Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris

Abstract

In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETDelta, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEDelta, reduced to the catalytic domain). We show that AChEH and AChET subunits are processed into the same molecular forms as in vivo or in transfected mammalian cells, but that lytic processes converting amphiphilic forms into non-amphiphilic derivatives appear to be more active in yeast. The production of glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion of monomers) demonstrates that P. pastoris can correctly process a mammalian C-terminal GPI-addition signal. Truncated rat and Bungarus AChE molecules, which exclusively generated non-amphiphilic monomers, were released more efficiently and thus produced more AChE activity. In the hope of increasing the production of AChE, we replaced the endogenous signal peptide by yeast prepeptides, with or without a propeptide. We found that the presence of a propeptide, which does not exist in AChE, does not prevent the proper folding of the enzyme, and that it may either increase or decrease the yield of secreted AChE, depending on the signal peptide. Surprisingly, the highest yield was obtained with the endogenous signal peptide. For all combinations, the yield was 2-3 times higher for Bungarus than for rat AChE, probably reflecting differences in the folding efficiency or stability of the polypeptides. The Michaelis constant (Km), the constant of inhibition by excess substrate (Kss) and the catalytic constant (kcat) values of the recombinant AChEs obtained both in P. pastoris and in COS cells, were essentially identical with those of the corresponding natural enzymes, and the Ki values of active-site and peripheral-site inhibitors (edrophonium, decamethonium, propidium) were similar.

The biological significance of substrate inhibition: A mechanism with diverse functions

“…The black curve is the rate of input of ACH into the cleft, consistent with known rapid input [33,35]. Typical apparent K m values are in the range 50-100 mM [28,36,37]; we used the value 58 mM in the model [28]. The blue curve in Fig.…”

The Binding Sites of Inhibitory Monoclonal Antibodies on Acetylcholinesterase

“…Chimeras El/165/Rt, El/266/Rt and El/337/Rt contain increasingly longer N-terminal regions of Electrophorus AChE, fused with the complementary rat peptide regions, beyond residues 165, 266, and 337. We introduced a stop codon at the end of the catalytic domain so that these constructs produced soluble AChE monomers (34,35). The distribution of Electrophorus and rat regions on the surface of the catalytic subunit is schematically illustrated in Fig.…”

Design of acetylcholinesterases for biosensor applications

“…One strategy involves the examination of the expression of recombinant rat or Bungarus AChE in Pichia pastoris by exchanging the native signal peptides for yeast signal peptides or by introducing propeptides which do usually not exist in AChE . A second approach arose from the observation that highly homologous AChEs from different species are produced at markedly different levels in the same expression systems (Cousin et al, 1996a;Morel and Massoulie, 1997). Rat/Bungarus and Bungarus/rat chimeras were expressed in COS cells (Morel and Massoulie, 2000) in order to elucidate the causes of the variability in the expression levels of different enzymes.…”