Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.     

Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization

In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.     

Granulin-like peptide in the mid-gut gland of the bivalve mollusk, Patinopecten yessoensis

A cysteine-rich polypeptide, termed CRP1, with a molecular mass of 5829 Da was found to occur in the mid-gut gland of the scallop Patinopecten yessoensis. CRP1 was purified by reverse phase and cation-exchange chromatographies. The amino acid sequence of CRP1 was deduced from its N-terminal amino acid sequence, amino acid composition and the sequence of a partial cDNA, indicating that CRP1 is a 57-amino-acid polypeptide containing 12 cysteine residues with a calculated molecular mass of 5841 Da (5829 Da when oxidized to form six disulfide bridges). A homology search of databases revealed that the deduced amino acid sequence of CRP1 displays significant similarity to those of granulin/epithelins, a family of growth-modulating factors; all cysteine residues in CRP1 are located at the same positions as those conserved characteristically in other known granulin/epithelins. Purified CRP1 inhibited the proliferation of mouse embryo cells. The results suggest that CRP1 functions as a growth-modulating factor in the scallop, and that granulin/epithelin family polypeptides and their precursors play physiologically important roles in invertebrates.     

Expression of Proteolipid Protein Gene Is Directly Associated with Secretion of a Factor Influencing Oligodendrocyte Development

Abstract: Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.     

Internodal elongation under submergence in the Amazonian wild rice species <Emphasis Type="Italic">Oryza glumaepatula</Emphasis>: the growth response is induced by hypoxia but not by ethylene

Phosphate (Pi) plays important roles in plant development and architecture. With the goal of identifying genomic regions that influence tolerance to Pi deficiency (TPDE) in hybrid rice (Oryza sativa L.), quantitative trait loci (QTL) were mapped using recombinant inbred lines (RILs) that were derived from a cross between tolerant ‘XieqingzaoB’ (XB) and susceptible ‘Zhonghui9308’. Six TPDE-related traits, including the root length, root dry weight, tillers number, shoot dry weight, total plant dry weight and root-to-shoot ratio, were evaluated for QTL analysis during both the tillering and heading stages. A correlation analysis showed that most of the traits were correlated with each other. Twenty-one additive QTL were detected and jointly explained between 10–49% of the trait variance, tending to cluster on chromosomes 4, 6, 10 and 11. Three QTL, qTPDE4 ^XB , qTPDE10 ^XB and qTPDE11.3 ^XB , were validated by the phenotypic evaluation using near isogenic lines (NILs, BC₄F₃) during the seedling stage. qTPDE4 ^XB showed the most stable tolerance against Pi deficiency. These QTL will enrich the genetic resources and accelerate hybrid rice breeding against Pi deficiency.