| The main contributions of this dissertation are: 1. Perform an intergrated transcriptomic and proteomic analyses on human CD34+ hematopoietic stem/progenitorcells (HSC) and identify 370 proteins of these cells, which is the maximum number of proteins identified from CD34+ cells by proteomics to date; 133 of these proteins were not identified by any previous transcriptomic analyses; CD34+ cells have plasticity; make the finding that anti-sense RNA do not abolish the expression of their corresponding genes; the heterogeneities of some of proteins are due to post-translational modifications. 2. Perform an intergrated transcriptomic and proteomic analyses on the human parasite, Schistosoma japonicum; find -15000 genes of S. japonicum, covering 90% of its total genes; find 8400 protein-coding genes; set up the largest functional gene database and clone libraries in the world; for the first time, identify as many as 3260 proteins from variety of life stages of S. japonicum; analyse the eggshell proteins and tegument proteins, which were the putative targets for drugs and candidates of vaccines; reveal a large quantity of genetic polymorphisms in the genes of S. japonicum, with confirmations by DNA sequencing and mass spectrometry; identify the conserved and specific proteins of S. japonicum; shed light to the biological characteristics of S. japonicum; facilitate the progress in developing anti-schistosome drugs and vaccines. 3. identify and analyze the host proteins in purified S. japonicum samples including adults and eggs. It's the first time that so many host proteins were revealed interacting with schistosome closely. These host proteins were associated with the immune responses of host against the infection of schistosome, the immuno-evasion of schistosome and granuloma formation. These findings extend the information on host-parasite interplay.This dissertation consists of three parts. The first is the optimization of the proteomic technologies, the basis for subsequent studies. The second part is the transcnptomic/proteomic research upon the human CD34+ hematopoietic stem/progenitor cells and the last part is the transcriptomic/proteomic research of the human blood fluke. The studies on HSC and blood fluke are independent to each other.Part I: Establishment and optimization of proteomic platform.We optimized the methods of 2DE-matrix-assisted laser desorption/ionization (MALDI)-MS, including the solublization and extraction of protein samples, 2DE, in-geldigestion, MALDI-MS, the establishment of searching databases and the manual interpretation of mass spectra. In addition, we setup and optimize the work flow of isolation by LC and identification by MS, and the tools for large scale data analyses. Based on the well-established local proteomic working platform, we performed the large scale proteomic analyses on CD34+ cells and S. japonicum.Part II: The intergrated transcriptomic and proteomic study on the human CD34+ hematopoietic stem/progenitor cells.Hematopoietic stem cells are capable of self-renewal and differentiation into different hematopoietic lineages. Whether or not that HSCs having possibility of transdifferentiating into multiple non-hematopoietic cell types remains further evaluation. These cells have been studied comprehensive in cellular level, molecular level and transcriptomic level and have also been investigated by proteomics recently. However, a comprehensively systematic investigation of these cells by intergration of transcriptomic and proteomic approaches is now warranted. Now we collect the CD34+ cells from human cord blood (CB) and perform a comprehensive study on the proteome of these cells by combining the transcriptomic and proteomic technologies including 2DE and nono-LC for separation and bio-mass spectrometry for identification and quantification of the proteins.Three hundred and seventy proteins were identified from CD34+ cells, among which 52 proteins were associated with metabolism and cell skeleton, with the remaining ones involving cell division, chromatin modulation, RNA editing, and synthesizing, folding, modification and degradation of proteins.We also identified 46 unknown proteins by interrogation of the human genome database. To verify the results of our proteomic study, we analyzed the four representative proteins (membrane protein, plasma protein, and nuclear protein) by indirect immunofluorescence assay, confirming the reliability of our proteomic data.We identified the molecular hallmarks associated with multiple hematopoietic lineages in this study, which suggesting that CD34+ cells from CB composed of hematopoietic stem cells with pluripotent potential of differentiation into multiple hematopoietic lineages.We found that CD34+ cells could express some proteins associated with neuron, eye and gonad, suggesting hematopoietic stem cells have plasticity and might transdifferentiate into many non-hematopoietic lineages. The plasticity of stem cells is one of the hot spot in this area today and is very important for the clinic use. The hormone and cytokine-likemoleculars synthesized by CD34+ cells may exert important influences on the stem cells for self-renewal and differentiation by the autocrine or paracrine pathways.By this proteomic study, we identified 133 proteins in CD34+ cells, which have no reported transcripts by previous transcriptomic analyses, although their transcripts could be detected by RT-PCR.There were 33 proteins identified in CD34+ cells having two or even more spots on the 2DE gel. The enolase has the most number of spots, 20, on the gel. We performed a comprehensive analyses on the transcriptomic data and compared various alternative splicings of genes with the proteomic data. We found that this heterogeneity of proteins on 2DE gel might generated by the post-translational modification, not the transcriptional alternative splicing.By transcriptomic analysis, we found that there were 128 genes had both the sense and anti-sense transcripts our previous CB EST data. Unexpectedly, fifteen proteins with the corresponding antisense RNAs were identified in the proteomic study, implying that these sense RNAs could be translationally expressed, without being completely interrupted by the antisense RNAs. Whether the gene expressed or not might depend on the ratio of sense and anti-sense transcripts.Part III: Transcriptomic and proteomic analyses of S. japonicum.The schistosomiasis causes serious public health problem. We performed an integrated analysis upon the transcriptome and proteome of S. japonicum. In this study, we sequenced about 84000 EST and obtained 8420 putative protein-coding genes, setting up the largest functional gene database of S. japonicum in the world.We found that among 8420 proteins, there were 419 putative secreted proteins and 583 membrane proteins. Some of the secreted proteins may be released to the body fluid of host by schistosome. The tegument membrane proteins may associate the host-parasite interplay. We catalog the 5077 proteins similar to known proteins according to the Gene Ontology (GO). The cataloging patterns of proteins throughout different life stages of schistosome are similar.We analyzed the protein profiles of the different life cycles including cercariae, hepatic schistosomula (intact body and tegument), adult (mix-sex adult, female, male and their tegument), egg (intact egg and the eggshell) and miracidia, by two-dimensional LC-MS, onedimensional PAGE, together with tandem mass spectrometry (MSMS). More than 420000 MSMSs were collected from >400 fractions of S. japonicum. The identification of 26484 peptides represented 3260 distinctive proteins, including ～900-1400 proteins from cercaria, hepatic schistosomulum, adults, eggs, and miracidia. The life stage-enriched proteins are ranging from ～230-470, reflecting that the schistosome of different life stages adapted to different environments.We compared the EST numbers and protein abundances throughout the different life stages and we found the overlappings between these two datasets were high than expected with a peak of 86.8% in adult, better than the average overlapping of 30% of normal proteomic studies.Three hundred and seventy three proteins were identified from the prepared teguments of females, males, mix-sex adult worms and hepatic schistosomula. Except for some known tegument proteins, many of them were new teguent proteins, offering many putative targets for developing new drugs and new candidates for vaccines.We detected 520 eggshell proteins, defining the composition of eggshell for the first time. There were eggshell proteins involving anti-oxidation, immune, adhesion, calcium pathway and granuloma formation and these informations were very important for understanding the host-parasite interplay.It is the first time that as many as ～7000 SNPs were identified from S. japonicum genes, and some of them might lead to amino acid mutations or cause extending or truncation of the proteins. We also found that many schistosome genes were under the pressue of purifying selection and few were under positive selection. Some SNPs were verified by genomic PCR and sequencing and MSMS analyses. We also found lots of insertion and deletion (INDEL) and microsatellites in schistosome genes. The polymorphisms of schistosome genes may associated with the differences in infection, the development of schistosome in intermediate host and final host, the sensitivity to drugs, the infectivity and immunogenicity. All of the new findings will provide evidences for describing the immuno-evasion of schistosome and the ability of schistosome to modulate or attenuate the host immune responses.We identified 1336 conserved proteins of S. japonicum after comparing the schistosome proteins with those of other model animals. This finding may be useful to understand the mechanisms of schistosome in antigen mimicry, immuno-evasion, and immune-dependinggrowth. We also found 1323 putative schistosome-specific proteins, which might be the potential targets for developing vaccines, drugs and diagnostic reagents for deal with schistosomiasis.The adult worms of S. japonicum avoid the attack of their host by adsorbing host proteins onto their teguments. We analyzed the contaminant host proteins in the schistosome samples. In this study, we identified 63 host proteins, including 1, 2, 27, and 40 from cercaria, hepatic schistosomula, adults and eggs, respectively, by performing a large scale proteomic analyses on the schistosome. This is the first time that so many host proteins might interact with schistosome in a close proximity. These host proteins were associated with the immune reaction of host against schistosome infection, the immune evasion of adult worms and the formation of granuloma. These findings might shed light on the host-parasite interplay... |
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