Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii

Abstract

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 T. 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.

Cryptosporidium Pathogenicity and Virulence

“…gp40 is localized at the surface and the apical region of the parasite and is shed from the surface, while gp15 is on the surface of sporozoites and is shed in trails during gliding movement (135,172). Proteins present in the trails of gliding zoites have been shown to play a role in parasite attachment, invasion, and motility (133,172,201,(212)(213)(214)(215). In C. parvum, gp15 is attached to the membrane via a GPI anchor (216).…”

Cryptosporidiuminfections: molecular advances

“…n.a. (Nelson et al 2006) GP40/15 Glycoproteins that have a predicted 60 kDa precursor that is proteolytically cleaved into a 15 and 40 kDa protein probably by a subtilisin-like serine protease (CpSUB1); the GP60 locus is a highly polymorphic cluster and can be divided in two allelic groups Ia-Id (C. hominis), II (C. parvum); GalNAc-specific lectins such as HPA, AIA, and MPA prevent cell attachment and invasion indicating GPs might bind via GalNAc to cells; moreover Nac could reduce binding of the mAb against GP15 but not glucose, mannose or galactose; immunization of mice with mAb against GP15 could reduce oocyst shedding by 67.5%; GP15/GP40 are shed during gliding GP40: predicted N-terminal signal peptide, a polyserine domain, multiple predicted glycosylation sites, a single potential N-glycosylation site GP15: glycosylated, GPI anchor GP15 is localized on the surface of sporozoites and merozoites; GP40 is localized in the apical region whereas GP15 is found on the complete surface; since GP40 does not have a transmembrane domain or GPI anchor it is proposed that GP15 and GP40 form a complex (Tilley et al 1991;Jenkins et al 1993;Cevallos et al 2000a, b;Sestak et al 2002;O'Connor et al 2003O'Connor et al , 2007aWanyiri et al 2009Wanyiri et al , 2007 GP900 Ab against GP900 or subdomains reduced infection of cells in a concentration-dependent manner; GP900 mRNA levels peak at 14-26 h p.i. of cells; comparison of Ab reactivity between C. parvum and C. hominis against GP900 revealed that Ab did not cross-react between the species, indicating that morphological differences of surface molecules might account for species-specific infectivity Mucin-like glycoprotein composed of distal cysteine-rich domains separated by polythreonine domains and a large membrane proximal N-glycosylated core region; deglycosylated protein app.…”

The State of Research for AIDS-Associated Opportunistic Infections and the Importance of Sustaining Smaller Research Communities

“…Some of these contain core O-glycans such as terminal GalNAc␣1-Ser/Thr and/or Gal␤1a3GalNAc␣1-Ser/Thr, the so-called "Tn and T antigens" which are not normally exposed in mammalian cells because they are masked by additional sugars such as sialic acid (11). This had resulted in difficulties in generating appropriately glycosylated recombinant proteins, but this problem was overcome by heterologous expression of Cryptosporidium glycoproteins in T. gondii (55,57) which utilizes similar enzymes for O-glycosylation (74). The T. gondii system has also been used for genetic complementation approaches (73).…”