Dataset Information

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum.

ABSTRACT: The small Cryptosporidium genome (?9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of ?78 kDa, smaller fragments of ?70, ?55, and ?30 kDa were simultaneously generated. After purification through a nickel-nitrilotriacetic acid affinity column, the full recombinant protein obtained was used to prepare polyclonal antibodies. Antibodies raised against INS-5 recognized the recombinant protein and native protein in sporozoite extracts. Further characterization of INS-5 included qRT-PCR assessment of gene expression; immunofluorescence localization of the protein expression in sporozoites, merozoites, and other developmental stages; and neutralization of invasion of C. parvum in vitro. The results obtained indicated that although INS-5 was expressed in sporozoites and merozoites, the high gene expression was from 36 to 48 h of the in vitro culture after invasion. Anti-INS-5 antibodies partially neutralized the invasion (inhibition rate = 38.5%). Results of this study suggest that INS-5 plays some role in the invasion and growth of C. parvum.

SUBMITTER: Ni N

PROVIDER: S-EPMC7225287 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum.

Ni Ni N Jia Ruilian R Guo Yaqiong Y Li Na N Wu Haizhen H Feng Yaoyu Y Xiao Lihua L

Frontiers in microbiology 20200508

The small Cryptosporidium genome (∼9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of ∼78 kDa, smaller fragments of ∼70, ∼55, and ∼30 kDa were simulta ...[more]

PMID: 32457703

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Project description:BackgroundHundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.ResultsThe C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC?? values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.ConclusionsIdentification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

Dataset Information

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum.

Publications

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in <i>Cryptosporidium parvum</i>.

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