestrosa et al, 1987;Roberts et al, 1988;. The asymmetric forms (A form) of AChE consist of catalytic tetramers linked to a collagen-like tail (Lee and Taylor, 1982;Tsim et al, 1988a); they may be attached to the synaptic basal lamina by ionic interaction (Bon et al, 1978;McMahan et al, 1978;Anglister, 1991).…”

Relationship of Collagen‐Tailed Acetylcholinesterase with Basal Lamina Components

“…In low-salt gradients in particular, collagen-tailed acetylcholinesterase formed fastsedimenting polydisperse aggregates [14] but labeled collagen, laminin or fibronectin remained as distinct, more slowly sedinienting peaks (data not shown).…”

“…Collagen-tailed forms of the enzyme are obvious candidates for such an association with the basal lamina and have been shown to aggregate with polyanionic compounds such as chondroitin sulfate, in physiological ionic conditions [14]. Their partial digestion by collagenase yields characteristic, fast-sedimenting derivatives, which no longer aggregate [7].…”