Mol Biochem Parasitol. 2007 Jan 7; [Epub ahead of print]
Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii
O'connor RM, Wanyiri JW, Wojczyk BS, Kim K, Ward H
Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Boston, MA, United States.
Cryptosporidium is a cause of diarrheal disease worldwide. Parasite glycoproteins involved in invasion of Cryptosporidium into host cells have been investigated as possible targets for effective interventions against this parasite. One of these, Cpgp40/15, is expressed as a precursor protein that is cleaved by a parasite-derived furin-like protease activity into gp15, a glycophosphatidyl inositol anchored surface protein, and gp40, that associates with gp15 and binds to host cells. Investigation of the functions of these glycoproteins requires an expression system that can produce similar glycosylation patterns to the native antigens. Previous work demonstrated that Cpgp40/15 transiently expressed in Toxoplasma gondii was appropriately localized and glycosylated. In this study, T. gondii stable transfectants expressing gp40/15, gp15, gp40 and hemagglutinin (HA) tagged gp40 were generated. T. gondii recombinant gp40HA and gp40/15 (recTggp40HA and recTggp40/15) were isolated from infected cells by HA affinity chromatography and Helix pomatia lectin affinity chromatography, respectively. Mass spectrometry confirmed that recTggp40-HA and native Cpgp40 were similarly glycosylated. Like native Cpgp40/15, recTggp40/15 could be cleaved into the gp40 and gp15 products by human furin or by a furin-like protease activity in T. gondii tachyzoite lysates. However, processing was inefficient in intact tachyzoites. Unlike the N-terminus of native Cpgp40/15, which appears to be processed following signal peptide cleavage, the N-terminus of recTggp40/15 began at the predicted signal sequence cleavage site, 11 amino acids upstream of the N-terminus of native Cpgp40. The ability to express and isolate appropriately glycosylated Cryptosporidium glycoproteins will enable further investigations into host-parasite interactions of this important pathogen.
PMID: 17275106 [PubMed - as supplied by publisher]