In vivo activities of heparan sulfate differentially modified by NDSTs during development.

Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for growth factor signaling during development. It is well known that the level and patterns of sulfate groups of heparan sulfate (HS) chains, or HS fine structures, have a major impact on HSPG function. On the other hand, the physiological significance of other structural features of HS, including NS/NA domain organization, remains to be elucidated. A blueprint of the HS domain structures is mainly controlled by HS N-deacetylase/N-sulfotransferases (NDSTs). To analyze in vivo activities of differentially modified HS, we established two knock-in (KI) Drosophila strains with the insertion of mouse Ndst1 (mNdst1) or Ndst2 (mNdst2) in the locus of sulfateless (sfl), the only Drosophila NDST. In these KI lines, mNDSTs are expressed from the sfl locus, in the level and patterns identical to the endogenous sfl gene. Thus, phenotypes of Ndst1 KI and Ndst2KI animals reflect the ability of HS structures made by these enzymes to rescue sfl mutation. Remarkably, we found that mNdst1 completely rescued the loss of sfl. mNdst2 showed a limited rescue ability, despite a higher level of HS sulfation compared to HS in mNdst1 KI. Our study suggests that independent of sulfation levels, additional HS structural features controlled by NDSTs play key roles during tissue patterning.

Endogenous chondroitin extends the lifespan and healthspan in C. elegans.

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans. However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans. Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.

Chondroitin sulfate is required for follicle epithelial integrity and organ shape maintenance in Drosophila.

Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies.

This report describes the isolation and characterization of two new antibodies, R-6C (IgM) and R-13E (IgM), which were generated in C57BL/6 mice (Mus musculus) using the Tic (JCRB1331) human induced pluripotent cell (hiPSC) line as an antigen, and their comparisons with two existing antibodies, R-10G (IgG1) and R-17F (IgG1). Their epitopes were studied by western blotting after various glycosidase digestions, binding analyses using enzyme-linked immunosorbent assays (ELISAs) and microarrays with various synthetic oligosaccharides. The minimum epitope structures identified were: Siaα2-3Galß1-3GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)ß1 (R-6C), Fucα1-2Galß1-3GlcNAcß1-3Galß1 (R-13E), Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)ß1 (R-10G), and Fucα1-2Galß1-3GlcNAß1-3Galß1-4Glc (lacto-N-fucopentaose I) (R-17F). Most glycoprotein epitopes are expressed as O-glycans. The common feature of these epitopes is the presence of an N-acetyllactosamine type 1 structure (Galß1-3GlcNAc) at their nonreducing termini, followed by a type 2 structure (Galß1-4GlcNAc); this arrangement comprises a type 1-type 2 motif. This motif is also shared by TRA-1-60, a traditional onco-fetal antigen. In contrast, the R-10G epitope has a type 2-type 2 motif. Among these antibodies, R-17F and R-13E exhibit cytotoxic activity toward hiPSCs. R-17F and R-13E exhibit extremely high similarity in the amino acid sequences in their complementarity-determining regions (CDRs), which is consistent with their highly similar glycan recognition. These antibodies are excellent tools for investigating the biological functions of glycoconjugates in hiPSCs/hESCs; they could be useful for the selection, isolation and selective killing of such undifferentiated pluripotent stem cells.

Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies.

We developed two human-induced pluripotent stem cell (hiPSC)/human embryonic stem cell (hESC)-specific glycan-recognizing mouse antibodies, R-10G and R-17F, using the Tic (JCRB1331) hiPSC line as an antigen. R-10G recognizes a low-sulfate keratan sulfate, and R-17F recognizes lacto-N-fucopentaose-1. To evaluate the general characteristics of stem cell glycans, we investigated the hiPSC line 201B7 (HPS0063), a prototype iPSC line. Using an R-10G affinity column, an R-10G-binding protein was isolated from 201B7 cells. The protein yielded a single but very broad band from 480 to 1236 kDa by blue native gel electrophoresis. After trypsin digestion, the protein was identified as podocalyxin by liquid chromatography/mass spectrometry. According to Western blotting, the protein reacted with R-10G and R-17F. The R-10G-positive band was resistant to digestion with glycan-degrading enzymes, including peptide N-glycanase, but the intensity of the band was decreased significantly by digestion with keratanase, keratanase II, and endo-ß-galactosidase, suggesting the R-10G epitope to be a keratan sulfate. These results suggest that keratan sulfate-type epitopes are shared by hiPSCs. However, the keratan sulfate from 201B7 cells contained a polylactosamine disaccharide unit (Galß1-4GlcNAc) at a significant frequency, whereas that from Tic cells consisted mostly of keratan sulfate disaccharide units (Galß1-4GlcNAc(6S)). In addition, the abundance of the R-10G epitope was significantly lower in 201B7 cells than in Tic cells.

Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants.

Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson's Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors.

Effects of Cu2+ on conformational change and aggregation of hPrP180-192 with a V180I mutation of the prion protein.

Prion diseases are neurodegenerative disorders caused by misfolding of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc). However, the aggregation mechanism is not entirely understood because of the physical properties of PrP, such as its solubility or aggregation in vitro and conformational or mutation diversity. Recently, we reported the physical and physiological properties of a synthetic fragment peptide. In the present study, we assessed the importance of a point mutation at the C-terminal region of PrP in structural conversion and aggregation and evaluated the physical and physiological properties of the point-mutated human-PrP180-192 V180I (hPrP180-192 V180I) using circular dichroism spectra, high-performance liquid chromatography, Affinix QNµ, and thioflavin-T staining, including the effects of Cu2+. The secondary structure of hPrP180-192 V180I changed from a random coil to a ß-sheet in Cu2+ free buffer. In addition, we observed molecular interactions in hPrP180-192 V180I and aggregation with itself, which were inhibited by Cu2+. We conclude that the point mutation in the C-terminal region of PrP, including hPrP180-192 V180I, and Cu2+ may play an important role in the conversion of PrPC to PrPSc.

Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes.

A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.

Microdetermination of Sialic Acids in Blood Samples by Hydrophilic Interaction Chromatography Coupled to Post-column Derivatization and Fluorometric Detection.

An analytical method for the determination of sialic acids in biological samples has been developed and applied to fetal bovine serum (FBS), newborn calf serum and adult bovine serum. The hydrolysis of sera was carried out and the liberated sialic acids were quantified using a rapid and sensitive HPLC. The HPLC includes the separation and detection of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) using hydrophilic interaction liquid chromatography and a fluorometric post-column reaction with 2-cyanoacetamide. The calibration graphs for Neu5Ac and Neu5Gc were linear over the range of 10 pmol - 5 nmol. The concentrations of sialic acids in FBS, newborn calf serum and adult bovine serum were 5.06, 3.79 and 1.64 mM, respectively. The ratios of Neu5Gc and Neu5Ac changed dramatically according to the development stages. The present method has a satisfactory sensitivity in the quantification of Neu5Ac and Neu5Gc in serum samples. It seems that this analytical system can therefore be applied for routine use in clinical investigations of serum sialylation changes in cancer patients.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides.

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galß1 - 3GlcNAc (type 1) or Galß1 - 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)3-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galß1 - 4GlcNAc(6S)ß1 - 3Galß1 - 4GlcNAc(6S)ß1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galß1 - 3GlcNAcß1 - 3Galß1 - 4GlcNAcß1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125-1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galß1 - 3GlcNAc(6S)ß1 - 3Galß1 - 4GlcNAc(6S)ß1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galß1 - 3GlcNAcß1 - 3Galß1 - 4GlcNAcß1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only "type 2-type 2 keratan sulfate" but also "type 1-type 2 keratan sulfate", significantly.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

Characterization of glycoproteins expressing the blood group H type 1 epitope on human induced pluripotent stem (hiPS) cells.

Recently, we established two mouse monoclonal antibodies (R-10G and R-17F). The R-17F antibody (IgG1 subtype) exhibited a strong cytotoxic effect on hiPS/ES cells. The R-17F antigen isolated from a total lipid extract of hiPS (Tic) cells was identified as LNFP I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc). In the present study, R-17F binding proteins were isolated from hiPS (Tic) cell lysates with an affinity column of R-17F. They gave one major R-17F positive band around 250 kDa, and several minor bands between 150 kDa and 25 kDa. The former band was identified as podocalyxin by LC/MS/MS after SDS-PAGE. Hapten inhibition studies on R-17F binding to R-17F column-purified proteins with various synthetic oligosaccharides revealed that the blood group H type 1 triaose structure (Fucα1-2Galß1-3GlcNAc) was the predominant epitope on all the R-17F binding proteins. These bands disappeared completely on digestion with α1-2 fucosidase, but not with α1-3/4 fucosidase. Upon PNGase F digestion, the R-17F positive band around and above 250 kDa did not show any change, while the minor bands between 150 kDa and 25 kDa disappeared completely, suggesting that the epitope is expressed on N-glycans in the latter and probably on O-glycans in the former. These results, together with those obtained in our previous studies on R-10G (Kawabe et al. Glycobiology, 23, 322-336 (2013)), indicated that both R-10G and R-17F epitopes are carried on the same podocalyxin molecule. The R-17F epitopes on these glycoproteins expressed on hiPS cells could be associated with the molecular mechanism underlying the carbohydrate-mediated cytotoxic activity of R-17F.

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

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 MS(n) 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 (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc). 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.

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, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-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.

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.

Analysis of Drosophila glucuronyl C5-epimerase: implications for developmental roles of heparan sulfate sulfation compensation and 2-O-sulfated glucuronic acid.

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.

The role of Drosophila heparan sulfate 6-O-endosulfatase in sulfation compensation.

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.

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-ß-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.