Cevallos, A. -GalNAc determinants on the recombinant gp40 was confirmed by reactivity with lectin and the monoclonal antibody 4E9, which recognizes -GalNAc residues, and digestion with -apparently processes the gp40/15 precursor into the gp40 and gp15 component glycopolypeptides, albeit inefficiently. These results suggest that a surrogate system using for the study of biology may be useful. pathogenesis hampers the development of effective vaccines and treatments. Identification of virulence factors and understanding the natural biology of are especially difficult because of the lack of an in vitro culture system for propagation of the parasite and, consequently, the inability to manipulate the genome by transfection. completes its whole life PQM130 cycle within the host gut epithelium (30). When oocysts present in the environment are ingested, sporozoites are released in the small intestine, where they attach to and invade intestinal epithelial cells. Parasite replication occurs in a unique intracellular location that is beneath the host cell microvillous membrane but is segregated from the cytoplasm. After two rounds of merogony, sexual stages are produced that fuse Stx2 and form oocysts that are either released into the environment or excyst and reinitiate infection. isolates that are classified as genotype II have an unrestricted host range, commonly infecting neonatal ruminants and humans, whereas genotype I isolates are restricted to human hosts (22). Despite an apparent lack of reservoir hosts, most human infections are caused by genotype I isolates (7). One approach to the development of anticryptosporidial agents has been to identify sporozoite and merozoite surface antigens involved in recognition, attachment, and invasion of the host epithelial cells in order to block these interactions. The glycoprotein products of the gene are PQM130 two of several antigens that are implicated in these processes (1, 2, 23, 29, 34). gp40 (also referred to as gp45 [29] and S45 [34]) and gp15 (also referred to as Cp17 [23] and S16 [34]) are produced as a single preprotein, proteolytically processed and localized to the surface of zoite stages, from where they are shed in trails during sporozoite locomotion. The soluble gp40 adhesin appears to attach to the sporozoite surface membrane through association with the glycophosphatidylinositol (GPI)-anchored gp15 (34; O’Connor and Ward, unpublished data). Antibodies to gp40 block infection in vitro, and partially purified gp40 binds to intestinal epithelial cells in a manner suggestive of a specific receptor-ligand interaction (2). gp40 and gp15 display O-linked -infection is the observation that the gene exhibits extensive polymorphism among genotype I isolates (19, 29). Although the functional significance of this variation is unknown, these data suggest that the antigens may be important targets of anticryptosporidial immunity. Since these data support the hypothesis that gp40 and gp15 are integral to the establishment of infection, further molecular characterization of the antigens is warranted. However, because of the difficulty of obtaining large quantities of native antigen, particularly from type I isolates, an appropriate eukaryotic expression system that could generate recombinant glycoproteins that would mimic the function of the native antigens is needed. To address this problem, PQM130 we explored the possibility of expressing these antigens in is a closely related apicomplexan that is easily propagated and genetically manipulated and has been shown to produce glycoproteins that display glycans similar to those observed on the gp40 and gp15 antigens (35). If a system to successfully exploit for heterologous expression of the gene could be developed, then not only could localization, processing, and identification of adhesive domains be investigated, but the potential functional significance of diversity could also be explored. In this paper, the appearance is normally reported by us from the gene in and glycosylation, partial digesting, and localization of its proteins products towards the tachyzoite surface area. These experiments claim that a heterologous appearance program using for the analysis of glycoproteins and their function in host-parasite connections may verify useful. METHODS and MATERIALS PQM130 Parasites. Iowa isolate (genotype II) oocysts had been extracted from Pleasant Hill Plantation, Troy, Idaho, and GCH1 oocysts (genotype II) had been extracted from Saul Tzipori, Tufts School College of Veterinary Medication, Grafton, Mass. lysates had been ready in phosphate-buffered saline, pH 7,.