Browsing by Author "Brümmer, Franz (Prof. Dr. rer. nat)"

Filter results by typing the first few letters
Now showing 1 - 1 of 1
  • Thumbnail Image
    ItemOpen Access
    Molecular studies on genes and proteins involved in biomineralization and development of the sea urchin Paracentrotus lividus
    (2014) Karakostis, Konstantinos; Brümmer, Franz (Prof. Dr. rer. nat)
    This study is focused on the Mediterranean sea urchin species, Paracentrotus lividus (P. lividus). The major part of the study covered the characterization of genes and proteins involved in biomineralization of P. lividus embryo and adult. The aim was to establish P. lividus as a working model for biomineralization studies. The primary goal was to develop a set of molecular tools involving plasmids, labeling probes, recombinant proteins and antibodies which subsequently were used in developmental and biomineralization studies. Candidate genes were selected based on previous findings in other species and on in silico analysis and comparison to the invertebrates and the sea urchin. The selected biomolecules involved acidic proteins (p16, p19), lectins (advillin, sm30α, sm50, galectin-8), a signaling protein (tetraspanin) and an enzyme (carbonic anhydrase). All the above groups of encoded proteins are known to participate in biomineralization in various species and therefore they were selected for the study of their role in the formation of the sea urchin skeleton. The protein-coding mRNA sequences p16, p19, advillin, tetraspanin, sm30α, sm50, carbonic anhydrase and galectin-8 were identified and cloned, following molecular biology techniques. The putative protein domains were analyzed and a phylogenetic comparison among the sea urchin species of P. lividus, Strongylocentrotus purpuratus (S. purpuratus) and Lytechinus variegatus (L. variegatus) revealed their evolution relation. Furthermore, the localization of the temporal and spatial expression of each transcript throughout the embryo development was characterized by comparative qPCR and whole mount in situ hybridization (WMISH). The acquired experimental data, revealed the expression profile of each one of these genes in the developing embryo and the insights on the role of each gene in development and skeletogenesis were discussed. Additionally, a functional characterization of recombinant carbonic anhydrase and galectin-8 proteins, cloned and expressed in vitro in E. coli and purified by affinity chromatography, gave insights of the role of each protein in development. Specific polyclonal antibodies were prepared and used to identify by Western Blot and ELISA, Pl-CA and Pl-Galectin-8-like proteins, in both the embryo and in the occluded matrix proteins of adult P. lividus, showing the importance of the two proteins in development. Functional assays, involving the in vitro esterase activity of Pl-CA and the lactose-specific hemagglutination activity of the lectin Pl-GALECTIN-8, confirmed that both recombinant proteins were active. Additionally, the biological role of Pl-GALECTIN-8 in cell adhesion was tested in vivo, in a human cell adhesion assay. Furthermore, aiming towards a complete characterization of the biomineralization proteins both in the embryo and in adult, the proteome of the occluded matrix proteins from adult P. lividus mineralized tests, was examined. Proteins were extracted from purified calcareous tests and identified by Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS). For the first time, the effect of the sea urchin occluded matrix protein on the formation of calcite is studied by an in vitro crystallization assay.