Search

Heparan sulfate proteoglycans (HSPGs) regulate a wide range of biological activities in both physiological and pathological conditions. Altered expression or deregulated function of HSPGs and their heparan sulfate (HS) chains significantly contribute to carcinogenesis as well and crucially depends on the functioning of the complex system of HS biosynthetic/modifying enzymes termed as "GAGosome". Here, we aimed at investigating the expression profile of the system in a cell culture model of stroma-epithelial crosstalk and searching for transcription factors potentially related to the regulation of expression of the genes involved. Coculture of BjTERT-fibroblasts with normal PNT2 human prostate epithelial cells resulted in significant downregulation (2-4-fold) of transcriptional activity of HS metabolism-involved genes ( EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE ) in both cell types, whereas coculture with prostate cancer cells (LNCaP, PC3, DU145) demonstrated no significant interchanges. Human Transcription Factor RT 2 Profiler PCR array and manual RT-PCR verification supposed FOS, MYC, E2F, SRF, NR3C1 as potential candidates for regulation and/or coordination of HS biosynthesis. Taken together, transcriptional activity of HS biosynthetic system in normal fibroblasts and prostate epithelial cells during their coculture might be controlled by their intercellular communication, reflecting of adaptation of these cells to each other. The regulation is attenuated or abrogated if normal fibroblasts interact with prostate cancer cells making the cancer cells independent of the limiting effects of fibroblasts, thus contributing to possibility of unlimited growth and progression. Overall, these data demonstrate an ability of cell-cell interactions to affect transcriptional activity of HS biosynthesis-involved genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Grigorieva, Strokotova, Ernberg and Kashuba.)

Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N -acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N -, 2- O -, 6- O -, and 3- O - are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.

Heparan sulfate (HS) is a linear polysaccharide found in almost all animal cells and plays an important role in various biological processes. HS functions mainly via covalently binding to core proteins to form HS proteoglycans (HSPGs), which are heterogeneous in the lengths of the HS chain, the modifications on HS and the core proteins. The molecular mechanisms underlying HSPG heterogeneity, although widely studied, are not yet fully defined. The expression profiles of HS biosynthesis enzymes and HSPG core proteins likely contribute to the HSPG heterogeneity, but these expression profiles remain poorly characterized. To investigate the expression profiles of genes encoding HS biosynthesis enzymes and HSPG core proteins, we systematically integrated the publicly available RNA sequencing data in mice. To reveal the spatial expression of these genes, we analyzed their expression in 21 mouse tissues. To reveal the temporal expression of these genes, we analyzed their expression at 17 time points during the mouse forebrain development. To determine the cell-type-specific expression of these genes, we obtained their expression profiles in 23 cell types in the mouse cerebral cortex by integrating single nucleus RNA sequencing data. Our findings demonstrate the spatial, temporal and cell-type-specific expression of genes encoding HS biosynthesis enzymes and HSPG core proteins and represent a valuable resource to the HS research community., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac 4 GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac 4 GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.

Several studies reported the concerted and mutual communication between the prostate epithelium and stroma, which determines the final organ architecture and function, but gets awry in cancer. Deciphering the mechanisms involved in this communication is crucial to find new therapeutic strategies. HS sequesters a number of secreted growth factors and cytokines, controlling their bioavailability to the target cells, suggesting that HS is an important regulator of the extracellular matrix (ECM) and a key player in the cell-cell and cell-microenvironment communication during prostate morphogenesis and physiology. We propose that by controlling HS biosynthesis and sulfation pattern, as well as the cleavage of the HS chain and/or the shedding of proteoglycans, epithelial and stromal cells are able to precisely tune the availability of signaling molecules and modulate ligand-receptor interaction and intracellular signal transduction., (© 2020 American Association of Anatomists.)

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Thymus organogenesis and T cell development are coordinated by various soluble and cell-bound molecules. Heparan sulfate (HS) proteoglycans can interact with and immobilize many soluble mediators, creating fields or gradients of secreted ligands. While the role of HS in the development of many organs has been studied extensively, little is known about its function in the thymus. Here, we examined the distribution of HS in the thymus and the effect of its absence on thymus organogenesis and T cell development. We found that HS was expressed most abundantly on the thymic fibroblasts and at lower levels on endothelial, epithelial, and hematopoietic cells. To study the function of HS in the thymus, we eliminated most of HS in this organ by genetically disrupting the glycosyltransferase Ext1 that is essential for its synthesis. The absence of HS greatly reduced the size of the thymus in fetal thymic organ cultures and in vivo, in mice, and decreased the production of T cells. However, no specific blocks in T cell development were observed. Wild-type thymic fibroblasts were able to physically bind the homeostatic chemokines CCL19, CCL21, and CXCL12 ex vivo. However, this binding was abolished upon HS degradation, disrupting the CCL19/CCL21 chemokine gradients and causing impaired migration of dendritic cells in thymic slices. Thus, our results show that HS plays an essential role in the development and growth of the thymus and in regulating interstitial cell migration., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Heparosan, the capsular polysaccharide of E. coli K5 is currently used as the starting material in the chemoenzymatic synthesis of heparan sulfate and the structurally related anticoagulant drug heparin. Base hydrolysis of N-acetyl groups and their subsequent N-sulfonation, are used to prepare N-sulfoheparosan an intermediate of biosynthesis. In the present study, when excess sulfonation reagent was used during N-sulfonation, some O-sulfation also took place in the N-sulfoheparosan product. After a nearly full digestion, a hexasaccharide fraction exhibited resistance to heparin lyase II. Excessive digestion by heparin lyase II and structural identification by NMR and mass spectroscopy indicated that the resistant hexasaccharide fraction has two structures, ΔUA-GlcNS-GlcA2S-GlcNS-GlcA-GlcNS and ΔUA-GlcNS-GlcA- GlcNS3S-GlcA-GlcNS in similar amounts. The 2-sulfated structure exhibited partial resistance to heparin lyase II; however the structure of ΔUA-GlcNS-GlcA-GlcNS3S was completely resistant to heparin lyase II.

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263 , a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1 , a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3- O -sulfotransferase expressed in cells. Enhanced 3- O -sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells., Competing Interests: Competing interest statement: The University of California San Diego and J.D.E. have a financial interest in TEGA Therapeutics, Inc. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies.

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., (Copyright © 2020 Reynolds-Peterson et al.)

Background: Glycosaminoglycans (GAGs) are essential in many infections, including recurrent bacterial respiratory infections, the main cause of mortality in cystic fibrosis (CF) patients., Methods: Using a cellular model of healthy and CF lung epithelium, a comparative transcriptomic study of GAG encoding genes was performed using qRT-PCR, and their differential involvement in the adhesion of bacterial pathogens analyzed by enzymatic degradation and binding competition experiments., Results: Various alterations in gene expression in CF cells were found which affect GAG structures and seem to influence bacterial adherence to lung epithelium cells. Heparan sulfate appears to be the most important GAG species involved in bacterial binding., Conclusions: Adherence to lung epithelial cells of some of the main pathogens involved in CF is dependent on GAGs, and the expression of these polysaccharides is altered in CF cells, suggesting it could play an essential role in the development of infectious pathology., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Global production of pharmaceutical heparin (Hp) is increasing, and the production process from raw mucosal material results in large amounts of waste by-products. These contain lower sulfated Hp-like and heparan sulfate (HS), as well as other glycosaminoglycans, which are bioactive entities with pharmaceutical potential. Here we describe the first purification, structural and functional characterisation of Hp-like and HS polysaccharides from the four major by-product fractions of standard heparin production. Analysis of the by-products by disaccharide composition analysis and NMR demonstrated a range of structural characteristics which differentiate them from Hp (particularly reduced sulfation and sulfated disaccharide content), and that they are each distinct. Functional properties of the purified by-products varied, each displaying distinct anticoagulant profiles in different assays, and all exhibiting significantly lower global and specific inhibition of the coagulation pathway than Hp. The by-products retained the ability to promote cell proliferation via fibroblast growth factor receptor signalling, with only minor differences between them. These collective analyses indicate that they represent an untapped and economical source of structurally-diverse Hp-like and HS polysaccharides with the potential for enhancing future structure-activity studies and uncovering new biomedical applications of these important natural products.

Heparan sulfate (HS), is a glycosaminoglycan (GAG) involved in various biological processes, including blood coagulation, wound healing and embryonic development. HS 3-O-sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, is a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a His tagged-3-OST-1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. The 3-OST-1 was purified by Ni affinity column and refolded by dialysis. The activity of obtained 3-OST-1 was 0.04 U/mL with a specific activity of 0.55 U/mg after renaturation. Furthermore, a co-expressed recombinant plasmid pET-28a-3-OST-1 with the chaperone expression system (pGro7) was constructed and transferred to E. coli BL21 (DE3) to co-express recombinant strain E. coli BL21 (DE3)/pET-28a-3-OST-1 + pGro7. The soluble expression of 3-OST-1 was significantly improved in the co-expressed recombinant strain, with enzyme activity reaching 0.06 U/mL and having a specific activity of 0.83 U/mg. N-sulfo, N-acetylheparosan (NSNAH) was modified by the recombinant expressed 3-OST-1 and the product was confirmed by 1 H NMR showing the sulfo group was successfully transferred to NSNAH., (Copyright © 2018. Published by Elsevier Inc.)

Heparan Sulfate (HS) is a cell signaling molecule linked to pathological processes ranging from cancer to viral entry, yet fundamental aspects of its biosynthesis remain incompletely understood. Here, the binding preferences of the uronyl 2-O-sulfotransferase (HS2ST) are examined with variably-sulfated hexasaccharides. Surprisingly, heavily sulfated oligosaccharides formed by later-acting sulfotransferases bind more tightly to HS2ST than those corresponding to its natural substrate or product. Inhibition assays also indicate that the IC 50 values correlate simply with degree of oligosaccharide sulfation. Structural analysis predicts a mode of inhibition in which 6-O-sulfate groups located on glucosamine residues present in highly-sulfated oligosaccharides occupy the canonical binding site of the nucleotide cofactor. The unexpected finding that oligosaccharides associated with later stages in HS biosynthesis inhibit HS2ST indicates that the enzyme must be separated temporally and/or spatially from downstream products during biosynthesis in vivo, and highlights a challenge for the enzymatic synthesis of lengthy HS chains in vitro.

A chemoenzymatic approach has been developed for the preparation of diverse libraries of heparan sulfate (HS) oligosaccharides. It employs chemically synthesized oligosaccharides having a chemical entity at a GlcN residue, which in unanticipated manners influences the site of modification by NST, C5-Epi/2-OST and 6-OST 1 /6-OST 3 , thus resulting in oligosaccharides differing in N/O-sulfation and epimerization pattern. The enzymatic transformations defined fine substrate requirements of NST, C5-Epi, 2-OST, and 6-OST., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling., Methods: We investigated heparan sulfate biosynthesis and FGF2 signaling using four cell lines, EXT1-deficient cells, EXT2-, EXTL2-, or EXTL3-knockdown cells, by HPLC, qRT-PCR, flow cytometry, and western blotting., Results: Reduced expression of either EXT1, EXT2, or EXTL3 decreased heparan sulfate biosynthesis, and consequently suppressed the FGF2-dependent proliferation of mouse L fibroblasts. In contrast, although knockdown of EXTL2 increased the amounts of heparan sulfate, FGF2-dependent proliferation was significantly inhibited because the increased heparan sulfate enhanced the incorporation of FGF2 into the cells., Conclusions: EXTL2 controls FGF2 signaling through regulation of heparan sulfate biosynthesis in a manner distinct from that of other exostosins., General Significance: This study provides new insights into the regulatory mechanisms of FGF2 signaling by EXTL2., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heparin has been used clinically as an anti-coagulant for more than 100 y and the major source of this therapeutic is still animal tissues. Contamination issues in some batches of heparin over 10 y ago have highlighted the need to develop alternative methods of production of this essential drug. 1 Bioengineering heparin by expressing serglycin in mammalian cells is a promising approach that was recently reported by the authors. 2 This addendum explores the approaches that the authors are taking to increase the yield of recombinantly expressed serglycin decorated with heparin/heparan sulfate focusing on cell culture and bioreactor conditions and proposes that the cell microenvironment is a key modulator of heparin biosynthesis.

3-O-Sulfotransferase enzyme (sHS) from Litopenaeus vannamei was cloned and its substrate specificity was investigated against a number of GAG structures, including modified heparin polysaccharides and model oligosaccharides. For the heparin polysaccharides, derived from porcine intestinal mucosa heparin, sulfate groups were incorporated into glucosamine residues containing both N-sulfated and N-acetylated substitution within the regions of the predominant repeating disaccharide, either I-A NS or I-A NAc . However, the resulting polysaccharides did not stabilize antithrombin, which is correlated with anticoagulant activity. It was also shown that the enzyme was able to sulfate disaccharides, I 2S -A NS and G-A NAc . The results further illustrate that 3-O-sulfation can be induced outside of the classical heparin-binding pentasaccharide sequence, show that 3-O-sulfation of glucosamine is not a sufficient condition for antithrombin stabilization and suggest that the use of this enzyme during HS biosynthesis may not occur as the final enzymatic step.

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development., Competing Interests: The authors have declared that no competing interests exist.

Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2- but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2(-/-) mice, which contained reduced levels of the polysaccharide. Detailed disaccharide composition analysis showed no major structural difference between heparan sulfate from control and Ndst2(-/-) tissues, with the exception of heparan sulfate from spleen where the relative amount of trisulfated disaccharides was lowered in the absence of NDST2. In vivo transcript expression levels of the heparan sulfate-polymerizing enzymes Ext1 and Ext2 were also largely unaffected by NDST2 levels, pointing to a mode of regulation other than increased gene transcription. Size estimation of heparan sulfate polysaccharide chains indicated that increased chain lengths in NDST2-overexpressing cells alone could explain the increased heparan sulfate content. A model is discussed where NDST2-specific substrate modification stimulates elongation resulting in increased heparan sulfate chain length., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.

Heparan sulfate (HS) is widely distributed in mammalian tissues in the form of HS proteoglycans, which play essential roles in various physiological and pathological processes. In contrast to the template-guided processes involved in the synthesis of DNA and proteins, HS biosynthesis is not believed to involve a template. However, it appears that the final structure of HS chains was strictly regulated. Herein, we report research based hypothesis that two major steps, namely "coding" and "decoding" steps, are involved in the biosynthesis of HS, which strictly regulate its chemical structure and biological activity. The "coding" process in this context is based on the distribution of sulfate moieties on the amino groups of the glucosamine residues in the HS chains. The sulfation of these amine groups is catalyzed by N-deacetylase/N-sulfotransferase, which has four isozymes. The composition and distribution of sulfate groups and iduronic acid residues on the glycan chains of HS are determined by several other modification enzymes, which can recognize these coding sequences (i.e., the "decoding" process). The degree and pattern of the sulfation and epimerization in the HS chains determines the extent of their interactions with several different protein factors, which further influences their biological activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multipotent neural stem cells (NSCs) provide a model to investigate neurogenesis and develop mechanisms of cell transplantation. In order to define improved markers of stemness and lineage specificity, we examined self-renewal and multi-lineage markers during long-term expansion and under neuronal and astrocyte differentiating conditions in human ESC-derived NSCs (hNSC H9 cells). In addition, with proteoglycans ubiquitous to the neural niche, we also examined heparan sulfate proteoglycans (HSPGs) and their regulatory enzymes. Our results demonstrate that hNSC H9 cells maintain self-renewal and multipotent capacity during extended culture and express HS biosynthesis enzymes and several HSPG core protein syndecans (SDCs) and glypicans (GPCs) at a high level. In addition, hNSC H9 cells exhibit high neuronal and a restricted glial differentiative potential with lineage differentiation significantly increasing expression of many HS biosynthesis enzymes. Furthermore, neuronal differentiation of the cells upregulated SDC4, GPC1, GPC2, GPC3 and GPC6 expression with increased GPC4 expression observed under astrocyte culture conditions. Finally, downregulation of selected HSPG core proteins altered hNSC H9 cell lineage potential. These findings demonstrate an involvement for HSPGs in mediating hNSC maintenance and lineage commitment and their potential use as novel markers of hNSC and neural cell lineage specification., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Heparan sulfate (HS) proteoglycans (PGs) are ubiquitously expressed on cell surfaces and in the extracellular matrix of most animal tissues, having essential functions in development and homeostasis, as well as playing various roles in disease processes. The functions of HSPGs are mainly dependent on interactions between the HS-side chains with a variety of proteins including cytokines, growth factors, and their receptors. In a given HS polysaccharide, negatively charged sulfate and carboxylate groups are arranged in various types of domains, generated through strictly regulated biosynthetic reactions and with enormous potential for structural variability. The mode of HS-protein interactions is assessed through binding experiments using saccharides of defined composition in vitro, signaling assays in cell models where HS structures are manipulated, and targeted disruption of genes for biosynthetic enzymes in animals (mouse, zebrafish, Drosophila, and Caenorhabditis elegans) followed by phenotype analysis. Whereas some protein ligands appear to require strictly defined HS structure, others bind to variable saccharide domains without apparent dependence on distinct saccharide sequence. These findings raise intriguing questions concerning the functional significance of regulation in HS biosynthesis and the potential for development of therapeutics targeting HS-protein interactions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.

Unlabelled: Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients., Importance: Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry., (Copyright © 2015 John Wiley & Sons, Inc.)

Heparan sulfate (HS) proteoglycans are key components of cell microenvironment and fine structure of their polysaccharide HS chains plays an important role in cell-cell interactions, adhesion, migration and signaling. It is formed on non-template basis, so, structure and functional activity of HS biosynthetic machinery is crucial for correct HS biosynthesis and post-synthetic modification. To reveal cancer-related changes in transcriptional pattern of HS biosynthetic system, the expression of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, 3OST1/HS3ST1, SULF1/2, HPSE) in human normal (fibroblasts, PNT2) and cancer (MCF7, LNCaP, PC3, DU145, H157, H647, A549, U2020, U87, HT116, KRC/Y) cell lines and breast, prostate, colon tumors was studied. Real-time RT-PCR and Western-blot analyses revealed specific transcriptional patterns and expression levels of HS biosynthetic system both in different cell lines in vitro and cancers in vivo. Balance between transcriptional activities of elongation- and post-synthetic modification- involved genes was suggested as most informative parameter for HS biosynthetic machinery characterization. Normal human fibroblasts showed elongation-oriented HS biosynthesis, while PNT2 prostate epithelial cells had modification-oriented one. However, cancer epithelial cells demonstrated common tendency to acquire fibroblast-like elongation-oriented mode of HS biosynthetic system. Surprisingly, aggressive metastatic cancer cells (U2020, DU145, KRC/Y) retained modification-oriented HS biosynthesis similar to normal PNT2 cells, possibly enabling the cells to keep like-to-normal cell surface glycosylation pattern to escape antimetastatic control. The obtained results show the cell type-specific changes of HS-biosynthetic machinery in cancer cells in vitro and tissue-specific changes in different cancers in vivo, supporting a close involvement of HS biosynthetic system in carcinogenesis.

Heparan sulfate proteoglycans are important modulators of cellular processes where the negatively charged polysaccharide chains interact with target proteins. The sulfation pattern of the heparan sulfate chains will determine the proteins that will bind and the affinity of the interactions. The N-deacetylase/N-sulfotransferase (NDST) enzymes are of key importance during heparan sulfate biosynthesis when the sulfation pattern is determined. In this chapter, metabolic labeling of heparan sulfate with [(35)S]sulfate or [(3)H]glucosamine in cell cultures is described, in addition to characterization of polysaccharide chain length and degree of N-sulfation. Methods to measure NDST enzyme activity are also presented.

Heparan sulfate (HS) and heparin (Hp) are linear polysaccharide chains composed of repeating (1→4) linked pyrosulfuric acid and 2-amino-2-deoxy glucopyranose (glucosamine) residue. Mentioned glycosaminoglycans chains are covalently O-linked to serine residues within the core proteins creating heparan sulfate/heparin proteoglycans (HSPG). The latter ones participate in many physiological and pathological phenomena impacting both the plethora of ligands such as cytokines, growth factors, and adhesion molecules and the variety of the ECM constituents. Moreover, HS/Hp determine the effective wound healing process. Initial growth of HS and Hp amount is pivotal during the early phase of tissue repair; however heparan sulfate and heparin also participate in further stages of tissue regeneration.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner. Reversible and selective inhibition of GAG biosynthesis would allow us to examine the importance of endogenous GAGs to specific cellular, tissue, or organ systems. In this chapter, we describe the chemical synthesis and biological evaluation of 4-deoxy-4-fluoro-xylosides as selective inhibitors of heparan sulfate and chondroitin/dermatan sulfate proteoglycan biosynthesis.

Heparan sulfate is a polysaccharide that plays essential physiological functions in the animal kingdom. Heparin, a highly sulfated form of heparan sulfate, is a widely prescribed anticoagulant drug worldwide. The heparan sulfate and heparin isolated from natural sources are highly heterogeneous mixtures differing in their polysaccharide chain lengths and sulfation patterns. The access to structurally defined heparan sulfate and heparin is critical to probe the contribution of specific sulfated saccharide structures to the biological functions as well as for the development of the next generation of heparin-based anticoagulant drugs. The synthesis of heparan sulfate and heparin, using a purely chemical approach, has proven extremely difficult, especially for targets larger than octasaccharides having a high degree of site-specific sulfation. A new chemoenzymatic method has emerged as an effective alternative approach. This method uses recombinant heparan sulfate biosynthetic enzymes combined with unnatural uridine diphosphate-monosaccharide donors. Recent examples demonstrate the successful synthesis of ultra-low molecular weight heparin, low-molecular weight heparin and bioengineered heparin with unprecedented efficiency. The new method provides an opportunity to develop improved heparin-based therapeutics.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions., (© 2014 by The American Society of Hematology.)

In this issue of Blood, Saez et al demonstrate that inhibition of heparan sulfate proteoglycan production by bone marrow osteolineage stromal cells results in hematopoietic stem cell egress from the bone marrow niche into the peripheral circulation.

Intrinsic and extrinsic signals as well as the extracellular matrix (ECM) tightly regulate stem cells for tissue homeostasis and regenerative capacity. Little is known about the regulation of tissue homeostasis by the ECM. Heparan sulfate proteoglycans (HSPGs), important components of the ECM, are involved in a variety of biological events. Two heparin sulfate 3-O sulfotransferase (Hs3st) genes, Hs3st-A and Hs3st-B, encode the modification enzymes in heparan sulfate (HS) biosynthesis. Here we demonstrate that Hs3st-A and Hs3st-B are required for adult midgut homeostasis. Depletion of Hs3st-A in enterocytes (ECs) results in increased intestinal stem cell (ISC) proliferation and tissue homeostasis loss. Moreover, increased ISC proliferation is also observed in Hs3st-B null mutant alone, or in combination with Hs3st-A RNAi. Hs3st-A depletion-induced ISC proliferation is effectively suppressed by simultaneous inhibition of the EGFR signaling pathway, suggesting that tissue homeostasis loss in Hs3st-A-deficient intestines is due to increased EGFR signaling. Furthermore, we find that Hs3st-A-depleted ECs are unhealthy and prone to death, while ectopic expression of the antiapoptotic p35 is able to greatly suppress tissue homeostasis loss in these intestines. Together, our data suggest that Drosophila Hs3st-A and Hs3st-B are involved in the regulation of ISC proliferation and midgut homeostasis maintenance., (Copyright © 2014. Published by Elsevier Inc.)

Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Heparan sulfate (HS), a long linear polysaccharide of alternating disaccharide residues, interacts with a wide variety of proteins, including many angiogenic factors. The involvement of HS in signaling of pro-angiogenic factors (e.g. vascular endothelial growth factor and fibroblast growth factor 2), as well as interaction with anti-angiogenic factors (e.g. endostatin), warrants its role as an important modifier of (tumor) angiogenesis. This review summarizes our current understanding of the role of HS in angiogenic growth factor signaling, and discusses therapeutic strategies to target HS and modulate angiogenesis.

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1(-/-) embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect., (Copyright © 2013 International Society of Matrix Biology. All rights reserved.)

The identification and validation of gene-gene interactions is a major challenge in human studies. Here, we explore an approach for studying epistasis in humans using a Drosophila melanogaster model of neonatal diabetes mellitus. Expression of the mutant preproinsulin (hINS(C96Y)) in the eye imaginal disc mimics the human disease: it activates conserved stress-response pathways and leads to cell death (reduction in eye area). Dominant-acting variants in wild-derived inbred lines from the Drosophila Genetics Reference Panel produce a continuous, highly heritable distribution of eye-degeneration phenotypes in a hINS(C96Y) background. A genome-wide association study (GWAS) in 154 sequenced lines identified a sharp peak on chromosome 3L, which mapped to a 400-bp linkage block within an intron of the gene sulfateless (sfl). RNAi knockdown of sfl enhanced the eye-degeneration phenotype in a mutant-hINS-dependent manner. RNAi against two additional genes in the heparan sulfate (HS) biosynthetic pathway (ttv and botv), in which sfl acts, also modified the eye phenotype in a hINS(C96Y)-dependent manner, strongly suggesting a novel link between HS-modified proteins and cellular responses to misfolded proteins. Finally, we evaluated allele-specific expression difference between the two major sfl-intronic haplotypes in heterozygtes. The results showed significant heterogeneity in marker-associated gene expression, thereby leaving the causal mutation(s) and its mechanism unidentified. In conclusion, the ability to create a model of human genetic disease, map a QTL by GWAS to a specific gene, and validate its contribution to disease with available genetic resources and the potential to experimentally link the variant to a molecular mechanism demonstrate the many advantages Drosophila holds in determining the genetic underpinnings of human disease.

The glycosaminoglycan (heparin and heparan sulfate) are polyanionic sulfated polysaccharides mostly recognized for its anticoagulant activity. In many countries, low-molecular-weight heparins have replaced the unfractionated heparin, owing to its high bioavailability, half-life, and less adverse effect. The low-molecular-weight heparins differ in mode of preparation (chemical or enzymatic synthesis and chromatography fractionations) and as a consequence in molecular weight distribution, chemical structure, and pharmacological activities. Bovine and porcine body parts are at present used for manufacturing of commercial heparins, and the appearance of mad cow disease and Creutzfeldt-Jakob disease in humans has limited the use of bovine heparin. Consequently, marine organisms come across the new resource for the production of low-molecular-weight heparin and heparan sulfate. The importance of this chapter suggests that the low-molecular-weight heparin and heparan sulfate from marine species could be alternative sources for commercial heparin., (© 2014 Elsevier Inc. All rights reserved.)

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.

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.

Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analogue of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analogue was activated intracellularly into UDP-4-deoxy-GlcNAc, and HS expression was inhibited up to ∼96% (IC50 = 16 μM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analogue, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors such as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Microinjection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All of these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis, which hampers pro-angiogenic signaling and neo-vessel formation.

Heparan sulfate and heparin are highly sulfated polysaccharides consisting of repeating disaccharide units of glucuronic acid or iduronic acid that is linked to glucosamine. Heparan sulfate displays a range of biological functions, and heparin is a widely used anticoagulant drug in hospitals. It has been known to organic chemists that the chemical synthesis of heparan sulfate and heparin oligosaccharides is extremely difficult. Recent advances in the study of the biosynthesis of heparan sulfate/heparin offer a chemoenzymatic approach to synthesize heparan sulfate and heparin. Compared to chemical synthesis, the chemoenzymatic method shortens the synthesis and improves the product yields significantly, providing an excellent opportunity to advance the understanding of the structure and function relationships of heparan sulfate. In this review, we attempt to summarize the progress of the chemoenzymatic synthetic method and its application in heparan sulfate and heparin research., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by multistep interactions between lymphocytes and high endothelial venules (HEVs). Heparan sulfate (HS) has been implicated in the presentation of chemokines on the surface of HEVs during this process. However, it remains unclear whether this cell surface presentation is a prerequisite for lymphocyte homing. In this study, we generated conditional knockout (cKO) mice lacking Ext1, which encodes a glycosyltransferase essential for HS synthesis, by crossing Ext1(flox/flox) mice with GlcNAc6ST-2-Cre transgenic mice expressing Cre recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated in PLN HEVs but retained in other blood vessels in the cKO mice. The accumulation of a major secondary lymphoid tissue chemokine, CCL21, on HEVs was also abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated that lymphocyte homing to PLNs was diminished in the cKO mice by 30-40%. Consistent with this result, contact hypersensitivity responses were also diminished in the cKO mice. The residual lymphocyte homing to PLNs in the cKO mice was dependent on pertussis toxin-sensitive Gi protein signaling, in which lysophosphatidic acid-mediated signaling was partly involved. These results suggest that chemokine presentation by HS on the surface of HEVs facilitates but is not absolutely required for lymphocyte homing.

Heparanase (Hpse) is an endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Heparan sulfate (HS) belongs to a class of glycosaminoglycans and is a highly sulfated, linear polysaccharide. HS biosynthesis and modification involves numerous enzymes. HS exists as part of glycoproteins named HS proteoglycans, which are expressed abundantly on the cell surface and in the extracellular matrix. HS interacts with numerous proteins, including growth factors, morphogens, and adhesion molecules, and thereby regulates important developmental processes in invertebrates and vertebrates. Embryonic stem cells (ESCs) are distinguished by their characteristics of self-renewal and pluripotency. Self-renewal allows ESCs to proliferate indefinitely in their undifferentiated state, whereas pluripotency implies their capacity to differentiate into the three germ layers and ultimately all cell types of the adult body. Both traits are tightly regulated by numerous cell signaling pathways. Recent studies have highlighted the importance of HS in the modulation of ESC functions, specifically their lineage fate. Here, we review the current advances that have been made in understanding the structural changes of HS during ESC differentiation and in deciphering the molecular mechanisms by which HS modulates cell fate. Finally, we discuss the applications of heparinoids and chemical inhibitors of HS biosynthesis for the manipulation of ESC culture and directed differentiation.