Mice deficient in heparan sulfate 3-O-sulfotransferase-1: normal hemostasis with unexpected perinatal phenotypes

Heparan sulfate that contains antithrombin binding sites is designated as anticoagulant heparan sulfate (HS^act) since, in vitro, it dramatically enhances the neutralization of coagulation proteases by antithrombin. Endothelial cell production of HS^act is controlled by the Hs3st1 gene, which encodes the rate limiting enzyme—heparan sulfate 3-O-sulfotransferase-1 (Hs3st1). It has long been proposed that levels of endothelial HS^act may tightly regulate hemostatic tone. This potential in vivo role of HS^act was assessed by generating Hs3st1 ^–/– knockout mice. Hs3st1 ^–/– and Hs3st1 ^+/+ mice were evaluated with a variety of methods, capable of detecting altered hemostatic tone. However, both genotypes were indistinguishable. Instead, Hs3st1 ^–/– mice exhibited lethality on a specific genetic background and also showed intrauterine growth retardation. Neither phenotypes result from a gross coagulopathy. So although this enzyme produces the majority of tissue HS^act, Hs3st1 ^–/– mice do not show an obvious procoagulant phenotype. These results suggest that the bulk of HS^act is not essential for normal hemostasis and that hemostatic tone is not tightly regulated by total levels of HS^act. Moreover, the unanticipated non-thrombotic phenotypes suggest structure(s) derived from this enzyme might serve additional/alternative biologic roles. Published in 2003.     

Heparan sulfate proteoglycan,integrin, and syndecan-4 are mechanosensors mediating cyclic strain-modulated endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

It is widely believed that the differentiation of embryonic stem cells (ESCs) into viable endothelial cells (ECs) for use in vascular tissue engineering can be enhanced by mechanical forces. In our previous work, we reported that shear stress enhanced important EC functional genes on a CD31⁺/CD45⁻ cell population derived from mouse ESC committed to the EC lineage. In the present study, in contrast to the effects of shear stress on this cell population, we observed that cyclic strain significantly reduced the expression of EC-specific marker genes (vWF, VE-cadherin, and PECAM-1), tight junction protein genes (ZO-1, OCLD, and CLD5), and vasoactive genes (eNOS and ET1), while it did not alter the expression of COX2. Taken together, these studies indicate that only shear stress, not cyclic strain, is a useful mechanical stimulus for enhancing the properties of CD31⁺/CD45⁻ cells for use as EC in vascular tissue engineering. To begin examining the mechanisms controlling cyclic strain-induced suppression of gene expression in CD31⁺/CD45⁻ cells, we depleted the heparan sulfate (HS) component of the glycocalyx, blocked integrins, and silenced the HS proteoglycan syndecan-4 in separate experiments. All of these treatments resulted in the reversal of cyclic strain-induced gene suppression. The current study and our previous work provide a deeper understanding of the mechanisms that balance the influence of cyclic strain and shear stress in endothelial cells.     

High-performance liquid chromatographic identification of eight constitutional disaccharides from heparan sulfate isomers digested with heparitinases

Identification with specific heparan sulfate-lyases, heparitinase I and heparinase of the constitutional unsaturated disaacharide (ΔDi-S_HS) derived from heparan sulfate (HS) isomers and heparin was achieved using high-performance liquid chromatography (HPLC) with a sulfonated styrene-divinylbenzene copolymer. Eight ΔDi-S_HS products derived from HS isomers were identified. Enzymatic digestion with heparitinase I and heparinase converts heterogeneous sulfated HS isomers and heparin into different ΔDi-S_HS. The practical application of these enzymes was examined using specific enzymes and HPLC. In a patient with Hurler syndrome, eight individual Δi-S_HS were identified in urinary HS isomers.     

Heparan Sulfate Is Required for Embryonic Stem Cells to Exit from Self-renewal

Pluripotent embryonic stem cells (ESCs) must select between alternative fates of self-renewal and lineage commitment at each division during continuous proliferation. Heparan sulfate (HS) is a highly sulfated polysaccharide and is present abundantly on the ESC surface. In this study, we investigated the role of HS in ESC self-renewal by examining Ext1^−/− ESCs that are deficient in HS. We found that Ext1^−/− ESCs retained their self-renewal potential but failed to transit from self-renewal to differentiation upon removal of leukemia inhibitory factor. Furthermore, we found that the aberrant cell fate commitment is caused by defects in fibroblast growth factor signaling, which directly retained high expression of the pluripotency gene Nanog in Ext1^−/− ESCs. Therefore, our studies identified and defined HS as a novel factor that controls ESC fate commitment and also delineates that HS facilitates fibroblast growth factor signaling, which, in turn, inhibits Nanog expression and commits ESCs to lineage differentiation.     

The principal neuronal gD-type 3-O-sulfotransferases and their products in central and peripheral nervous system tissues.

Within the nervous system, heparan sulfate (HS) of the cell surface and extracellular matrix influences developmental, physiologic and pathologic processes. HS is a functionally diverse polysaccharide that employs motifs of sulfate groups to selectively bind and modulate various effector proteins. Specific HS activities are modulated by 3-O-sulfated glucosamine residues, which are generated by a family of seven 3-O-sulfotransferases (3-OSTs). Most isoforms we herein designate as gD-type 3-OSTs because they generate HS(gD+), 3-O-sulfated motifs that bind the gD envelope protein of herpes simplex virus 1 (HSV-1) and thereby mediate viral cellular entry. Certain gD-type isoforms are anticipated to modulate neurobiologic events because a Drosophila gD-type 3-OST is essential for a conserved neurogenic signaling pathway regulated by Notch. Information about 3-OST isoforms expressed in the nervous system of mammals is incomplete. Here, we identify the 3-OST isoforms having properties compatible with their participation in neurobiologic events. We show that 3-OST-2 and 3-OST-4 are principal isoforms of brain. We find these are gD-type enzymes, as they produce products similar to a prototypical gD-type isoform, and they can modify HS to generate receptors for HSV-1 entry into cells. Therefore, 3-OST-2 and 3-OST-4 catalyze modifications similar or identical to those made by the Drosophila gD-type 3-OST that has a role in regulating Notch signaling. We also find that 3-OST-2 and 3-OST-4 are the predominant isoforms expressed in neurons of the trigeminal ganglion, and 3-OST-2/4-type 3-O-sulfated residues occur in this ganglion and in select brain regions. Thus, 3-OST-2 and 3-OST-4 are the major neural gD-type 3-OSTs, and so are prime candidates for participating in HS-dependent neurobiologic events.     

Biological Activity of the Isomeric Forms of Helminthosporium sacchari Toxin and of Homologs Produced in Culture

The effect of Helminthosporium sacchari (HS) toxin isomers and related, pathogen-produced compounds on dark CO₂ fixation in HS-susceptible sugar cane leaf slices was investigated. HS toxin consists of a mixture of three isomeric bis-5-O-(β-galactofuranosyl)-β-galactofuranosides (A, B, and C) differing in the position of one double bond in the sesquiterpene aglycone. Maximum inhibition of dark CO₂ fixation in susceptible sugar cane (CP52-68) occurred within 30 to 40 minutes, and amounts necessary to reach 50% inhibition values typically were approximately 1.7 micromolar for natural toxin mixture ( 2:3:5 mixture of isomers A:B:C) and 4, 6, and 0.7 micromolar for isomers A, B, and C, respectively. Other fractions from cultures of the pathogen consist of comparable mixtures of sesquiterpene isomers but have only 1, 2, or 3 galactofuranose units (HS₁, HS₂, HS₃) or two α-glucopyranose units as well as four β-galactofuranose units (HS₆). The lower toxin homologs were not toxic to clone CP52-68, but protected sugar cane from the effects of toxin. Minimum ratios of protectant: toxin giving 95% protection were approximately 50:1, 6:1, and 12:1 for HS₁, HS₂, and HS₃, respectively. HS₂ and HS₃ protected when added up to 12 minutes after toxin as well as when added with or before toxin. Some common plant galactopyranosides were not toxic and did not protect at 500:1 molar excess. The sample of HS₆ was toxic at 500 micromolar, and did not protect against HS toxin. With the availability of purified, homogeneous preparations of HS toxin, homologs, and chemically modified or synthetic analogs, the dark CO₂ fixation assay should prove to be a useful tool for understanding the mode of action of HS toxin.     

Integration of molecular and physiological models to explain time of anthesis in wheat

Background and Aims

A model to predict anthesis time of a wheat plant from environmental and genetic information requires integration of current concepts in physiological and molecular biology. This paper describes the structure of an integrated model and quantifies its response mechanisms.

Methods

Literature was reviewed to formulate the components of the model. Detailed re-analysis of physiological observations are utilized from a previous publication by the second two authors. In this approach measurements of leaf number and leaf and primordia appearance of near isogenic lines of spring and winter wheat grown for different durations in different temperature and photoperiod conditions are used to quantify mechanisms and parameters to predict time of anthesis.

Key Results

The model predicts the time of anthesis from the length of sequential phases: 1, embryo development; 2, dormant; 3, imbibed/emerging; 4, vegetative; 5, early reproductive; 6, pseudo-stem extension; and 7, ear development. Phase 4 ends with vernalization saturation (VS), Phase 5 with terminal spikelet (TS) and Phase 6 with flag leaf ligule appearance (FL). The durations of Phases 4 and 5 are linked to the expression of Vrn genes and are calculated in relation to change in Haun stage (HS) to account for the effects of temperature per se. Vrn1 must be expressed to sufficient levels for VS to occur. Vrn1 expression occurs at a base rate of 0·08/HS in winter ‘Batten’ and 0·17/HS in spring ‘Batten’ during Phases 1, 3 and 4. Low temperatures promote expression of Vrn1 and accelerate progress toward VS. Our hypothesis is that a repressor, Vrn4, must first be downregulated for this to occur. Rates of Vrn4 downregulation and Vrn1 upregulation have the same exponential response to temperature, but Vrn4 is quickly upregulated again at high temperatures, meaning short exposure to low temperature has no impact on the time of VS. VS occurs when Vrn1 reaches a relative expression of 0·76 and Vrn3 expression begins. However, Vrn2 represses Vrn3 expression so Vrn1 must be further upregulated to repress Vrn2 and enable Vrn3 expression. As a result, the target for Vrn1 to trigger VS was 0·76 in 8-h photoperiods (Pp) and increased at 0·026/HS under 16-h Pp as levels of Vrn2 increased. This provides a mechanism to model short-day vernalization. Vrn3 is expressed in Phase 5 (following VS), and apparent rates of Vrn3 expression increased from 0·15/HS at 8-h Pp to 0·33/HS at 16-h Pp. The final number of leaves is calculated as a function of the HS at which TS occurred (TS^HS): 2·86 + 1·1 × TS^HS. The duration of Phase 6 is then dependent on the number of leaves left to emerge and how quickly they emerge.

Conclusions

The analysis integrates molecular biology and crop physiology concepts into a model framework that links different developmental genes to quantitative predictions of wheat anthesis time in different field situations.     

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4⁺ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.     

Changes in composition and sulfation patterns of glycoaminoglycans in renal cell carcinoma

Glycosaminoglycans (GAGs) are heterogeneous, linear, highly charged, anionic polysaccharides consisting of repeating disaccharides units. GAGs have some biological significance in cancer progression (invasion and metastasis) and cell signaling. In different cancer types, GAGs undergo specific structural changes. In the present study, in depth investigation of changes in sulfation pattern and composition of GAGs, heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate and hyaluronan (HA) in normal renal tissue (NRT) and renal cell carcinoma tissue (RCCT) were evaluated. The statistical evaluation showed that alteration of the HS (HS_NRT = 415.1 ± 115.3; HS_RCCT = 277.5 ± 134.3), and CS (CS_NRT = 35.3 ± 12.3; CS_RCCT = 166.7 ± 108.8) amounts (in ng/mg dry tissue) were statistically significant (p < 0.05). Sulfation pattern in NRT and RCCT was evaluated to reveal disaccharide profiles. Statistical analyses showed that RCCT samples contain significantly increased amounts (in units of ng/mg dry tissue) of 4S_CS (NRT = 25.7 ± 9.4; RCCT = 117.1 ± 73.9), SE_CS (NRT = 0.7 ± 0.3; RCCT = 4.7 ± 4.5), 6S_CS (NRT = 6.1 ± 2.7; RCCT = 39.4 ± 34.7) and significantly decreased amounts (in units of ng/mg dry tissue) of NS6S_HS (RCCT = 28.6 ± 6.5, RCCT = 10.2 ± 8.0), NS2S_HS (RCCT = 44.2 ± 13.8; RCCT = 27.2 ± 15.0), NS_HS (NRT = 68.4 ± 15.8; RCCT = 50.4 ± 21.2), 2S6S_HS (NRT = 1.0 ± 0.4; RCCT = 0.4 ± 0.3), and 6S_HS (NRT = 60.6 ± 17.5; RCCT = 24.9 ± 12.3). If these changes in GAGs are proven to be specific and sensitive, they may serve as potential biomarkers in RCC. Our findings are likely to help us to show the direction for further investigations to be able to bring different diagnostic and prognostic approaches in renal tumors.     

Endothelial Heparan Sulfate 6-O-Sulfation Levels Regulate Angiogenic Responses of Endothelial Cells to Fibroblast Growth Factor 2 and Vascular Endothelial Growth Factor

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF₁₆₅) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF₁₆₅/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1–40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF₁₆₅-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF₁₆₅-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents.     

Heparan sulfate S-domains and extracellular sulfatases (Sulfs): their possible roles in protein aggregation diseases

Highly sulfated domains of heparan sulfate (HS), also known as HS S-domains, consist of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)–]. The expression of HS S-domains at the cell surface is determined by two mechanisms: tightly regulated biosynthetic machinery and enzymatic remodeling by extracellular endoglucosamine 6-sulfatases, Sulf-1 and Sulf-2. Intracellular or extracellular deposits of misfolded and aggregated proteins are characteristic of protein aggregation diseases. Although proteins can aggregate alone, deposits of protein aggregates in vivo contain a number of proteinaceous and non-protein components. HS S-domains are one non-protein component of these aggregated deposits. HS S-domains are considered to be critical for signal transduction of several growth factors and several disease conditions, such as tumor progression, but their roles in protein aggregation diseases are not yet fully understood. This review summarizes the current understanding of the possible roles of HS S-domains and Sulfs in the formation and cytotoxicity of protein aggregates.     

Involvement of heparan sulfate in the renoprotective effects of imidapril,an angiotensin-converting enzyme inhibitor,in diabetic db/db mice

Abstract

We investigated the renoprotective effects of imidapril hydrochloride ((-)-(4?S)-3-[(2?S)-2-[[(1?S)-1-ethoxycarbonyl-3-phenylpropyl] amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril), an angiotensin-converting enzyme inhibitor, in a diabetic animal model. We used BKS.Cg-+Lepr^db/+Lepr^db (db/db) mice, a genetic animal model of obese type 2 diabetes. Diabetic db/db mice suffered from glomerular hyperfiltration, albuminuria and hypoalbuminemia. Oral administration of 5?mg/kg/day of imidapril for 3 weeks suppressed renal hyperfiltration, reduced albuminuria and normalized hypoalbuminemia. Imidapril did not influence body weights, blood pressure or blood glucose concentrations in db/db mice. Urinary excretion of heparan sulfate (HS) in non-treated 11-week-old db/db mice was significantly lower than that in age-matched non-diabetic db/+m mice. HS is a component of HS proteoglycans, which are present in glomerular basement membranes and glycocalyx of cell surfaces. Reduced urinary HS excretion indicated glomerular HS loss in db/db mice. Imidapril increased urinary excretion of HS to concentrations observed in db/+m mice, indicating that imidapril prevented the loss of renal HS. These results suggest that imidapril ameliorates renal hyperfiltration and loss of renal contents of HS. Improvement of filtration function and maintenance of HS, which is an important structural component of glomeruli, may contribute to renoprotective effects of imidapril.     

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.     

Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 is generated in endosomes by an endogenously or ascorbate-induced S-nitrosothiol-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid β (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We showed by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as ³⁵S labeling experiments that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (WT MEFs), Tg2576 MEFs, and N2a neuroblastoma cells but not in APP^−/− and APLP2^−/− MEFs. Transfection of APP^−/− cells with a vector encoding APP restored nuclear import of anMan-containing HS. In WT MEFs and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEFs, the β-inhibitor blocked transport, but the γ-inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of WT MEFs. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine, indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.     

Blockage of undesirable endocytosis of recombinant human growth/differentiation factor-5 in Chinese hamster ovary cell cultures requires heparin analogs with specific chain lengths

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis lowers the yield of recombinant human bone morphogenetic proteins (rhBMPs), such as rhBMP-2 and rhBMP-4, from Chinese hamster ovary (CHO) cell cultures. Exogenous recombinant human growth/differentiation factor-5 (rhGDF-5), a member of the BMP family, bound to cell surface HSPGs and was actively internalized into CHO cells. Knockdown of heparan sulfate (HS) synthesis enzymes in CHO cells revealed that the chain length and N-sulfation of HS affected the binding of rhGDF-5 to HSPGs and subsequent rhGDF-5 internalization. To increase product yield by minimizing rhGDF-5 internalization in recombinant CHO (rCHO) cell cultures, heparin, and dextran sulfate (DS) of various polysaccharide chain lengths, which are structural analogs of HS, were examined for blockage of rhGDF-5 internalization. Heparin fragments of four monosaccharides (MW of 1.2 kDa) and DS (MW of 15 kDa) did not inhibit rhGDF-5 internalization whereas unfractionated heparin and DS of 200 kDa could significantly inhibit it. Compared to the control cultures, supplementation with unfractionated heparin or DS of 200 kDa at 1 g L^-1 resulted in more than a 10-fold increase in the maximum rhGDF-5 concentration. Taken together, the supplementation of structural HS analogs improved rhGDF-5 production in rCHO cell cultures by inhibiting rhGDF-5 internalization.     

Lack ofl-Iduronic Acid in Heparan Sulfate Affects Interaction with Growth Factors and Cell Signaling

HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi^−/− mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi^−/− MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi^−/− cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi^−/− compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi^−/− but not to WT HS. glia-derived neurotropic factor has a key role in kidney development, defective in Hsepi^−/− mice. By contrast, Hsepi^−/− and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.Signaling activities of numerous growth factors and morphogens during development involve cell surface receptor systems consisting of a tyrosine kinase-type receptor along with a heparan sulfate proteoglycan (HSPG)2 co-receptor (1, 2). The complex and heterogeneous HSPG macromolecules occur in the extracellular matrix and on the surfaces of virtually all animal cells (3). HS side chains of HSPGs show great structural variability; hence, they interact with a multitude of proteins and influence a variety of biological processes, including growth factor signaling (4).The structural diversity of HS is best envisaged through an account of its biosynthesis (Fig. 1). The process is initiated by glycosylation reactions that generate saccharide sequences composed of alternating GlcA and GlcNAc units covalently bound to a core protein (through a specific “linker” tetrasaccharide sequence). The resulting polymer of (GlcAβ1,4-GlcNAcα1,4-)_n disaccharide repeats is modified through a series of reactions, including N-deacetylation/N-sulfation of GlcNAc residues, C5-epimerization of GlcA to l-iduronic acid (IdoA) units, and O-sulfation at various positions of the hexuronic acid and glucosamine residues (4, 5). Modulation of these modification reactions, through as yet poorly understood mechanisms, yields HS chains of strictly regulated saccharide composition, varying with tissue source and age (6, 7). Interactions between protein ligands and HS involve selective ionic binding of peptide sequences containing basic amino acid residues to saccharide domains with clustered sulfate groups and IdoA residues. The latter components are considered to promote ligand apposition through their conformational flexibility (8).Open in a separate windowFIGURE 1.Schematic display of HS biosynthesis. A precursor structure composed of alternating GlcA and GlcNAc residues linked to a core protein is modified through the series of enzymatic reactions indicated and outlined under“Results.” The modifications yield domains of consecutive N-acetylated or N-sulfated disaccharide units, along with mixed sequences. Wild type HS contains both GlcA and IdoA residues, whereas Hsepi^−/− HS lacks IdoA units but shows increased N- and 6-O-sulfation. In particular, the mutant HS features extended sequences of -GlcA-GlcNS6S- disaccharide units.Targeted disruption of genes encoding HS biosynthesis enzymes demonstrated critical roles for HSPGs in developmental processes (9–11). The observed phenotypes vary dramatically in severity, from gastrulation failure to subtle disturbance of organ development. This variability may reflect redundancy due to the occurrence of isoforms for some of the enzymes. Alternatively, developmental events might be critically dependant on HS involvement but not on the fine structure of the polysaccharide chain, such that even structurally deranged HS would fulfill a functional role (12). The glucuronyl C5-epimerase (HSEPI) that catalyzes the conversion of GlcA to IdoA in HS biosynthesis is encoded by a single gene (13). Targeted disruption of this gene in mice resulted in an abnormal HS structure, completely lacking IdoA residues and with severely distorted sulfation pattern. The Hsepi-deficient mice die shortly after birth with multiple developmental defects, such as skeletal malformations and kidney agenesis. Intriguingly, however, other major organ systems, such as the brain and vascular system, known to depend on HS-supported signaling processes, developed seemingly normally (14).The present study was undertaken to obtain insight into the mechanisms behind the developmental defects in the Hsepi mutant mice, using a model system based on murine embryonic fibroblast (MEF) cell lines. The mutant MEF cells responded poorly to FGF2 in proliferation and migration experiments and showed defective FGF2-dependent intracellular signaling. Moreover, binding studies revealed an aberrant mode of interaction between mutant HS chains and certain growth factors, of potential relevance to growth factor signaling.     

A 4-deoxy analogue of N-acetyl-d-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro

Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -β1-4GlcA-α1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-d-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect. 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.     

Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.