Dataset Information

Overexpression in Escherichia coli and functional reconstitution of the liposome binding ferriheme protein nitrophorin 7 from the bloodsucking bug Rhodnius prolixus.

ABSTRACT: A number of ferriheme proteins, termed nitrophorins (NPs), occur in the saliva of the bloodsucking insect Rhodnius prolixus ('kissing bug'), which is a vector for Chagas' disease. Nitrophorins bind the heme b cofactor in the beta-barrel of their lipocalin fold, which is further anchored through a proximal histidine-Fe(III) bond. The distal Fe(III) coordination site then binds nitric oxide (NO) for delivery into a host's tissues during blood feeding, where, upon NO release, the distal Fe(III) site acts as a histamine trap to delay the victim's immune response. Previously, four nitrophorins from R. prolixus, NP1 to NP4, have been extensively characterized. Recently, another nitrophorin, NP7, was discovered in a cDNA library derived from the same insect. Among the R. prolixus nitrophorins, NP7 was found to be unique in its ability to bind to negatively charged cell surfaces. However, the yield of functional recombinant NP7 was rather low when the established protocol for NP1-4 was followed. Here, we report on a novel expression and reconstitution method for NP7 that yields sufficient amounts of pure protein for extensive characterization (28-fold increase). This method may prove useful for the reconstitution of other proteins with a lipocalin fold.

SUBMITTER: Knipp M

PROVIDER: S-EPMC2094717 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Overexpression in Escherichia coli and functional reconstitution of the liposome binding ferriheme protein nitrophorin 7 from the bloodsucking bug Rhodnius prolixus.

Knipp Markus M Zhang Hongjun H Berry Robert E RE Walker F Ann FA

Protein expression and purification 20070307 1

A number of ferriheme proteins, termed nitrophorins (NPs), occur in the saliva of the bloodsucking insect Rhodnius prolixus ('kissing bug'), which is a vector for Chagas' disease. Nitrophorins bind the heme b cofactor in the beta-barrel of their lipocalin fold, which is further anchored through a proximal histidine-Fe(III) bond. The distal Fe(III) coordination site then binds nitric oxide (NO) for delivery into a host's tissues during blood feeding, where, upon NO release, the distal Fe(III) sit ...[more]

PMID: 17428677

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Project description:The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

Project description:Due to an abundance of repetitive DNA, the annotation of heterochromatic regions of the genome such as the Y chromosome is problematic. The Y chromosome is involved in key biological functions such as male-fertility and sex-determination and hence, accurate identification of its sequences is vital. The hemipteran insect Rhodnius prolixus is an important vector of Chagas disease, a trypanosomiasis affecting 6-7 million people worldwide. Here we report the identification of the first Y-linked genes of this species.The R. prolixus genome was recently sequenced using separate libraries for each sex and the sequences assembled only with male reads are candidates for Y linkage. We found 766 such candidates and PCR tests with the ten largest ones, confirmed Y-linkage for all of them, suggesting that "separate libraries" is a reliable method for the identification of Y-linked sequences. BLAST analyses of the 766 candidate scaffolds revealed that 568 scaffolds contained genes or part of putative genes. We tested Y-linkage for 36 candidates and found that nine of them are Y-linked (the PCR results for the other 25 genes were inconclusive or revealed autosomal/X-linkage). Hence, we describe in this study, for the first time, Y-linked genes in the R. prolixus genome: two zinc finger proteins (Znf-Y1 and Znf-Y2), one metalloproteinase (Met-Y), one aconitase/iron regulatory protein (Aco-Y) and five genes devoid of matches in any database (Rpr-Y1 to Rpr-Y5). Expression profile studies revealed that eight genes are expressed mainly in adult testis (some of which presented a weak expression in the initial developmental stages), while Aco-Y has a gut-restricted expression.In this study we showed that the approach used for the R. prolixus genome project (separate sequencing of male and female DNA) is key to easy and fast identification of sex-specific (e.g. Y chromosome sequences). The nine new R. prolixus Y-linked genes reported here provide unique markers for population and phylogenetic analysis and further functional studies of these genes may answer some questions about sex determination, male fertility and Y chromosome evolution in this important species.

Dataset Information

Overexpression in Escherichia coli and functional reconstitution of the liposome binding ferriheme protein nitrophorin 7 from the bloodsucking bug Rhodnius prolixus.

Publications

Overexpression in Escherichia coli and functional reconstitution of the liposome binding ferriheme protein nitrophorin 7 from the bloodsucking bug Rhodnius prolixus.

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