Mamoru Nomura

Cyclic AMP- and calmodulin-dependent phosphorylation of 21 and 26 kDa proteins in axoneme is a prerequisite for SAAF-induced motile activation in ascidian spermatozoa

Sperm activating and ‐attracting factor (SAAF), derived from the egg of the ascidian Ciona, activates sperm motility through adenosine 3′:5′‐cyclic monophosphate (cAMP)‐synthesis. A demembranated preparation of intact immotile sperm without SAAF was shown to require cAMP for reactivation. However, a demembranated preparation of intact motile sperm treated with SAAF did not require cAMP for reactivation, suggesting that cAMP is a prerequisite factor for SAAF‐dependent activation of sperm motility. Furthermore, a cAMP‐dependent protein kinase (PKA) inhibitor, H‐89, was found to inhibit sperm motility. During in vivo or in vitro activation of sperm motility by SAAF or cAMP, a 26 kDa axonemal protein and 21 kDa dynein light chain were phosphorylated, respectively, suggesting the involvement of PKA‐dependent phosphorylation of these proteins in sperm activation. The calmodulin antagonist, W‐7, and an inhibitor of calmodulin‐dependent myosin light chain kinase, ML‐7, also inhibited the activation of sperm motility. Inhibition was reversed by the addition of phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine. Demembranated preparations of immotile sperm in the presence of W‐7 or ML‐7 were reactivated by cAMP, suggesting that calmodulin participated in sperm activation and that cAMP synthesis was followed by activation of a calmodulin‐dependent mechanism.

Proteomic profiles of embryonic development in the ascidian Ciona intestinalis.

We report here proteomics-based protein profiles of three embryonic stages of the ascidian Ciona intestinalis. Two-dimensional gel electrophoresis revealed 416, 539, and 695 protein spots in the unfertilized eggs, 16 cell-stage embryos, and tadpole larvae, respectively. Comparative and quantitative analyses of the spot patterns identified proteins showing an increase or decrease in amount during embryonic development. Protein identification by MALDI-TOF/MS indicated not only the abundance and importance of metabolic enzymes and translation elongation factors but also the functional importance of actin-binding proteins and molecular chaperones during ascidian development. Global changes in spots for vitellogenin-like protein suggested post-translational modification or proteolytic digestion of this protein during embryogenesis. Comparison between mRNA and protein levels among unfertilized eggs, 16 cell-stage embryos and tadpole larvae indicated nonparallel expression patterns of genes and proteins. Ascidians provide an excellent system for studying gene expression and cell differentiation during development, and the present study should shed light on the associated molecular mechanism at the protein level.

CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5.

Functional proteomics in Ciona intestinalis: a breakthrough in the exploration of the molecular and cellular mechanism of ascidian development.

Ascidians have been providing a unique experimental system for a variety of fields, including reproductive biology, developmental biology, neurobiology, immunology, and evolutional biology. Recent progress in the genome sequencing of Ciona intestinalis has led to the development of a great tool for investigating the gene functions and expressions involved in several biological events in ascidians. The disclosure of genomic information has ushered in the postgenomic era, spearheaded by extensive protein analysis. The characterization of the function, localization, and molecular interaction of cellular proteins results in a more direct description of the molecular mechanism underlying several biological processes. Proteomics in ascidians, however, has just recently appeared and is not well established yet. In this study, we give an outline of the technical processes used in proteomics and review the recent status of ascidian proteomics. Developmental Dynamics 236:1782–1789, 2007.

Proteomic profiling reveals compartment‐specific, novel functions of ascidian sperm proteins

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X‐100‐soluble and ‐insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS–PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non‐redundant proteins were identified by mass spectrometry (MALDI‐TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm‐specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca2+‐binding proteins in axonemes, localization of testis‐specific serine/threonine kinase, and the presence of G‐protein coupled signaling and ubiquitin pathway in sperm flagella. Mol. Reprod. Dev. 78:529–549, 2011.

CIPRO 2.5: Ciona intestinalis Protein Database - a unique integrated repository of large-scale omics data, bioinformatic analyses, and curated annotation, with ability for user rating and comments

CIPRO database (http://cipro.ibio.jp/2.5) is an integrated protein database for a tunicate species Ciona intestinalis that is part of the Urochordata. Although CIPRO provides proteomic and transcriptomic data on a single species, the animal is considered unique in the evolutionary tree, representing a possible origin of the vertebrates and is a good model for understanding chordate evolution, including human evolution. Furthermore, C. intestinalis has been one of the favorites of developmental biologists; therefore, a lot of amount of accumulated knowledge on its development, morphology, in addition to the recent genome sequence and gene expression data exists. The CIPRO database aims to collect published data and to present unique information, including the unpublished transcriptomic and proteomic data and human curated annotation, for the use of researchers in biology and bioinformatics. The current database contains 89,673 unique sequences covering all the proteins from all the gene models on this species; the number was reduced to 70,493 by similarity clustering. Of these sequences, more than 5,000 proteins are manually annotated based on the large-scale transcriptomic, proteomic and bioinformatic data. Those annotations can be subjected to be qualification by rating, curation, and comments by named and anonymous users through the web site of CIPRO database. Unique features of CIPRO database include: (i) Original experimental data Unpublished experimental data, including 2D-PAGE with the identified protein spots by protein mass fingerprint (PMF) MS analysis, expressions or localizations of protein and RNA across developmental stages and tissues, altogether summarized in a single chart for the comparison among status and methods. RNA expressions are observed by microarray and EST. Each protein is linked to an independent Ascidian Proteome Database summarizing large-scale MS-based proteomic analyses. (ii) Whole Ciona intestinalis proteome database Proteins across gene models are presented: all protein models derived from published gene models are incorporated, including Kyoto model (KG), KH (successor of KG model), PROCITS, JGIs versions 1 and 2, and Ensembl (version 58.2) are incorporated. Identical sequences across gene models are shown. (iii) Original comprehensive user-friendly interfaces Bioinformatic analyses and prediction results are summarized in pictures for grasp at a glance: homology search, cytolocalization, secondary structure prediction combined with modification sites, such include phosphorylation and three-dimensional structures. (iv) Comparative analysis data for disease association Comparison with human genome: map location of human homologues is graphically shown with associated disease information. Comparative data for other model organisms are also included. (v) Community-wide curation capability opened to users To facilitate progressive improvement of annotation by visited users, users can place additional annotation for the protein name and/or comments, which will be subjected to rating by the followed data viewers. To aid curation by wide community, information for literature and essence of matched motif patterns and other related protein information are shown with the links. (vi) Useful search facilities Various search methods are provided including blast homology, free text, partial sequence, protein mass fragment, and cross item searches.