Identification, baculoviral expression and biochemical characterization of a novel cholinesterase of Amblyomma americanum (Acari: Ixodidae)

Authors: Temeyer, Kevin; Schlechte, Kristie; GROSS, AARON - Virginia Polytechnic Institution & State University; Lohmeyer, Kimberly - Kim

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2023
Publication Date: 4/22/2023
Citation: Temeyer, K.B., Schlechte, K.G., Gross, A.D., Lohmeyer, K.H. 2023. Identification, baculoviral expression and biochemical characterization of a novel cholinesterase of Amblyomma americanum (Acari: Ixodidae). International Journal of Molecular Sciences. https://doi.org/10.3390/ijms24097681.
DOI: https://doi.org/10.3390/ijms24097681

Interpretive Summary: The lone star tick is prevalent across the Eastern United States, is aggressive at locating host animals or humans on which to feed, and is capable of transmitting a number of pathogens causing vector-borne diseases. In addition, bites from the lone star tick appear to be responsible for the development of alpha-gal syndrome, an allergy to meat that can cause potentially life-threatening anaphylactic shock after eating meat or meat products. The allergy appears to develop in humans following a bite from lone star ticks because their saliva contains the alpha-gal allergen also present in red meat. New research conducted at U.S. Department of Agriculture laboratories has identified a novel cholinesterase enzyme that also appears to be present in saliva of the lone star tick. The enzyme was expressed as a recombinant protein (rAaChE1) and biochemically characterized. Antibodies specifically targeting the rAaChE1 enzyme were found to bind to protein components present in the tick brain (synganglion) and tick salivary glands, providing evidence for the presence of the AaChE1 enzyme in tick saliva. Previous USDA research demonstrated that cholinesterase enzymes were present in saliva of several blood-feeding parasites, including ticks, mosquitoes, sand flies, and biting midges, that are capable of transmitting pathogens causing vector-borne diseases. Cholinesterase enzymes break down acetylcholine important in function of animal nervous and immune systems, and may play a role in in altering the host immune response helping blood-feeding parasites to successfully feed, and might also alter host immune responses to pathogens, allergens, and vaccinations.

Technical Abstract: A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide(iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 M, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 M and 14.18 M, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.