Abstract
Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.
Highlights
Junctions, whereas complexes containing PRiMA are anchored in cell membranes and represent the major AChE species in the brain [3]
We have previously shown that the t peptide forms an amphiphilic ␣-helix, with a sector containing seven aromatic residues that are strictly conserved in all vertebrate cholinesterases (AChE, as well as butyryl cholinesterase (BChE)) [4]
The assembly of AChET with collagen Q (ColQ) has been studied in detail using site-directed mutagenesis and biochemical analyses of oligomers produced in transfected cells [6, 7], as well as crystallographic studies of a complex formed between synthetic t and proline-rich attachment domains (PRADs) peptides [8]
Summary
CDNA Constructs and Site-directed Mutagenesis—All constructs were expressed in the pEF-BOS vector [13, 14]. In the QN-HC construct, which was described previously [16], the N-terminal region of ColQ, containing the PRAD, is fused to a GPI addition signal derived from the Torpedo AChEH subunit. This construct produces GPI-anchored AChE tetramers, which can be compared with PRiMA-anchored tetramers. Deglycosylation—Samples of cell extracts and culture media containing about 5 mOD/min of AChE activity were mixed with a 1 l of N-glycanase (Glyco enzymatic deglycosylation kit GK80110 from PROzyme), completed to 20 l with extraction buffer, and incubated at 37 °C overnight after addition of 10 l of denaturation buffer (4% SDS, 0.125 M Tris-HCl, pH 6.8, 20% glycerol)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
