Cloning and Characterization of Ancylostoma-secreted Protein
Hawdon, John M.; Jones, Brian F.; Hoffman, Donald R.; Hotez, Peter J.
The developmentally arrested third stage infective larva of hookworms resumes development upon entry into the definitive host. This transition to parasitism can be modeled in vitro by stimulating infective larvae with a low molecular weight ultrafiltrate of host serum together with methylated glutathione analogues. When stimulated to resume development in vitro, activated larvae of the hookworm Ancylostoma caninum released a 42-kDa protein, termed Ancylostoma-secreted protein (ASP). ASP was the major protein released by activated hookworm larvae. Degenerate oligonucleotide primers, based on a partial internal amino acid sequence of the protein, were used together with flanking vector sequence primers to amplify a fragment from a third stage larval cDNA library by polymerase chain reaction. The fragment was used as a probe to isolate a longer clone from the larval cDNA library. The full-length ASP cDNA was found to encode a 424-amino acid protein with homology to the antigen 5/antigen 3 family of proteins from hymenopteran venoms and a family of cysteine-rich secretory proteins. ASP was expressed in bacterial cells, and a polyclonal antiserum against purified recombinant ASP was produced. The antiserum, which was demonstrated to be specific for ASP, was used as a probe to measure the kinetics of ASP release by hookworm larvae. ASP is released within 30 min of stimulation, with the majority released by 4 h. Low levels of ASP were released continuously following activation, but only if the stimuli were present in the incubation medium. The compound 4,7-phenanthroline, previously shown to inhibit larval activation, also inhibited release of ASP. The specific, rapid release of ASP by activated infective larvae suggests that this molecule occupies a critical and central role in the transition from the external environment to parasitism.
Hawdon, John M., & Jones, Brian F., & Hoffman, Donald R., & Hotez, Peter J.. (March 1996). Cloning and Characterization of Ancylostoma-secreted Protein. Journal of Biological Chemistry, (271:12), p.6672-6678. Retrieved from http://hdl.handle.net/10342/8880
Hawdon, John M., and Jones, Brian F., and Hoffman, Donald R., and Hotez, Peter J.. "Cloning and Characterization of Ancylostoma-secreted Protein". Journal of Biological Chemistry. 271:12. (6672-6678.), March 1996. July 24, 2021. http://hdl.handle.net/10342/8880.
Hawdon, John M. and Jones, Brian F. and Hoffman, Donald R. and Hotez, Peter J., "Cloning and Characterization of Ancylostoma-secreted Protein," Journal of Biological Chemistry 271, no. 12 (March 1996), http://hdl.handle.net/10342/8880 (accessed July 24, 2021).
Hawdon, John M., Jones, Brian F., Hoffman, Donald R., Hotez, Peter J.. Cloning and Characterization of Ancylostoma-secreted Protein. Journal of Biological Chemistry. March 1996; 271(12) 6672-6678. http://hdl.handle.net/10342/8880. Accessed July 24, 2021.