Transcriptomic analysis reveals cell apoptotic signature modified by heparanase in melanoma cells.

Heparanase has been implicated in many pathological conditions, especially inflammation and cancer, attributed to its degradation of heparan sulfate, a crucial component maintaining the integrity of the extracellular matrix. By silencing the heparanase gene (HPSE) in MDA-MB-435s melanoma cells, we investigated the impact of this protein on gene transcription. Transcriptome sequencing yielded a list of 279 differentially expressed genes, of which 140 were up-regulated and 239 down-regulated. The 140 up-regulated genes were classified into a substantial set of gene ontology defined functions, for example, positive regulation of cell death, apoptotic process, response to cytokine, while 239 down-regulated genes classify only into the two categories: nucleosome and nucleosome assembly. Our focus was drawn to an array of 28 pro-apoptotic genes regulated by heparanase: real-time PCR experiments further validated up-regulation of EGR1, TXNIP, AXL, CYR61, LIMS2 and TNFRSF12A by at least 1.5-fold, among which EGR1, CYR61, and TNFRSF12A were confirmed on protein level. We demonstrated significantly increased apoptotic cells by TUNEL staining upon HPSE silencing, mediated by activation of caspase 3/PARP1 pathway. The pro-apoptotic gene expression and observation of apoptosis were extended to another melanoma cell line, MV3 cells, thus consolidating the anti-apoptosis effect of heparanase in melanoma cells.

Melanoma Cell Adhesion and Migration Is Modulated by the Uronyl 2-O Sulfotransferase.

Although the vast majority of melanomas are characterized by a high metastatic potential, if detected early, melanoma can have a good prognostic outcome. However, once metastasised, the prognosis is bleak. We showed previously that uronyl-2-O sulfotransferase (Ust) and 2-O sulfation of chondroitin/dermatan sulfate (CS/DS) are involved in cell migration. To demonstrate an impact of 2-O sulfation in metastasis we knocked-down Ust in mouse melanoma cells. This significantly reduced the amount of Ust protein and enzyme activity. Furthermore, in vitro cell motility and adhesion were significantly reduced correlating with the decrease of cellular Ust protein. Single cell migration of B16VshUst(16) cells showed a decreased cell movement phenotype. The adhesion of B16V cells to fibronectin depended on α5ß1 but not αvß3 integrin. Inhibition of glycosaminoglycan sulfation or blocking fibroblast growth factor receptor (FgfR) reduced α5 integrin in B16V cell lines. Interestingly, FgfR1 expression and activation was reduced in Ust knock-down cells. In vivo, pulmonary metastasis of B16VshUst cells was prevented due to a reduction of α5 integrin. As a proof of concept UST knock-down in human melanoma cells also showed a reduction in ITGa5 and adhesion. This is the first study showing that Ust, and consequently 2-O sulfation of the low affinity receptor for FgfR CS/DS, reduces Itga5 and leads to an impaired adhesion and migration of melanoma cells.

A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis.

Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.

Targeting Serglycin Prevents Metastasis in Murine Mammary Carcinoma.

In hematopoietic cells, serglycin proteoglycans mainly contribute to proper storage and secretion of inflammatory mediators via their negatively charged glycosaminoglycans. Serglycin proteoglycans are also expressed in cancer cells where increased expression has been linked to poor prognosis. However, the serglycin-dependent mediators promoting cancer progression remain to be determined. In the present study we report that genetic ablation of serglycin proteoglycan completely blocks lung metastasis in the MMTV-PyMT-driven mouse breast cancer model, while serglycin-deficiency did not affect primary tumour growth or number of mammary tumours. Although E-cadherin expression was higher in the serglycin-deficient primary tumour tissue, indicating reduced invasiveness, serglycin-deficient tumour cells were still detected in the circulation. These data suggest that serglycin proteoglycans play a role in extravasation as well as colonization and growth of metastatic cells. A microarray expression analysis and functional annotation of differentially expressed genes identified several biological pathways where serglycin may be important. Our results suggest that serglycin and serglycin-dependent mediators are potential drug targets to prevent metastatic disease/dissemination of cancer.

Chondroitinase AC: A host-associated genetic feature of Helicobacter bizzozeronii.

Investigating mechanisms involved in host adaptation is crucial to understand pathogen evolution. Helicobacter species appear to have a host species-specific tropism, coevolving with their natural hosts, and to develop several strategies allowing the colonization of the stomach throughout lifetime of their hosts. However, little is known about genetic features associated with the adaptation to a specific animal host. In this study we discovered a polysaccharide lyase that is expressed by the canine-associated species H. bizzozeronii and acts as chondroitinase AC-type lyase of broad specificity. Except for its low pH-optimum between pH 4.0 and pH 5.5, the properties of the H. bizzozeronii chondroitin lyase AC resemble the ones from Arthrobacter aurescens. However, homologues of this gene have been detected only in Helicobacter species colonizing the canine and feline gastric mucosa. Since a unique feature of the canine stomach is the secretion of chondroitin-4-sulphate in the gastric juice of the fundus mucosa by chief cells, the expression of chondroitinase AC by H. bizzozeronii is likely the consequence of adaptation of this bacterium to its host and a potential link to gastric disorders in dogs.

Uronyl 2-O sulfotransferase potentiates Fgf2-induced cell migration.

Fibroblast growth factor 2 (Fgf2) is involved in several biological functions. Fgf2 requires glycosaminoglycans, like chondroitin and dermatan sulfates (hereafter denoted CS/DS) as co-receptors. CS/DS are linear polysaccharides composed of repeating disaccharide units [-4GlcUAb1-3-GalNAc-b1-] and [-4IdoUAa1-3-GalNAc-b1-],which can be sulfated. Uronyl 2-O-sulfotransferase (Ust)introduces sulfation at the C2 of IdoUA and GlcUA resulting inover-sulfated units. Here, we investigated the role of Ust-mediated CS/DS 2-O sulfation in Fgf2-induced cell migration. We found that CHO-K1 cells overexpressing Ust contain significantly more CS/DS2-O sulfated units, whereas Ust knockdown abolished CS/DS 2-O sulfation. These structural differences in CS/DS resulted in altered Fgf2 binding and increased phosphorylation of ERK1/2 (also known as MAPK3 and MAPK1, respectively). As a functional consequence of CS/DS 2-O sulfation and altered Fgf2 binding, cell migration and paxillin activation were increased. Inhibition of sulfation, knockdown of Ust and inhibition of FgfR resulted in reduced migration. Similarly, in 3T3 cells Fgf2 treatment increased migration, which was abolished by Ust knockdown. The proteoglycan controlling the CHO migration was syndecan 1. Knockdown of Sdc1 in CHO-K1 cells overexpressing Ust abolished cell migration.We conclude that the presence of distinctly sulfated CS/DS can tune the Fgf2 effect on cell migration.

Characterization of glycosaminoglycan (GAG) sulfatases from the human gut symbiont Bacteroides thetaiotaomicron reveals the first GAG-specific bacterial endosulfatase.

Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973-25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.

An automated mass spectrometry-based screening method for analysis of sulfated glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear polysaccharides, consisting of repeated disaccharide units, attached to core proteins in all multicellular organisms. Chondroitin sulfate (CS) and dermatan sulfate (DS) constitute a subgroup of sulfated GAGs for which the degree of sulfation varies between species and tissues. One major goal in GAG characterization is to correlate structure to function. A common approach is to exhaustively degrade the GAG chains and thereafter determine the amount of component disaccharide units. In large-scale studies, there is a need for high-throughput screening methods since existing methods are either very time- or samples consuming. Here, we present a new strategy applying MALDI-TOF MS in positive ion mode for semi-qualitative and quantitative analysis of CS/DS derived disaccharide units. Only a few picomoles of sample are required per analysis and 10 samples can be analyzed in 25 min, which makes this approach an attractive alternative to many established assay methods. The total CS/DS concentration in 19 samples derived from Caenorhabditis elegans and mammalian tissues and cells was determined. The obtained results were well in accordance with concentrations determined by a standard liquid chromatography-based method, demonstrating the applicability of the method for samples from various biological matrices containing CS/DS of different sulfation degrees.

HS3ST2 modulates breast cancer cell invasiveness via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and cadherin expression.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.

A visualizable chain-terminating inhibitor of glycosaminoglycan biosynthesis in developing zebrafish.

Heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAG) are proteoglycan-associated polysaccharides with essential functions in animals. They have been studied extensively by genetic manipulation of biosynthetic enzymes, but chemical tools for probing GAG function are limited. HS and CS possess a conserved xylose residue that links the polysaccharide chain to a protein backbone. Here we report that, in zebrafish embryos, the peptide-proximal xylose residue can be metabolically replaced with a chain-terminating 4-azido-4-deoxyxylose (4-XylAz) residue by administration of UDP-4-azido-4-deoxyxylose (UDP-4-XylAz). UDP-4-XylAz disrupted both HS and CS biosynthesis and caused developmental abnormalities reminiscent of GAG biosynthesis and laminin mutants. The azide substituent of protein-bound 4-XylAz allowed for rapid visualization of the organismal sites of chain termination in vivo through bioorthogonal reaction with fluorescent cyclooctyne probes. UDP-4-XylAz therefore complements genetic tools for studies of GAG function in zebrafish embryogenesis.

Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling.

Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

MicroRNA-24 suppression of N-deacetylase/N-sulfotransferase-1 (NDST1) reduces endothelial cell responsiveness to vascular endothelial growth factor A (VEGFA).

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.

On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis.

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

The mutual impact of syndecan-1 and its glycosaminoglycan chains--a multivariable puzzle.

Proteoglycans, with their core proteins and attached glycosaminoglycan chains, are recognized as important partners in many biological processes, yet often experimental analysis of their molecular action is considered for only part of these molecules: either the protein or the carbohydrate unit. In this article, we have tried to summarize, with an example of the syndecan family in general and more specifically with syndecan-1, what is known considering the mutual influence of these different components, and we follow whether the nature of the glycosaminoglycan chains matters for these effects.

Functional overlap between chondroitin and heparan sulfate proteoglycans during VEGF-induced sprouting angiogenesis.

OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis. METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, transforming growth factor ß, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of transforming growth factor ß and platelet-derived growth factor B signal transduction. CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

Heparan sulfate proteoglycans as multifunctional cell regulators: cell surface receptors.

Proteoglycans are macromolecules expressed on the cell surfaces and in the extracellular matrix of most animal tissues (Annu Rev Biochem 68:729-777, 1999; Int Rev Cell Mol Biol 276:105-159, 2009). Heparan sulfate proteoglycans (HSPGs) are essential for animal development and homeostasis, and are involved in various pathological processes. The functions of HSPGs are largely exerted through interaction of the heparan sulfate (HS) side chains with different types of ligands, including diverse molecules such as cytokines, enzymes, and pathogens. One of the important roles of cell surface HSPGs is to mediate cytokine-induced cell signaling through interaction with growth factors (GFs) and their cognate receptors. A selective dependence of GFs for different structural features of HS has been demonstrated by applying cell models that are mutated variously in HS structure due to deficiency in enzymes involved in the biosynthesis of HS chains.

Structural basis of oligosaccharide receptor recognition by human papillomavirus.

High risk human papillomavirus types 16 (HPV16) and 18 (HPV18) can cause cervical cancer. Efficient infection by HPV16 and HPV18 pseudovirions requires interactions of particles with cell-surface receptor heparan sulfate oligosaccharide. To understand the virus-receptor interactions for HPV infection, we determined the crystal structures of HPV16 and HPV18 capsids bound to the oligosaccharide receptor fragment using oligomeric heparin. The HPV-heparin structures revealed multiple binding sites for the highly negatively charged oligosaccharide fragment on the capsid surface, which is different from previously reported virus-receptor interactions in which a single type of binding pocket is present for a particular receptor. We performed structure-guided mutagenesis to generate mutant viruses, and cell binding and infectivity assays demonstrated the functional role of viral residues involved in heparin binding. These results provide a basis for understanding virus-heparan sulfate receptor interactions critical for HPV infection and for the potential development of inhibitors against HPV infection.

Serglycin-independent release of active mast cell proteases in response to Toxoplasma gondii infection.

Earlier studies identified serglycin proteoglycan and its heparin chains to be important for storage and activity of mast cell proteases. However, the importance of serglycin for secretion and activity of mast cell proteases in response to parasite infection has been poorly investigated. To address this issue, we studied the effects on mast cell proteases in serglycin-deficient and wild type mice after peritoneal infection with the obligate intracellular parasite Toxoplasma gondii. In line with previous results, we found severely reduced levels of cell-bound mast cell proteases in both noninfected and infected serglycin-deficient mice. However, serglycin-deficient mice secreted mast cell proteases at wild type levels at the site of infection, and enzymatic activities associated with mast cell proteases were equally up-regulated in wild type and serglycin-deficient mice 48 h after infection. In both wild type and serglycin-deficient mice, parasite infection resulted in highly increased extracellular levels of glycosaminoglycans, including hyaluronan and chondroitin sulfate A, suggesting a role of these substances in the general defense mechanism. In contrast, heparan sulfate/heparin was almost undetectable in serglycin-deficient mice, and in wild type mice, it was mainly confined to the cellular fraction and was not increased upon infection. Furthermore, the heparan sulfate/heparin population was less sulfated in serglycin-deficient than in wild type mice indicative for the absence of heparin, which supports that heparin production is dependent on the serglycin core protein. Together, our results suggest that serglycin proteoglycan is dispensable for normal secretion and activity of mast cell proteases in response to peritoneal infection with T. gondii.

The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

Heparan sulfate domain organization and sulfation modulate FGF-induced cell signaling.

Heparan sulfates (HSs) modulate various developmental and homeostatic processes by binding to protein ligands. We have evaluated the structural characteristics of porcine HS in cellular signaling induced by basic fibroblast growth factor (FGF2), using CHO745 cells devoid of endogenous glycosaminoglycans as target. Markedly enhanced stimulation of cell signaling, measured as phosphorylation of ERK1/2 and protein kinase B, was only observed with the shortest HS chains isolated from liver, whereas the longer chains from either liver or intestine essentially prolonged duration of signals induced by FGF2 in the absence of polysaccharide. Structural analysis showed that contiguous sulfated domains were most abundant in the shortest HS chains and were more heavily sulfated in HS from liver than in HS from intestine. Moreover, the shortest chains from either source entered into ternary complexes with FGF2 and FGF receptor-1c more efficiently than the corresponding longer chains. In addition to authentic HSs, decasaccharide libraries generated by chemo-enzymatic modification of heparin were probed for effect on FGF2 signaling. Only the most highly sulfated decamers, previously found most efficient in ternary complex formation (Jastrebova, N., Vanwildemeersch, M., Rapraeger, A. C., Giménez-Gallego, G., Lindahl, U., and Spillmann, D. (2006) J. Biol. Chem. 281, 26884-26892), promoted FGF2 cellular signaling as efficiently as short HS chains from liver. Together these results suggest that the effects of HS on FGF2 signaling are determined by both the structure of the highly sulfated domains and by the organization/availability of such domains within the HS chain. These findings underpin the need for regulation of HS biosynthesis in relation to control of growth factor-induced signaling pathways.