Abstract
Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3′ and 5′rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His57 and Ser195 conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.
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Allingham P.G., Kerlin R.L., Tellam R.L., Briscoe S. and Standfast H.A. 1992. Passage of host immunoglobulin across the mid-gut epithelium into the haemolymph of blood-fed buffalo flies (Heamatobia irritans exigua). J. Insect Physiol. 38: 9–17.
Baron R.W. and Cowell D.D. 1991. Mammalian immune responses to myasis. Parasitol. Today 7: 353–355.
Ben-Yakir D. 1989. Quantitative studies of host immunoglobulin in the haemolymph of ticks (Acari). J. Med. Entomol. 26: 243–246.
Casu R.E., Jarmey J.M., Elvin C.M. and Eisemann C.H. 1994. Isolation of a trypsin-like serine protease gene family from the sheep blowfly Lucilia cuprina. Insect Mol. Biol. 3: 159–170.
Coombs G.H. and Mottram J.C. 1997. Parasite proteinases and amino acid metabolism: possibilities for chemotherapeutic exploitation. Parasitology 114: S80–S114.
Dubremetz J.F. and McKerrow J.H. 1995. Invasion mechanisms. In: Marr J.J. and Muller M. (eds), Biochemistry and Molecular Biology of Parasites. Academic Press, Tokyo, Japan, pp. 307–322.
Elvin C.M. and Kemp D.H. 1994. Generic approaches to obtaining efficacious antigens from vector arthropods. Int. J. Parasitol. 24: 67–79.
Elvin C.M., Vuocolo T., Smith W.J.M., Eiseman C.H. and Riddles P.W. 1994. An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina). Insect. Mol. Biol. 3: 105–115.
Elvin C.M., Whan V. and Riddles P.W. 1993. A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua). Mol. Gen. Genet. 240: 132–139.
Greer J. 1990. Comparative modeling methods: Application to the family of the mammalian serine proteases. Proteins Structures, Functions, and Genetics 7: 317–334.
Jiang Q., Hall M., Noriega F.G. and Wells M. 1997. cDNA cloning and pattern of expression of an adult, female-specific chymotrypsin from Aedes aegypti midgut. Insect Biochem. Mol. Biol. 27: 283–289.
Kraut J. 1977. Serine protease: structure and mechanism of catalysis. Ann. Rev. Biochem. 46: 331–358.
Krem M.M. and Di Cera E. 2001. Molecular markers of serine proteinase evolution. EMBO J. 20: 3036–3045.
Lehane M.J. 1994. Digestive enzymes, haemolysins and symbionts in the search for vaccine against blood-sucking insects. Int. J. Parasitol. 24: 27–32.
Mukhebi A.W. 1992. Economic impact of theileriosis and its control in Africa. In: Norval R.A.I., Perry B.D. and Young A.S. (eds), In the epidemiology of theileriosis in Africa. Academic press, London, pp. 380–402.
Mulenga A., Sugimoto C., Ingram G., Ohashi K. and Onuma M. 1999. Molecular cloning of two Haemaphysalis longicornis cathepsin L-like cysteine proteinase genes. J. Vet. Med. Sci. 61: 497–503.
Mulenga A., Sugimoto C., Ingram G., Ohashi K. and Onuma M. 2001. Characterization of two cDNAs encoding serine proteinases from the hard tick Haemaphysalis longicornis. Insect Biochem. Mol. Biol. 31: 817–825.
Mulenga A., Sugimoto C. and Onuma M. 2000. Issues in tick vaccine development: identification and characterization of potential candidate vaccine antigens. Microbe Infect. 2: 1353–1361.
Mulenga A., Sugimoto C., Sako Y., Ohashi K., Musoke A., Shubash M. et al. 1999. Molecular characterization of a Haemaphysalis longicornis tick salivary gland-associated 29 kilodalton protein and its effect as a vaccine against tick infestation in rabbits. Infect. Immun. 67: 1652–1658.
Muller H.M., Crampton J.M., della Tore A., Sinden R. and Crisanti A. 1993. Members of a trypsin gene family in Anopheles gambiae are induced in the gut by blood meal. EMBO. J. 12: 2891–2900.
Neurath H. 1984. Evolution of proteolytic enzymes. Science 224: 350–357.
Neurath H. 1986. The versatility of proteolytic enzymes. J. Cell Biochem. 32: 35–49.
Noriega F.G., Wang X.Y., Pennington J.E., Barrilas-Murry C.V. and Wells M.A. 1996. Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, aminoterminal sequence determination, and cloning and sequencing of the gene. Insect Biochem. Mol. Biol. 26: 119–126.
Rao M.B., Tanskale A.M., Ghate M.S. and Deshpande V.V. 1998. Molecular and biotechnology aspects of microbial proteases. Microb. Mol. Biol. Rev. 62: 597–635.
Ribeiro J.M.C. 1987. Role of saliva in blood feeding of arthropods. Annu. Rev. Entomol. 32: 463–478.
Ribeiro J.M.C. 1988. The midgut hemolysin of Ixodes dammini (Acari: Ixodidae). J. Parasitol. 74: 532–537.
Rosenthal P.J. 1999. Proteases of protozoan parasites. Adv. Parasitol. 43: 105–159.
Sakanari J.A., Staunton C.E., Eakin A.E., Craik C.S. and McKerrow J.H. 1989. Serine proteases from nematode and protozoan parasites: Isolation of sequence homologues using generic molecular probes. Proc. Nat. Acad. Sci. USA 86: 4863–4867.
Sauer J.R., Bowman A.S., McSwain J.L. and Essenberg R.C. 1996. Salivary gland physiology of bloodfeeding arthropods. In: Wikel S.K. (ed.), The Immunology of Host-ectoparasitic Relationships. CAB International, Wallingford.
Sauer J.R., McSwain J.L., Bowman A.S. and Essenberg R.C. 1995. Tick salivary gland physiology. Annu. Rev. Entomol. 40: 267–345.
Sonenshine D.E. 1993. Biology of ticks. Oxford University Press, Oxford.
Spates G.E. 1979. Fecundity of the stable fly: effects of soybean trypsin inhibitor and phospholipidae A inhibitor on the fecundity. Ann. Entomol. Soc. Amer. 72: 845–852.
Tellam R.L., Smith D., Kemp D.H. and Willadsen P. 1992. Vaccination against ticks. In: Wong W.K. (ed.), Animal parasite control utilizing biotechnology. CRC Press Inc, Boca Raton, pp. 303–331.
Vaughan J.A. and Azad A.F. 1988. Passage of host immunoglobulin G from blood meal into haemolymph of selected mosquito species (Diptera: culicidae). J. Med. Entomol. 25: 472–474.
Wang H. and Nuttal P. 1995. Immunoglobin G binding proteins in male Rhipicephalus appendiculatus ticks. Parasite Immunol. 17: 517–524.
Wang S., Young F. and Hickley D.D. 1995. Genomic organization and expression of a trypsin from spruce budworm Choristoneura fumiferana. Insect Biochem. Mol. Biol. 25: 899–908.
Willadsen P. 1987. Immunological approaches to the control of ticks. Int. J. Parasitol. 17: 671–677.
Willadsen P., Bird J., Cobon G.S. and Hungerford J. 1995. Commercialization of a recombinant vaccine against Boophilus microplus. Parasitology 110: S43–S50, (suppl).
Willadsen P. and Jongejan F. 1999. Immunology of the tick-host interaction and the control of ticks and tick-borne diseases. Parasitology Today 15: 258–262.
Willadsen P., Smith D., Cobon G. and McKenna R.V. 1996. Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91. Parasite Immunol. 18: 241–246.
Xiong B. and Jacobs-Lorena M. 1995. The black fly Simulium vittatum trypsin gene: characterization of the 5_-upstream region and induction by the blood meal. Exp. Parasitol. 81: 363–370.
Zhu Y. and Baker J.E. 1999. Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer. Rhyzopertha dominica (Coleoptera: Bostrichidae) 29: 1053–1063.
Zhu Y. and Baker J.E. 2000. Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica. Arch. Insect Biochem. Physiol. 43: 173–184.
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Mulenga, A., Misao, O. & Sugimoto, C. Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization. Exp Appl Acarol 29, 151–164 (2003). https://doi.org/10.1023/A:1024278402288
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DOI: https://doi.org/10.1023/A:1024278402288