Molecular architecture of acetylcholinesterase collagen-tailed forms; construction of a glycolipid-tailed tetramer.

Asymmetric forms of Torpedo acetylcholinesterase (AChE) are produced in COS cells by the simultaneous expression of collagenic subunits (Q) and catalytic T subunits (AChET). Truncated AChET delta subunits, from which most of the C-terminal peptide (TC) had been deleted by mutagenesis, did not associate with Q subunits. The TC peptide is therefore necessary for the association of the AChET and Q subunits. In order to determine the orientation of the Q subunit in the collagen-tailed forms, we have developed an antiserum against its non-collagenic C-terminal domain, expressed as a fusion protein in Escherichia coli. This antiserum, which recognized the Q subunit in Western blots, was found to react with intact asymmetric forms, but not with collagenase-treated forms, from which the distal part of the tail had been cleaved, suggesting that the N-terminal non-collogenic domain (QN) is responsible for the interaction with the AChET subunits. This was confirmed by creating a chimeric subunit (QN/HC), in which QN was linked to the C-terminal peptide of the H subunit of Torpedo AChE, which contains the glycophosphatidylinositol (GPI) cleavage/attachment signal: co-expression of AChET and QN/NC produced GPI-anchored tetramers, which were sensitive to PI-PLC and largely exposed to the external surface of the cells. We thus demonstrate that: (i) the HC peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the QN domain is sufficient to bind a catalytic AChET tetramer by interacting with the TC peptide.