Hidenao Toyoda

A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

The 3′-Phosphoadenosine 5′-Phosphosulfate Transporters, PAPST1 and 2, Contribute to the Maintenance and Differentiation of Mouse Embryonic Stem Cells

Recently, we have identified two 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent Km value of 1.54 µM or 1.49 µM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.

A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells.

Background: Carbohydrate epitopes are often used as markers for characterization of hiPS cells. Results: A mouse IgG1 antibody (R-17F) was raised using hiPS cells as an antigen. Conclusion: R-17F recognizes lacto-N-fucopentaose I on glycolipid and exhibits a cytotoxic effect on hiPS/ES cells. Significance: R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS/ES cells, which are a risk factor for carcinogenesis. We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MSn analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc). A critical role of the terminal Fucα1–2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1–2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.

Analysis of Drosophila Glucuronyl C5-Epimerase: IMPLICATIONS FOR DEVELOPMENTAL ROLES OF HEPARAN SULFATE SULFATION COMPENSATION AND 2-O-SULFATED GLUCURONIC ACID*

Background: The physiological significance of C5-epimerization of glucuronic acid (GlcA) remains elusive. Results: Drosophila Hsepi mutants are viable and fertile with only minor morphological defects, but they have a short lifespan. Conclusion: Sulfation compensation and 2-O-sulfated GlcA contribute to the mild phenotypes of Hsepi mutants. Significance: The findings suggest novel developmental roles of 2-O-sulfated GlcA. During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.

Phenotype‐based clustering of glycosylation‐related genes by RNAi‐mediated gene silencing

Glycan structures are synthesized by a series of reactions conducted by glycosylation‐related (GR) proteins such as glycosyltransferases, glycan‐modifying enzymes, and nucleotide‐sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide‐O‐xylosyltransferase, β1,4‐galactosyltransferase I, β1,3‐galactosyltransferase II, and β1,3‐glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N‐linked, O‐linked, Notch‐related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe‐dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N‐linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

The Role of Drosophila Heparan Sulfate 6-O-Endosulfatase in Sulfation Compensation

Background: HS sulfation compensation provides robustness to cellular signaling and animal development, but its mechanism is unknown. Results: Sulf1 is required for sulfation compensation in Hs2st mutants. Conclusion: Sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1. Significance: The finding that Sulf1 is a novel component of HS sulfation compensation machinery provides novel mechanistic insight into this poorly understood phenomenon. The biosynthesis of heparan sulfate proteoglycans is tightly regulated by multiple feedback mechanisms, which support robust developmental systems. One of the regulatory network systems controlling heparan sulfate (HS) biosynthesis is sulfation compensation. A previous study using Drosophila HS 2-O- and 6-O-sulfotransferase (Hs2st and Hs6st) mutants showed that loss of sulfation at one position is compensated by increased sulfation at other positions, supporting normal FGF signaling. Here, we show that HS sulfation compensation rescues both Decapentaplegic and Wingless signaling, suggesting a universal role of this regulatory system in multiple pathways in Drosophila. Furthermore, we identified Sulf1, extracellular HS 6-O-endosulfatase, as a novel component of HS sulfation compensation. Simultaneous loss of Hs2st and Sulf1 led to 6-O-oversulfation, leading to patterning defects, overgrowth, and lethality. These phenotypes are caused at least partly by abnormal up-regulation of Hedgehog signaling. Thus, sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1.

Generation of Rat Induced Pluripotent Stem Cells Using a Plasmid Vector and Possible Application of a Keratan Sulfate Glycan Recognizing Antibody in Discriminating Teratoma Formation Phenotypes

Induced pluripotent stem cells (iPSCs) offer an invaluable tool for biological research and regenerative medicine. We report establishment of rat iPSCs (riPSCs) using a plasmid vector encoding four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. Although all riPSC clones were generated and cultured under the same conditions, expressed hallmark pluripotency markers and differentiated successfully in vitro, the expression of a keratan sulfate glycan epitope with unique properties defined by R-10G antibody varied in the riPSC clones. In contrast, tumor rejection antigen (TRA)-1-81 epitope expression was comparable. A clone highly reactive to R-10G antibody formed teratomas in vivo consisting of cells from all three germ layers. However, clones expressing a lower level of the epitope defined by R-10G resulted in tumors with rapid growth consisting of undifferentiated cells. Additionally, riPSCs could be successfully differentiated into a neuronal lineage including glutamate neurons that responded to agonist stimulation. These observations demonstrate a glycophenotypic difference that may potentially serve as a useful probe for riPSC evaluation and to study the role of glycans in pluripotency and carcinogenesis in these cells.