Submitted to: World Association for the Advancement of Veterinary Parasitologists
Publication Type: Abstract Only
Publication Acceptance Date: July 20, 2007
Publication Date: August 19, 2007
Citation: Zarlenga, D.S., Gasbarre, L.C. 2007. A calcium-dependent, structural homologue of a novel salivary apyrase from blood-feeding arthropods has been identified in Ostertagia third stage larvae. World Association for the Advancement of Veterinary Parasitologists. pp.65 Technical Abstract: Apyrases (ATP diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyze extracellular ATP and ADP to orthophosphate and AMP. Most apyrases have been structurally linked to the heat shock70/sugar kinase/actin superfamily which is associated with motility, adhesion, secretion, regulation of hemostasis, non-synaptic information transfer and platelet formation. A second class of newly-described, calcium-dependent, salivary apyrases known to counteract blood-clotting, has been identified in several hematophagous arthropods. Herein, we have cloned an apyrase (OoAp) gene encoding a protein that structurally conforms to the calcium-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library with hyperimmune bovine serum. The full-length sequence was subcloned and the protein expressed (rOoAP). Hydrolysis of various dNTPs by rOoAP was monitored through the release of orthophosphate. Our results showed that the rOoAP is a Ca2+-dependent protein with greatest activity on ATP, ADP, UTP and UDP, and a pH maximum between 6.5 and 7.5. Hydrolysis was not observed on CTP or AMP. Host antibodies to OoAp appeared 14 dpi and increased through 30 dpi. Immunohistochemical and Western blot analyses demonstrated that OoAP is produced or collects within the muscle layer below the hypodermis, and is developmentally-regulated. Peak protein expression likely preceded L3 development given the absence of significant mRNA transcription in this stage. Mouse anti-rOoAP recognized conserved epitopes in protein extracts from Haemonchus, Cooperia, and Oesophagostomum. Currently, work is focusing on the relationship between apyrase activity and maintenance of the L3 and/or their arrested state of development.