Yeast cells harboring human alpha-1,3-fucosyltransferase at the cell surface engineered using Pir, a cell wall-anchored protein

Human alpha-1,3-fucosyltansferase (FucT) encoded by the FUT6 gene was displayed at the cell surface of yeast cells engineered using the yeast cell wall protein Pir1 or Pir2, and the FucT activity was detected at the surface of cells producing the Pir1-HA-FUT6 or Pir2-FLAG-FUT6 fusion proteins. To obtain higher activity, we engineered the host yeast cells in which endogenous PIR genes of the PIR1-4 gene family were disrupted. Among the disruptants, the pir1Delta pir2Delta pir3Delta strain with the PIR1-HA-FUT6 fusion gene showed the highest FucT activity, which was about three-fold higher than that of the wild-type strain. Furthermore, the co-expression of both the Pir1-HA-FUT6 and the Pir2-FLAG-FUT6 fusions showed an approximately 1.5-fold higher activity than that in the cell wall displaying Pir1-HA-FUT6 alone. The present method was thus effective for producing yeast cells that can easily synthesize various oligosaccharides, such as Le(x) and sLe(x), using Pir-glycosyltransferase fusions in combination with the deletion of endogenous PIR genes.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Sclerostin is a novel secreted osteoclast-derived bone morphogenetic protein antagonist with unique ligand specificity

Sclerosteosis is a progressive sclerosing bone dysplasia. Sclerostin (the SOST gene) was originally identified as the sclerosteosis-causing gene. However, the physiological role of sclerostin remains to be elucidated. Sclerostin was intensely expressed in developing bones of mouse embryos. Punctuated expression of sclerostin was localized on the surfaces of both intramembranously forming skull bones and endochondrally forming long bones. Sclerostin-positive cells were identified as osteoclasts. Recombinant sclerostin protein produced in cultured cells was efficiently secreted as a monomer. We examined effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for mouse preosteoblastic MC3T3-E1 cells. Sclerostin inhibited the BMP6 and BMP7 activity but not the BMP2 and BMP4 activity. Sclerostin bound to BMP6 and BMP7 with high affinity but bound to BMP2 and BMP4 with lower affinity. In conclusion, sclerostin is a novel secreted osteoclast-derived BMP antagonist with unique ligand specificity. We suggest that sclerostin negatively regulates the formation of bone by repressing the differentiation and/or function of osteoblasts induced by BMPs. Since sclerostin expression is confined to the bone-resorbing osteoclast, it provides a mechanism whereby bone apposition is inhibited in the vicinity of resorption. Our findings indicate that sclerostin plays an important role in bone remodeling and links bone resorption and bone apposition.     

Biological activities of homologous loop regions in the laminin alpha chain G domains

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.     

Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish

Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.     

Heparan sulfate is required for bone morphogenetic protein-7 signaling

Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling.     

Distinct roles of Rab3B and Rab13 in the polarized transport of apical,basolateral, and tight junctional membrane proteins to the plasma membrane

Regulated transport of proteins to distinct plasma membrane domains is essential for the establishment and maintenance of cell polarity in all eukaryotic cells. The Rab family small G proteins play a crucial role in determining the specificity of vesicular transport pathways. Rab3B and Rab13 localize to tight junction in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. Here we examined their roles in regulating the cell-surface transport of apical p75 neurotrophin receptor (p75NTR), basolateral low-density lipoprotein receptor (LDLR), and tight junctional Claudin-1 using transport assay in non-polarized fibroblasts. Overexpression of Rab3B mutants inhibited the cell-surface transport of LDLR, but not p75NTR and Claudin-1. In contrast, overexpression of Rab13 mutants impaired the transport of Claudin-1, but not LDLR and p75NTR. These results suggest that Rab3B and Rab13 direct the cell-surface transport of LDLR and Claudin-1, respectively, and may contribute to epithelial polarization.     

Coordinate induction of AMP deaminase in human atrium with mitochondrial DNA deletion

Despite the heteroplasmic lower population of mitochondrial (mt) DNA deletion, mtDNA deletion is significantly related to the loss of atrial adenine nucleotides. To elucidate its mechanism, we examined the frequency of a 7.4-kb mtDNA deletion, the concentration of adenine nucleotides, and the activity of AMP catabolic enzymes in 10 human right atria obtained from cardiac surgery, using quantitative PCR, HPLC, and immunoprecipitations. The atrial concentrations of ATP, ADP, AMP, and the total adenine nucleotides were significantly lower in patients with deletion than those in patients without deletion, despite the lower frequency of their deletion. The activities of total AMP deaminase (AMPD), liver-type (AMPD 2), and heart-type isoform (AMPD 3) were significantly higher in patients with deletion than in patients without deletion, although there was no significant difference in the cytosolic 5(')-nucleotidase among them. In conclusion, mtDNA deletion coordinately induces AMP deaminase to contribute to the loss of atrial adenine nucleotides through degrading AMP excessively.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.