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 (AChE(T)). Truncated AChE(T-DELTA) Subunits, from which most of the C-terminal peptide (T(C)) had been deleted by mutagenesis, did not associate with Q subunits. The T(C) peptide is therefore necessary for the association of the AChE(T) 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-collagenic domain (Q(N)) is responsible for the interaction with the AChE(T) subunits. This was confirmed by creating a chimeric subunit (Q(N)/H(C)), in which Q(N) 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 AChE(T) and Q(N)/N(C) 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 H(C) peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the Q(N) domain is sufficient to bind a catalytic AChE(T) tetramer by interacting with the T(C) peptide.