Acetylcholinesterases of the nematode Steinernema carpocapsae

Abstract

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine = 0.05 pM). The corresponding molecular forms are: an amphiphilic 14s form converted into a hydrophilic 14.5s form by mild proteolysis and two hydrophilic 12s and 7s forms. Reduction of the amphiphilic 14s form with 10 mM dithiothreitol produces hydrophilic 7s and 4s forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12s forms correspond to hydrophilic monomer, dimer, tetramer and that the 14s form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 =0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4s forms (monomers) and a major amphiphilic 7s form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7s form thus possesses a glycolipid anchor. It appears that Steinernemu (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.Acetylcholinesterase (AChE) and butyrylcholinesterase are highly polymorphic enzymes in vertebrates. Six different molecular forms can be expressed in a tissue-specific and agedependent manner. Three globular forms (Gl, G2 and G4 forms) correspond to monomers, dimers and tetramers of catalytic subunits, and three asymmetric forms (A forms) are associations of one, two or three tetramers to a collagen 'tail' [I].In invertebrates, only AChE has been described so far and its molecular diversity is more restricted : for example, there is no convincing evidence for the existence of collagen-tailed, asymmetric forms in these organisms. In insects, only G1 and G 2 forms have been characterized. The major molecular form is an amphiphilic dimer (2, 31 linked to the plasma membrane by a glycolipid anchor [4 -61. A glycolipid-anchored AChE has also been characterized in the trematode Schistosoma mansoni [7].

The molecular forms of acetylcholinesterase from Necator americanus (Nematoda), a hookworm parasite of the human intestine

“…Necator AChE seems to be more related to class B AChE of Celegans (product of ace-2) as far as molecular forms are concerned (hydrophilic and amphiphilic dimers). It has been shown that amphiphilic dimers of class B AChE are glycolipid-anchored in Steinernema [9]. The low yields of amphiphilic forms in crude DS extracts of Necator precluded such a demonstration.…”

“…sucrose gradients, in 1 M NaCI, 20mM Tris/HCI pH 7.5 (plus antiproteolytics as in extraction buffers), in the presence or absence of 1% Triton X-100 [9]. Samples (100-200 pl) were loaded and centrifuged at 36000 rpm for 15 h at 4°C.…”

“…Lyophilised adult N. americanus were differentially extracted as described by Arpagaus et al [9]. Whole worms were homogenised on ice in 20mM TrisMC1 pH7.5.…”

“…Class A forms sediment at 5.2 S, 11.3 S and 13 S, class B at 4.9 S and 6.7 S and class C (a minor component) sediments at 7 S. It was suggested that some of these forms were membrane-bound, on the basis of their requirement for detergent for solubilisation [8]. More recently, Arpagaus et al [9] have described two major AChE classes from Steinernemu carpcapsue, a nematode parasite of insects. These two AChE species differ in their catalytic properties and they are likely to be encoded by the equivalent of the C. elegans ace-1 and ace-2 genes.…”

Acetylcholinesterase in Dendrobaena veneta (Oligochaeta: Opisthopora) is Present with Forms Sensitive and Insensitive to Phosphatidylinositol Phospholipase C

“…In Invertebrata, asymmetric forms of cholinesterase have been detected in Cephalochordata (Pezzementi et al, 1991), while only globular forms have been found in Tnsecta (Gnagey et al, 1987;Toutant, 1989), Nematoda (Arpagaus et al, 1992(Arpagaus et al, , 1994, Sipunculida (Talesa et al, 1993a) and Mollusca (Talesa et al, 1994(Talesa et al, , 1995b. In addition, our previous reports regarding cholinesterases from Invertebrata (Talesa et al, 1990(Talesa et al, , 1994 often showed a lower substrate specificity in comparison with the acetylcholinesterases from Vertebrata, as well as a wide variability in the kinetic behaviour.…”