Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrP^C, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrP^C GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.     

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2–PTEN–AKT Pathway and Placental Cell Proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2–PTEN–AKT pathway and placental cell proliferation.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

Relationship Between Amino Acid Properties and Protein Stability: Buried Mutations

In order to understand the mechanism of protein stability and to develop a simple method for predicting mutation-induced stability changes, we analyzed the relationship between stability changes caused by buried mutations and changes in 48 amino acid properties. As expected from the importance of hydrophobicity, properties reflecting hydrophobicity are strongly correlated with the stability of proteins. We found that subgroup classification based on secondary structure increased correlations significantly, and mutations within -strand segments correlated better than did those in -helical segments, which may result from stronger hydrophobicity of the -strands. Multiple regression analyses incorporating combinations of three properties from among all possible combinations of the 48 properties increased the correlation coefficient to 0.88 and by an average of 13% for all data sets. Analyzing the stability of tryptophan synthase mutants with Glu49 replaced by all other residues except Arg revealed that combining buriedness, solvent-accessible surface area for denatured protein, and unfolding Gibbs free energy change increased the correlation to 0.95. Consideration of sequence and structural information (neighboring residues in sequence and in space) did not significantly strengthen the correlations in buried mutations, suggesting that nonspecific interactions dominate in the interior of proteins.     

Polymorphism of dopamine receptor D4 exon I corresponding region in chicken

In stockbreeding, there are indications that behavioral traits of livestock have an effect on breeding and production. If the variation in individual behavior is related to that in neurotransmitter-related genes such as in humans, it would be possible to breed pedigrees composed of individuals having behavioral traits that are useful to production and breeding using selection based on genotypes. In this study, we investigated the exon I region of dopamine receptor D4 (DRD4), in which variation is related to psychiatric disorder in humans, in major poultry species namely Japanese quail (Coturnix japonica), chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and helmeted guinea fowl (Numida meleagris). Furthermore, we investigated Japanese cormorant (Phalacrocorax capillatus) and Japanese jungle crow (Corvus macrorhynchos) as an out-group. In these species of birds, the repeat of proline was identified in the region corresponding to the human polymorphic region. The repeat number was 9 in Japanese quail, ring-necked pheasant and Japanese cormorant; 12 in helmeted guinea fowl; and 3 in Japanese jungle crow. However, no polymorphism was found in these species. In contrast, polymorphism was observed in chicken and two alleles with 8 and 9 repeats were identified. Although 9 repeats (allele 9) were predominant in most chicken breeds, Black Minorca had only 8 repeats (allele 8). Intra-breed polymorphism was found in 6 out of 12 breeds, and two alleles (alleles 8 and 9) were detected in these breeds. This polymorphism, which is the first to be reported on a neurotransmitter-related gene in birds, would contribute significant information for elucidation of differences in behavioral traits in chicken breeds.     

Inter-colony and sex differences in the effects of parental body condition and foraging effort on the brood growth of Adélie penguins

Among colonies with different foraging distances, central-place-foraging seabirds may change their foraging and reproductive efforts. We compared the body condition, meal frequency, and diving behavior of male and female Adélie penguins at two locations: Dumont d'Urville, where there was little sea ice and they foraged in open waters far from the colony; and Syowa, where there was heavy, fast sea ice and they foraged in ice cracks close to the colony. The parental mass decrease rate during the chick-rearing period was similar between colonies and between sexes. A large individual variation in meal frequency positively affected the brood growth rate, but daily underwater time did not. A weak but significant positive effect of body condition on brood growth rate was found only in males at Syowa. It was suggested that males work with better body condition than females. We propose the hypothesis that the regional difference in the distance to the feeding sites and the sex difference in body energy reserve might constrain the capacity to regulate reproductive effort.