Characterization of a Cryptosporidium parvum protein that binds single-stranded G-strand telomeric DNA

We have initiated a project to characterize telomere-associated proteins of Cryptosporidium parvum. Searching public databases with C. parvum expressed sequence tag (EST) sequences revealed one EST sequence that is highly similar to Gbp1p of Chlamydomonas reinhardtii (Cr Gbp1p), a protein that binds single-stranded telomeric DNA. This EST was used to clone a gene encoding a 198 amino acids long protein (CpGbp). Sequence analysis suggested that CpGbp contains two RNA recognition motif (RRMs) domains linked with a short hinge region. RT-PCR analysis showed that the mRNA expression of CpGbp was up- and down-regulated significantly comparing to that of CpDNAPol, suggesting a potential role of CpGbp playing in the parasite's life cycle. In Western blot analysis, monoclonal antibody against recombinant CpGbp identified one band (approximately 23kDa) specifically from cell extracts of C. parvum sporozoites. Confocal microscopy analysis with anti-CpGbp antibody localized CpGbp proteins to the nucleus, consistent with its potential role in telomere length regulation. In electrophoretic mobility shift assays (EMSAs), recombinant CpGbp bound oligonucleotide TG3 that bears three copies of C. parvum telomeric DNA G-strand repeat "TTTAGG", but not C-strand or double-stranded telomeric DNA sequences. To map the binding domain and to define the binding site of CpGbp, we constructed four CpGbp deletion mutants and synthesized ten TG3 mutants and tested their binding affinities by EMSAs. We found that only the RRM domain at N-terminus has oligonucleotide-binding ability in vitro. And the minimal sequence necessary for CpGbp's binding is "GTTTAGGTTTAG". These data support the notion that CpGbp represents a C. parvum single-stranded telomeric DNA binding protein.