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.
Duval
N
N
Laboratoire de Neurobiologie, CNRS URA 295, Ecole Normale Supérieure, France.
Krejci
E
E
Grassi
J
J
Coussen
F
F
Massoulié
J
J
Bon
S
S
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry* / Q000276:immunology; D004557:Electric Organ / Q000201:enzymology; D000939:Epitopes; D004926:Escherichia coli; D006017:Glycolipids / Q000737:chemistry* / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D003012:Microbial Collagenase / Q000378:metabolism; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014101:Torpedo; D014162:Transfection
D000939:Epitopes; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D003094:Collagen; D000110:Acetylcholinesterase; D003012:Microbial Collagenase