Hostname: page-component-586b7cd67f-2brh9 Total loading time: 0 Render date: 2024-11-27T21:41:06.580Z Has data issue: false hasContentIssue false
  • English
  • Français

Saposin-like proteins from the intestine of the blood-feeding hookworm, Ancylostoma caninum

Published online by Cambridge University Press:  17 November 2006

T. A. DON ,
Y. OKSOV ,
S. LUSTIGMAN  and
A. LOUKAS
T. A. DON
Affiliation:
Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Queensland 4006, Australia School of Molecular and Microbial Sciences, The University of Queensland, Queensland 4072, Australia
Y. OKSOV
Affiliation:
Lindsley F. Kimball Research Institute, The New York Blood Center, NY 10021, USA
S. LUSTIGMAN
Affiliation:
Lindsley F. Kimball Research Institute, The New York Blood Center, NY 10021, USA
A. LOUKAS
Affiliation:
Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Queensland 4006, Australia
Get access Rights & Permissions [Opens in a new window]

Abstract

Hookworms feed on blood, utilizing haemoglobin for nutrition, growth and reproduction. The haemoglobin digestion cascade has been partially elucidated, but the process immediately preceding this event, haemolysis, has received considerably less attention. We have cloned and expressed Ancylostoma caninum mRNAs encoding 2 proteins belonging to the saposin-like protein (SAPLIP) family, termed Ac-slp-1 and Ac-slp-2. The open reading frames of SLP-1 and SLP-2 were used to identify expressed sequence tags encoding SAPLIPs from the 4 major clades of animal parasitic nematodes. Both Ac-slp-1 and slp-2 mRNAs were shown to be expressed in all life stages assessed, with slp-1 predominantly being expressed in third-stage larvae (L3) before and after activation with dog serum. Recombinant SLP-1 and SLP-2 were expressed in insect cells and used to raise specific antisera in mice. These antisera were used as probes in fluorescence microscopy to localize the anatomic expression sites of both proteins to small, punctate organelles or vesicles within the intestinal cells of adult worms; weak staining was detected on the microvillar brush border of the intestine. Using transmission electron microscopy, both proteins were localized to similar vesicles in the intestinal cells of the L3. Recombinant proteins contained C-terminal purification tags that potentially precluded dimerization and possibly interfered with the subsequent detection of haemolytic activity. Their expression in the gut of the L3 and adult stages suggests a role for these hookworm SAPLIPs in the lysis of host cells during tissue migration and/or feeding.

Keywords

Ancylostoma caninumhookwormSAPLIPsaposin
Type
Research Article
Information
Parasitology , Volume 134 , Issue 3 , March 2007 , pp. 427 - 436
Copyright
© 2006 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Andersson, M., Gunne, H., Agerberth, B., Boman, A., Bergman, T., Sillard, R., Jornvall, H., Mutt, V., Olsson, B., Wigzell, H., Dagerlind, A., Boman, H. G. and Gudmandsson, G. H. ( 1995). NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity. The EMBO Journal 14, 1615–1625.Google Scholar
Andra, J. and Leippe, M. ( 1994). Pore-forming peptide of Entamoeba histolytica. Significance of positively charged amino acid residues for its mode of action. FEBS Letters 354, 97–102.Google Scholar
Bethony, J., Loukas, A., Smout, M., Brooker, S., Mendez, S., Plieskatt, J., Goud, G., Bottazzi, M. E., Zhan, B., Wang, Y., Williamson, A., Lustigman, S., Correa-Oliveira, R., Xiao, S. and Hotez, P. J. ( 2005). Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals. The FASEB Journal 19, 1743–1745.CrossRefGoogle Scholar
Blaxter, M. L., De Ley, P., Garey, J. R., Liu, L. X., Scheldeman, P., Vierstraete, A., Vanfleteren, J. R., Mackey, L. Y., Dorris, M., Frisse, L. M., Vida, J. T. and Thomas, W. K. ( 1998). A molecular evolutionary framework for the phylum Nematoda. Nature 392, 71–75.CrossRefGoogle Scholar
Brindley, P. J., Kalinna, B. H., Wong, J. Y., Bogitsh, B. J., King, L. T., Smyth, D. J., Verity, C. K., Abbenante, G., Brinkworth, R. I., Fairlie, D. P., Smythe, M. L., Milburn, P. J., Bielefeldt-Ohmann, H., Zheng, Y. and McManus, D. P. ( 2001). Proteolysis of human hemoglobin by schistosome cathepsin D. Molecular and Biochemical Parasitology 112, 103–112.CrossRefGoogle Scholar
Bruhn, H. ( 2005). A short guided tour through functional and structural features of saposin-like proteins. The Biochemical Journal 389, 249–257.CrossRefGoogle Scholar
Bruhn, H., Riekens, B., Berninghausen, O., Leippe, M., Herbst, R., Ott, C., Jacobs, T., Marti, T. and Marciano-Cabral, F. ( 2003). Amoebapores and NK-lysin, members of a class of structurally distinct antimicrobial and cytolytic peptides from protozoa and mammals: a comparative functional analysis of pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri. The Biochemical Journal 375, 737–744.CrossRefGoogle Scholar
De Silva, N. R., Brooker, S., Hotez, P. J., Montresor, A., Engels, D. and Savioli, L. ( 2003). Soil-transmitted helminth infections: updating the global picture. Trends in Parasitology 19, 547–551.CrossRefGoogle Scholar
Don, T. A., Jones, M. K., Smyth, D., O'Donoghue, P., Hotez, P. and Loukas, A. ( 2004). A pore-forming haemolysin from the hookworm, Ancylostoma caninum. International Journal for Parasitology 34, 1029–1035.CrossRefGoogle Scholar
Espino, A. M. and Hillyer, G. V. ( 2003). Molecular cloning of a member of the Fasciola hepatica saposin-like protein family. Journal of Parasitology 89, 545–552.CrossRefGoogle Scholar
Espino, A. M. and Hillyer, G. V. ( 2004). A novel Fasciola hepatica saposin-like recombinant protein with immunoprophylactic potential. Journal of Parasitology 90, 876–879.CrossRefGoogle Scholar
Grams, R., Adisakwattana, P., Ritthisunthorn, N., Eursitthichai, V., Vichasri-Grams, S. and Viyanant, V. ( 2006). The saposin-like proteins 1, 2, and 3 of Fasciola gigantica. Molecular and Biochemical Parasitology 148, 133–143.CrossRefGoogle Scholar
Hawdon, J. M. and Schad, G. A. ( 1991). Albumin and a dialyzable serum factor stimulate feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum. Journal of Parasitology 77, 587–591.CrossRefGoogle Scholar
Hecht, O., Van Nuland, N. A., Schleinkofer, K., Dingley, A. J., Bruhn, H., Leippe, M. and Grotzinger, J. ( 2004). Solution structure of the pore-forming protein of Entamoeba histolytica. The Journal of Biological Chemistry 279, 17834–17841.CrossRefGoogle Scholar
Hotez, P. J., Brooker, S., Bethony, J. M., Bottazzi, M. E., Loukas, A. and Xiao, S. ( 2004). Hookworm infection. The New England Journal of Medicine 351, 799–807.CrossRefGoogle Scholar
Hotez, P. J., Bethony, J., Bottazzi, M. E., Brooker, S., Diemert, D. and Loukas, A. ( 2006). New technologies for the control of human hookworm infection. Trends in Parasitology 22, 327–331.CrossRefGoogle Scholar
Kishimoto, Y., Hiraiwa, M. and O'Brien, J. S. ( 1992). Saposins: structure, function, distribution, and molecular genetics. Journal of Lipid Research 33, 1255–1267.Google Scholar
Lee, J. Y., Cho, P. Y., Kim, T. Y., Kang, S. Y., Song, K. Y. and Hong, S. J. ( 2002). Hemolytic activity and developmental expression of pore-forming peptide, clonorin. Biochemical and Biophysical Research Communications 296, 1238–1244.CrossRefGoogle Scholar
Leippe, M. ( 1995). Ancient weapons: NK-lysin, is a mammalian homolog to pore-forming peptides of a protozoan parasite. Cell 83, 17–18.CrossRefGoogle Scholar
Leippe, M. ( 1997). Amoebapores. Parasitology Today 13, 178–183.CrossRefGoogle Scholar
Leippe, M., Ebel, S., Schoenberger, O. L., Horstmann, R. D. and Muller-Eberhard, H. J. ( 1991). Pore-forming peptide of pathogenic Entamoeba histolytica. Proceedings of the National Academy of Sciences, USA 88, 7659–7663.CrossRefGoogle Scholar
Leippe, M., Tannich, E., Nickel, R., Van Der Goot, G., Pattus, F., Horstmann, R. D. and Muller-Eberhard, H. J. ( 1992). Primary and secondary structure of the pore-forming peptide of pathogenic Entamoeba histolytica. The EMBO Journal 11, 3501–3506.Google Scholar
Leippe, M., Andra, J., Nickel, R., Tannich, E. and Muller-Eberhard, H. J. ( 1994). Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes. Molecular Microbiology 14, 895–904.CrossRefGoogle Scholar
Liepinsh, E., Andersson, M., Ruysschaert, J. M. and Otting, G. ( 1997). Saposin fold revealed by the NMR structure of NK-lysin. Nature Structural Biology 4, 793–795.CrossRefGoogle Scholar
Liu, A., Wenzel, N. and Qi, X. ( 2005). Role of lysine residues in membrane anchoring of sapsoin C. Archives of Biochemistry and Biophysics 443, 101–112.CrossRefGoogle Scholar
Loukas, A., Bethony, J. M., Mendez, S., Fujiwara, R. T., Goud, G. N., Ranjit, N., Zhan, B., Jones, K., Bottazzi, M. E. and Hotez, P. J. ( 2005). Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs. PLoS Medicine 2, e295.CrossRefGoogle Scholar
Lustigman, S., Brotman, B., Huima, T., Prince, A. M. and McKerrow, J. H. ( 1992). Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus. The Journal of Biological Chemistry 267, 17339–17346.Google Scholar
Munford, R. S., Sheppard, P. O., O'Hara, P. J., Kishimoto, Y., Hiraiwa, M. and O'Brien, J. S. ( 1995). Saposin-like proteins (SAPLIP) carry out diverse functions on a common backbone structure. Journal of Lipid Research 36, 1653–1663.Google Scholar
Ranjit, N., Jones, M. K., Stenzel, D. J., Gasser, R. B. and Loukas, A. ( 2006). A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy. International Journal for Parasitology 36, 701–710.CrossRefGoogle Scholar
Roche, M. and Layrisse, M. ( 1966). The nature and causes of “hookworm anemia”. The American Journal of Tropical Medicine and Hygiene 15, 1031–1102.CrossRefGoogle Scholar
Tran, M. H., Pearson, M. S., Bethony, J. M., Smyth, D. J., Jones, M. K., Duke, M., Don, T. A., McManus, D. P., Correa-Oliveira, R. and Loukas, A. ( 2006). Tetraspanins on the surface of Schistosoma mansoni are protective vaccine antigens in mice and Sm-TSP-2 is selectively recognized by naturally resistant individuals. Nature Medicine 12, 835–840.CrossRefGoogle Scholar
Williamson, A. L., Brindley, P. J., Abbenante, G., Prociv, P., Berry, C., Girdwood, K., Pritchard, D. I., Fairlie, D. P., Hotez, P. J., Dalton, J. P. and Loukas, A. ( 2002). Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity. The FASEB Journal 16, 1458–1460.CrossRefGoogle Scholar
Williamson, A. L., Brindley, P. J., Abbenante, G., Datu, B. J., Prociv, P., Berry, C., Girdwood, K., Pritchard, D. I., Fairlie, D. P., Hotez, P. J., Zhan, B. and Loukas, A. ( 2003 a). Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion. Journal of Infectious Diseases 187, 484–494.Google Scholar
Williamson, A. L., Brindley, P. J., Knox, D. P., Hotez, P. J. and Loukas, A. ( 2003 b). Digestive proteases of blood-feeding nematodes. Trends in Parasitology 19, 417–423.Google Scholar
Williamson, A. L., Lecchi, P., Turk, B. E., Choe, Y., Hotez, P. J., Mckerrow, J. H., Cantley, L. C., Sajid, M., Craik, C. S. and Loukas, A. ( 2004). A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. The Journal of Biological Chemistry 279, 35950–35957.CrossRefGoogle Scholar
Zhai, Y. and Saier, M. H. Jr. ( 2000). The amoebapore superfamily. Biochimica et Biophysica Acta 1469, 87–99.CrossRefGoogle Scholar