Amino acid residues important for CMP-sialic acid recognition by the CMP-sialic acid transporter: analysis of the substrate specificity of UDP-galactose/CMP-sialic acid transporter chimeras.

In our previous studies, we demonstrated that chimeric molecules of the CMP-sialic acid (CMP-Sia) transporter (CST) and the UDP-galactose (Gal) transporter (UGT) in which the seventh transmembrane helix-containing segment was derived from the CST could transport both CMP-Sia and UDP-Gal and that the CST-derived seventh transmembrane helix segment was sufficient for the chimera to recognize CMP-Sia in the otherwise UGT context. In this study, we continued to more precisely define the submolecular region that is necessary for CMP-Sia recognition, and we demonstrated that the N-terminal half of the seventh transmembrane helix of CST is essential for the CMP-Sia transport mediated by the chimeric transporters. We further showed that Tyr214Gly and Ser216Phe mutations of a chimeric transporter that was capable of transporting both CMP-Sia and UDP-Gal led to the selective loss of CMP-Sia transport activity without affecting UDP-Gal transport activity. Conversely, when a residue in a chimeric transporter that was active for UDP-Gal transport but not CMP-Sia transport was replaced by Tyr, so that Tyr occupied the same position as in the CMP-Sia transporter, the resulting mutant chimera acquired the ability to transport CMP-Sia. These results demonstrated that Tyr214 and Ser216, located in the seventh transmembrane helix of the human CST, are critically important for the recognition of CMP-Sia as a transport substrate. Identification of determinants critical for the discrimination between relevant and irrelevant substrates will advance our understanding of the mechanisms of substrate recognition by nucleotide sugar transporters.

Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila.

Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch.

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human.

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

Translocation of activated heterotrimeric G protein Galpha(o) to ganglioside-enriched detergent-resistant membrane rafts in developing cerebellum.

The association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the alpha-subunit of a heterotrimeric G protein, G(o) (Galpha(o)). Using sucrose density gradient analysis of cerebellar membranes, Galpha(o), but not Gbetagamma, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5'-O-(thiotriphosphate) (GTPgammaS), which stabilizes G(o) in its active form. On the other hand, both Galpha(o) and Gbetagamma were excluded from the DRM raft fractions in the presence of guanyl-5'-yl thiophosphate, which stabilizes G(o) in its inactive form. Only Galpha(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Galpha(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Galpha(o) into the DRM rafts. Furthermore, Galpha(o) was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1alpha, a physiological ligand for the G protein-coupled receptor, stimulated [(35)S]GTPgammaS binding to Galpha(o) and caused Galpha(o) translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raft-disrupting agent methyl-beta-cyclodextrin. These results demonstrate the involvement of signal-dependent Galpha(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.

Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity.

Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFkappaB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D(3) and prostaglandin E(2) on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A (125)I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis.

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.

Notch deficiency implicated in the pathogenesis of congenital disorder of glycosylation IIc.

Congenital disorder of glycosylation IIc (CDG IIc), also termed leukocyte adhesion deficiency II, is a recessive syndrome characterized by slowed growth, mental retardation, and severe immunodeficiency. Recently, the gene responsible for CDG IIc was found to encode a GDP-fucose transporter. Here, we investigated the possible cause of the developmental defects in CDG IIc patients by using a Drosophila model. Biochemically, we demonstrated that a Drosophila homolog of the GDP-fucose transporter, the Golgi GDP-fucose transporter (Gfr), specifically transports GDP-fucose in vitro. To understand the function of the Gfr gene, we generated null mutants of Gfr in Drosophila. The phenotypes of the Drosophila Gfr mutants were rescued by the human GDP-fucose transporter transgene. Our phenotype analyses revealed that Notch (N) signaling was deficient in these Gfr mutants. GDP-fucose is known to be essential for the fucosylation of N-linked glycans and for O-fucosylation, and both fucose modifications are present on N. Our results suggest that Gfr is involved in the fucosylation of N-linked glycans on N and its O-fucosylation, as well as those of bulk proteins. However, despite the essential role of N O-fucosylation during development, the Gfr homozygote was viable. Thus, our results also indicate that the Drosophila genome encodes at least another GDP-fucose transporter that is involved in the O-fucosylation of N. Finally, we found that mammalian Gfr is required for N signaling in mammalian cultured cells. Therefore, our results implicate reduced N signaling in the pathology of CDG IIc.

Adhesion activity of fetal gonadal cells to EGF and discoidin domains of milk fat globule-EGF factor 8 (MFG-E8), a secreted integrin-binding protein which is transiently expressed in mouse early gonadogenesis.

MFG-E8, a secreted integrin-binding protein, consists of two EGF domains containing a RGD motif and two discoidin domains. In mouse embryogenesis, MFG-E8 is highly expressed in gonadal stromal cells near mesonephros at 11.5-12.5 dpc, but its function in gonadogenesis has not been characterized. To clarify a possible role of MFG-E8 in developing gonads, we analyzed the adhesion activity of 10.5-15.5 dpc gonadal cells to recombinant proteins of EGF or discoidin domains of MFG-E8. In EGF-coated wells, the gonadal cells at 11.5-12.5 dpc revealed a significantly higher adhesion activity as compared to those at 10.5 and 15.5 dpc, while discoidin domains showed a constant number of the adhered cells throughout these stages. To identify the adhesive cells of 11.5-dpc gonads, immunohistochemistry with anti-SF1/Ad4Bp antibody (a specific marker for supporting, steroidogenic, and coelomic epithelial cells) and staining for alkaline phosphatase (a germ cell marker) were carried out. As a result, EGF domains, as well as discoidin domains, were capable of binding to all three groups of SF1/Ad4Bp-positive and negative somatic cells, and germ cells of 11.5-dpc gonads. These findings therefore suggest that MFG-E8 mediates the cell-to-cell interaction among several somatic cell types and germ cells in mouse early gonadogenesis.

Distal myopathy with rimmed vacuoles: impaired O-glycan formation in muscular glycoproteins.

Distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Arachis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. alpha-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including alpha-dystroglycan, occurs in DMRV.

Identification and characterization of human Golgi nucleotide sugar transporter SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family.

We report the molecular cloning of SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family. The gene SLC35D2 maps to chromosome 9q22.33. SLC35D2 cDNA codes for a hydrophobic protein consisting of 337 amino acid residues with 10 putative transmembrane helices. Northern blot analysis revealed the SLC35D2 mRNA as a single major band corresponding to 2.0 kb in length. SLC35D2 was localized in the Golgi membrane and exhibited around 50% similarity with three nucleotide sugar transporters: human SLC35D1 (UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter), fruitfly fringe connection (frc) transporter, and nematode SQV-7 transporter, the latter two being involved in developmental and organogenetic processes. Heterologous expression of SLC35D2 protein in yeast indicated that UDP-N-acetylglucosamine is a candidate for the substrate(s) of the transporter. The sequence similarity, subcellular localization, and transporting substrate suggest that SLC35D2 is a good candidate for the ortholog of frc transporter, which is involved in the Notch signaling system by providing the fringe N-acetylglucosaminyltransferase with the substrate. We also describe the identification and categorization of the human SLC35 gene family.

Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta.

We have identified a novel phospholipase A1, named mPA-PLA1beta, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA1alpha. The sequence of mPAPLA1beta encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA1, mPA-PLA1alpha. mPA-PLA1beta contains a short lid and deleted beta9 loop, which are characteristics of PLA1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA1alpha and phosphatidylserine-specific PLA1. Both mPA-PLA1beta and mPA-PLA1alpha recombinant proteins exhibited PA-specific PLA1 activity and were vanadate-sensitive. When mPAPLA1beta-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA1alpha and beta-expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA1alpha protein was recovered from the cell supernatant. By contrast, mPA-PLA1beta was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA1beta has higher affinity to heparin than mPA-PLA1alpha. We also found that the membrane-associated mPA-PLA1s were insoluble in solubilization by 1% Triton X-100 and were detected in Triton X-100-insoluble buoyant fractions of sucrose gradients. The present study raises the possibility that production of LPA by mPA-PLA1alpha and -beta occurs on detergent-resistant membrane domains of the cells where they compete with lipid phosphate phosphatase for PA.

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.

Roles of HNK-1 carbohydrate epitope and its synthetic glucuronyltransferase genes on migration of rat neural crest cells.

HNK-1 carbohydrate epitope is localized on the surface of avian neural crest cells (NCCs), and is necessary for their migration. However, it is still disputed whether the epitope works in similar ways in mammalian embryos. In this study, we found that HNK-1 carbohydrate epitope was specifically detected in some of the cranial ganglia, migrating trunk NCCs and some non-NCC derivatives in the rat embryo. Two genes encoding glucuronyltransferases that synthesize the HNK-1 epitope in vitro (GlcAT-P and GlcAT-D) were recently identified in the rat. Interestingly, the NCCs in the cranial ganglia expressed the GlcAT-D gene, whereas the migrating trunk NCCs expressed the GlcAT-P gene. To investigate in vivo functions of the GlcATs in the NCC migration further, we overexpressed GlcAT genes by electroporation in the cranial NCCs in cultured rat embryos. Transfection of both GlcAT genes resulted in efficient synthesis of the HNK-1 epitope in the NCCs. GlcAT-P overexpression increased distance of cranial NCC migration, whereas GlcAT-D overexpression did not show this effect. Our data suggest that the HNK-1 epitope synthesized by different GlcATs is involved in migration in the sublineages of the NCCs in the rat embryo, and that GlcAT-P and GlcAT-D mediate different effects on the NCC migration.

Immunohistochemical and biochemical analyses of GD3, GT1b, and GQ1b gangliosides during neural differentiation of P19 EC cells.

In an earlier study, we showed that expressions of GD3, GT1b, and GQ1b gangliosides in P19 embryonic carcinoma (EC) cells were enhanced during their neural differentiation induced by retinoic acid. We now further demonstrated that this increase of the b-series gangliosides is due to an increase in their corresponding synthases (sialyltransferase-II, -IV, and -V) in the Golgi. Of the three gangliosides studied, GQ1b appeared to be the best candidate for monitoring such differentiation process. We also used fluorescence-labeled monoclonal antibodies and confocal fluorescence microscopy to obtain direct visual information about the relationship of gangliosides and neural specific proteins in neuron development. Again, GQ1b is the most interesting as it localizes with synaptophysin and neural cell adhesion molecules (NCAMs) on synaptic boutons or dendritic spines in RA-induced neurons (R/N). This suggests that GQ1b could be used as a marker for synapse formation during construction of the neural network.

Association of GPI-anchored protein TAG-1 with src-family kinase Lyn in lipid rafts of cerebellar granule cells.

We have demonstrated that antibody-mediated crosslinking of GPI-anchored TAG-1 induced activation of src-family kinase Lyn and rapid tyrosine phosphorylation of an 80-kDa protein (p80), a putative substrate for Lyn, in the lipid raft fraction prepared from primary cerebellar cultures, suggesting the functional association of TAG-1 with Lyn in lipid rafts of the rat cerebellum. In this study, the association was confirmed using a cDNA expression system. TAG-1-expressing CHO transfectants exhibited enhanced self-aggregation and promoted neurite outgrowth of primary cerebellar cultures as a culture substrate. The anti-TAG-1 antibody co-immunoprecipitated Lyn with TAG-1 and induced co-patching of TAG-1 with Lyn in both TAG-1 and Lyn-expressing CHO transfectants. Density gradient analysis revealed that TAG-1 is present in the lipid raft fraction of the CHO transfectants. Furthermore, pretreatment with a sphingolipid biosynthesis inhibitor ISP-1 reduced the extent of tyrosine phosphorylation of p80 by the antibody-mediated crosslinking of TAG-1. Immunocytochemical study showed that both TAG-1 and Lyn are present in cerebellar granule cells. These observations suggest that TAG-1 associates with Lyn in lipid rafts of rat cerebellar granule cells.

Depletion of definitive gut endoderm in Sox17-null mutant mice.

In the mouse, the definitive endoderm is derived from the epiblast during gastrulation, and, at the early organogenesis stage, forms the primitive gut tube, which gives rise to the digestive tract, liver, pancreas and associated visceral organs. The transcription factors, Sox17 (a Sry-related HMG box factor) and its upstream factors, Mixer (homeobox factor) and Casanova (a novel Sox factor), have been shown to function as endoderm determinants in Xenopus and zebrafish, respectively. However, whether the mammalian orthologues of these genes are also involved with endoderm formation is not known. We show that Sox17(-/-) mutant embryos are deficient of gut endoderm. The earliest recognisable defect is the reduced occupancy by the definitive endoderm in the posterior and lateral region of the prospective mid- and hindgut of the headfold-stage embryo. The prospective foregut develops properly until the late neural plate stage. Thereafter, elevated levels of apoptosis lead to a reduction in the population of the definitive endoderm in the foregut. In addition, the mid- and hindgut tissues fail to expand. These are accompanied by the replacement of the definitive endoderm in the lateral region of the entire length of the embryonic gut by cells that resemble the visceral endoderm. In the chimeras, although Sox17-null ES cells can contribute unrestrictedly to ectodermal and mesodermal tissues, few of them could colonise the foregut endoderm and they are completely excluded from the mid- and hindgut endoderm. Our findings indicate an important role of Sox17 in endoderm development in the mouse, highlighting the idea that the molecular mechanism for endoderm formation is likely to be conserved among vertebrates.

Pax6 controls the expression of Lewis x epitope in the embryonic forebrain by regulating alpha 1,3-fucosyltransferase IX expression.

Pax6 is a transcription factor involved in brain patterning and neurogenesis. Expression of Pax6 is specifically observed in the developing cerebral cortex, where Lewis x epitope that is thought to play important roles in cell interactions is colocalized. Here we examined whether Pax6 regulates localization of Lewis x using Pax6 mutant rat embryos. The Lewis x epitope disappeared in the Pax6 mutant cortex, and activity of alpha1,3-fucosyltransferase, which catalyzed the last step of Lewis x biosynthesis, drastically decreased in the mutant cortex as compared with the wild type. Furthermore, expression of a fucosyltransferase gene, FucT-IX, specifically decreased in the mutant, while no change was seen for expression of another fucosyltransferase gene, FucT-IV. These results strongly suggest that Pax6 controls Lewis x expression in the embryonic brain by regulating FucT-IX gene expression.