Analysis Of Two Types Of Acetylcholinesterase From The Silkworm, Bombyx Mori

Acetylcholinesterase (AChE, EC 3.1.1.7) catalyzes the hydrolysis of the neurotransmitter acetylcholine (ATCh) at cholinergic synapses to keep the normal transmission of nerve impulses. It is the target of the organophosphate and carbamate compounds. The intensive use of insecticide has led to the pollution of environment and many insects have developed resistance. As a kind of important economic insect, silkworm is susceptible to pesticides. Usually, it is used as a representative of non-target organisms in toxicological study of pesticides in China. About 70% of pests of agriculture and forestry belong to Lepidoptera. The silkworm, Bombyx mori is also a representative of Lepidoptera for a long history. So the study of the silkworm acetylcholinesterase may provide scientific basis for the development of new insecticides with less harm to the environment and with higher effectivity to resistant insect pests. The study may also help to shorten the time of research and development of new insecticides, lower the cost and perhaps find a new way to control insects. In this study having the acetylcholinesterase of the silkworm as the research object, we studied the physiological-biochemical characteristics of the enzyme. We amplified the two types of aces in the silkworm using the specific primers and provide the direct evidence for the two loci existed in the silkworm. By comparison of the cDNA sequence with the corresponding region of genomic DN A, the gene structures of the acetylcholinesterases were disclosed. The two genes were expressed in E. coli and insect cells, and the bioactivities of the expressed AChEs were detected. The ace gene from the housefly was also expressed in insect cells and purified by procainamide affinity chromatography in this study.The major results are summarized as following:1 The study of the biochemical properties of the AChE preparation from the silkworm showed that hydrolyzing substrate efficiency of AChE were ATCh>PTch>BTch. At pH8.0 and 37℃, the activity of the enzyme was highest. The I₅0 of paraoxon and eserine were 9.8×10^-7 M and 4.8×10^-7M. Comparison withAChEs from the honeybee and the housefly showed the activity of the AChE from the silkworm was relatively lower, perhaps this is the reason for the silkworm sensitive to insecticides.2 cDNA encoding two types of acetylcholinesterase from the silkworm were cloned and sequenced, and were designated as Bmacel and Bmace2, respectively. The results provide a direct evidence of two ace loci existed in the silkworm. The Bmacel is distributed by 2 introns and in the genome sequence of Bmace2 five introns are found. The whole open reading frame of Bmacel is 2052bp, potentially encoding a protein of 683 amino acids. A 17 amino acids signal peptide was predicted by SignalP at the N-terminal. The open reading frame of Bmace2 was 1917bp,encoding a protein of 638 amino acids with a 23 amino acids signal peptide. The alignment with other species indicated that the cloned genes have all the features of AChE: conserved active site triad, three pairs of cysteines putatively forming interchain disulfide bonding, conserved oxy-anino hole, conserved FGESAG sequence.3 The GST-AChEland GST-AChE2 were expressed in E. coli, and the antisera were prepared. ELISA result showed the titers of the two antisera were all 1:6.4 X 104.4 The genes were inserted into pFASTBacl to construct pFASTBacl-Bmacel and pFASTBacl-Bmace2. In order to benefit the expression, the C-truncated fragments of both Bmacel and Bmacel genes were also inserted into the donor plasmid. The four recombinant Bacmids were transfected into the insect cell line Tn-5Bl-4. SDS-PAGE and western blotting confirmed the successful expression of the expected proteins. The analysis of glycosylation revealed that the expressed AChEs were glycoslated. But the enzyme showed low activity. Comparison of the expressed AChEs with AChEs from other insects showed the same results. The protein expressed were more sensitve to paraoxon and eserine than the enzyme extracted from the heads of the silkworm. The I50 of AChE2 was lower than that of AChEl.5 The ace gene from the sensitive strain of Musca domestica was cloned and expressed in E. coli and Tn cells. The protein expressed in E. coli existed in inclusion body with no activity, after refolding the activity was still low. The protein expressedin Tn cells showed high bioactivity. We purified the expressed protein by procainamide affinity chromatagraphy. The purification fold was 41, and the yield was 17.94%. The inhibition of paraoxon and esrine to the enzyme revealed the purified protein was more sensitive than the crude protein.6 Phyiogenetic analysis of the insect and other species was carried out using the neighbor joining method with PAUP ver 4.0. The phyiogenetic tree inferred that insect AChEs were divided into 2 subgroups. The insects of Homoptera, Diptera, Lepidoptera, Blattaria and the Apis mellifera belong to the subgroup I. In the subgroup II, all the insects in subgroup I are involved besides housefly, fruitfly and some other insects. The Bmacel and Bmace2 belong to the two groups respectively.