Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

A Role for 3-O-Sulfated Heparan Sulfate in Promoting Human Cytomegalovirus Infection in Human Iris Cells

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3-O-sulfotransferase-3 (3-OST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.     

The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.     

A role for 3-O-sulfotransferase isoform-4 in assisting HSV-1 entry and spread

Many heparan sulfate (HS) 3-O-sulfotransferase (3-OST) isoforms generate cellular receptors for herpes simplex virus type-1 (HSV-1) glycoprotein D (gD). Interestingly, the ability of 3-OST-4 to mediate HSV-1 entry and cell-to-cell fusion has not been determined, although it is predominantly expressed in the brain, a primary target of HSV-1 infections. We report that expression of 3-OST-4 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and a mutant (Rid1) strain of HSV-1. Evidence for generation of gD receptors by 3-OST-4 was suggested by gD-mediated interference assay and the ability of 3-OST-4 expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases-II/III). In addition, 3-OST-4 expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together our results suggest a role of 3-OST-4 in HSV-1 pathogenesis.     

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.     

Role of 6-O-Sulfated Heparan Sulfate in Chronic Renal Fibrosis

Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-β induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.     

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane

Background

Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations.

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.     

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

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.     

Essential role of M1 macrophages in blocking cytokine storm and pathology associated with murine HSV-1 infection

Ocular HSV-1 infection is a major cause of eye disease and innate and adaptive immunity both play a role in protection and pathology associated with ocular infection. Previously we have shown that M1-type macrophages are the major and earliest infiltrates into the cornea of infected mice. We also showed that HSV-1 infectivity in the presence and absence of M2-macrophages was similar to wild-type (WT) control mice. However, it is not clear whether the absence of M1 macrophages plays a role in protection and disease in HSV-1 infected mice. To explore the role of M1 macrophages in HSV-1 infection, we used mice lacking M1 activation (M1^-/- mice). Our results showed that macrophages from M1^-/- mice were more susceptible to HSV-1 infection in vitro than were macrophages from WT mice. M1^-/- mice were highly susceptible to ocular infection with virulent HSV-1 strain McKrae, while WT mice were refractory to infection. In addition, M1^-/- mice had higher virus titers in the eyes than did WT mice. Adoptive transfer of M1 macrophages from WT mice to M1^-/- mice reduced death and rescued virus replication in the eyes of infected mice. Infection of M1^-/- mice with avirulent HSV-1 strain KOS also increased ocular virus replication and eye disease but did not affect latency-reactivation seen in WT control mice. Severity of virus replication and eye disease correlated with significantly higher inflammatory responses leading to a cytokine storm in the eyes of M1^-/- infected mice that was not seen in WT mice. Thus, for the first time, our study illustrates the importance of M1 macrophages specifically in primary HSV-1 infection, eye disease, and survival but not in latency-reactivation.     

Quantitative and Qualitative Alterations of Heparan Sulfate in Fibrogenic Liver Diseases and Hepatocellular Cancer

Heparan sulfate (HS), due to its ability to interact with a multitude of HS-binding factors, is involved in a variety of physiological and pathological processes. Remarkably diverse fine structure of HS, shaped by non-exhaustive enzymatic modifications, influences the interaction of HS with its partners. Here we characterized the HS profile of normal human and rat liver, as well as alterations of HS related to liver fibrogenesis and carcinogenesis, by using sulfation-specific antibodies. The HS immunopattern was compared with the immunolocalization of selected HS proteoglycans. HS samples from normal liver and hepatocellular carcinoma (HCC) were subjected to disaccharide analysis. Expression changes of nine HS-modifying enzymes in human fibrogenic diseases and HCC were measured by quantitative RT-PCR. Increased abundance and altered immunolocalization of HS was paralleled by elevated mRNA levels of HS-modifying enzymes in the diseased liver. The strong immunoreactivity of the normal liver for 3-O-sulfated epitope further increased with disease, along with upregulation of 3-OST-1. Modest 6-O-undersulfation of HCC HS is probably explained by Sulf overexpression. Our results may prompt further investigation of the role of highly 3-O-sulfated and partially 6-O-desulfated HS in pathological processes such as hepatitis virus entry and aberrant growth factor signaling in fibrogenic liver diseases and HCC. (J Histochem Cytochem 58:429–441, 2010)     

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

Epigenetic Inactivation of Heparan Sulfate (Glucosamine) 3-O-Sulfotransferase 2 in Lung Cancer and Its Role in Tumorigenesis

Background

This study was aimed at investigating the functional significance of heparan sulfate (glucosamine) 3-O-sulfotransferase 2 (HS3ST2) hypermethylation in non-small cell lung cancer (NSCLC).

Methodology/ Principal Findings

HS3ST2 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting) and EpiTYPER^TM assays showed substantial hypermethylation of CpG island at the promoter region of HS3ST2 in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2′-deoxycytidine (5-Aza-dC). A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation in vitro. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, P = 0.009; Spearman’s rank correlation). HS3ST2 hypermethylation was found in 95 (32%) of 298 primary NSCLCs. Patients with HS3ST2 hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25–3.58, P = 0.005) compared to those without HS3ST2 hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation.

Conclusions/ Significance

The present study suggests that HS3ST2 hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.     

HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ ^high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ ^high CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ ^highCD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.     

Glycanogenomics: a qPCR-approach to investigate biological glycan function

As an indirect approach towards glycan structures, qRT-PCR analyses using the ΔΔC_T method were performed to investigate changes in expression levels of heparan sulfate-synthesising enzymes of stimulated and unstimulated HMVECs. We chose NDSTs as early enzymes initiating sulfation and 3OSTs which act late generating specific binding sites. Major changes in expression patterns were found for the NDST3 and 3OST1 isoforms. Both enzymes were down-regulated 7- and 6-fold, respectively, following TNF-α stimulation, and 3.5- and 7.6-fold following LPS-stimulation suggesting a common restructuring process of HS in inflammation leading to a less diverse sulfation pattern. Immunostaining of TNF-α-stimulated cells using a phage display-derived antibody specific for 3-O-sulfation and unsulfated regions of HS resulted in significant fluorescence changes between unstimulated and stimulated.     

Chondroitin 4-O-sulfotransferase-1 regulates E disaccharide expression of chondroitin sulfate required for herpes simplex virus infectivity

We have demonstrated a defect in expression of chondroitin 4-O-sulfotransferase-1 (C4ST-1) in murine sog9 cells, which are poorly sensitive to infection by herpes simplex virus type 1 (HSV-1). Sog9 cells were previously isolated as CS-deficient cells from gro2C cells, which were partially resistant to HSV-1 infection and defective in the expression of heparan sulfate (HS) because of a splice site mutation in the EXT1 gene encoding the HS-synthesizing enzyme. Here we detected a small amount of CS chains in sog9 cells with a drastic decrease in 4-O-sulfation compared with the parental gro2C cells. RT-PCR revealed that sog9 cells had a defect in the expression of C4ST-1 in addition to EXT1. Gel filtration analysis showed that the decrease in the amount of CS in sog9 cells was the result of a reduction in the length of CS chains. Transfer of C4ST-1 cDNA into sog9 cells (sog9-C4ST-1) restored 4-O-sulfation and amount of CS, verifying that sog9 cells had a specific defect in C4ST-1. Furthermore, the expression of C4ST-1 rendered sog9 cells significantly more susceptible to HSV-1 infection, suggesting that CS modified by C4ST-1 is sufficient for the binding and infectivity of HSV-1. Analysis of CS chains of gro2C and sog9-C4ST-1 cells revealed a considerable proportion of the E disaccharide unit, consistent with our recent finding that this unit is an essential component of the HSV receptor. These results suggest that C4ST-1 regulates the expression of the E disaccharide unit and the length of CS chains, the features that facilitate infection of cells by HSV-1.     

The “in and out” of glucosamine 6-<Emphasis Type="Italic">O</Emphasis>-sulfation: the 6th sense of heparan sulfate

The biological properties of Heparan sulfate (HS) polysaccharides essentially rely on their ability to bind and modulate a multitude of protein ligands. These interactions involve internal oligosaccharide sequences defined by their sulfation patterns. Amongst these, the 6-O-sulfation of HS contributes significantly to the polysaccharide structural diversity and is critically involved in the binding of many proteins. HS 6-O-sulfation is catalyzed by 6-O-sulfotransferases (6OSTs) during biosynthesis, and it is further modified by the post-synthetic action of 6-O-endosulfatases (Sulfs), two enzyme families that remain poorly characterized. The aim of the present review is to summarize the contribution of 6-O-sulfates in HS structure/function relationships and to discuss the present knowledge on the complex mechanisms regulating HS 6-O-sulfation.