title: Biochemical and functional characterization of CpMuc4, a Cryptosporidium surface antigen that binds to host epithelial cells.
abstract: Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a Cryptosporidium parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in Escherichia coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in Toxoplasma gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States.
authors.forename: John
authors.initials: J
authors.lastname: Paluszynski
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Monahan
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States.
authors.forename: Maura
authors.initials: M
authors.lastname: Williams
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States.
authors.forename: Olivia
authors.initials: O
authors.lastname: Lai
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: Morris
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States.
authors.forename: Patrick
authors.initials: P
authors.lastname: Burns
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, United States. Electronic address: roconnor@tuftsmedicalcenter.org.
authors.forename: Roberta
authors.initials: R
authors.lastname: O'Connor
mesh_terms: D000595:Amino Acid Sequence; D000953:Antigens, Protozoan / Q000235:genetics / Q000378:metabolism*; D018938:Caco-2 Cells; D016785:Cryptosporidium parvum / Q000276:immunology* / Q000472:pathogenicity; D004847:Epithelial Cells / Q000469:parasitology; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006790:Host-Parasite Interactions; D006801:Humans; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D034101:Sporozoites / Q000276:immunology*; D014122:Toxoplasma / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D000939:Epitopes
keywords: Adhesins; Attachment; Cryptosporidium; Glycoproteins; Sporozoite; Toxoplasma