Heparan Sulfate Sulfotransferases Research Articles

Pancancer analysis of the correlations of HS6ST2 with prognosis, tumor immunity, and drug resistance

HS6ST2 has ability to encodes a member of the heparan sulfate (HS) sulfotransferase gene family, which catalyze the transfer of sulfate to HS and a crucial regulator of cell growth, differentiation, adhesion, and migration. Although mounting evidence supports a vital role for HS6ST2 in tumorigenesis of some cancers, no pan-cancer analysis of HS6ST2 has been reported. Therefore, we aimed to explore the prognostic value of HS6ST2 in 33 cancer types and investigate its potential immune function. Based on data from The Cancer Genome Atlas, Cancer Cell Lines Encyclopedia, Genotype Tissue Expression, and GSCA, we used a range of bioinformatics approaches to explore the potential carcinogenic role of HS6ST2, analysis of HS6ST2 and prognosis, DNA methylation, RNA methylation, microsatellite instability (MSI), tumor mutation burden (TMB), and immune cell infiltration in different tumors. The results show that HS6ST2 was highly expressed in most cancers but lower in Breast invasive carcinoma, Kidney Chromophobe, Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma, and Uterine Corpus Endometrial Carcinoma. Moreover, HS6ST2 is positively or negatively associated with prognosis in different cancers. HS6ST2 expression was not only associated with MSI in 5 cancer types and associated with TMB in 10 cancer types, and it's significantly correlated with DNA methylation in 13 types of cancer, but it's correlated with RNA methylation related genes in most cancer. HS6ST2 expression was correlated with immune cell infiltration, immune-related genes, tumor immune microenvironment, and drug resistance in various cancers. Eventually, HS6ST2 was validated in human lung adenocarcinoma tissues. Our study reveals that HS6ST2 can function as a prognostic marker in various malignant tumors because of its role in tumorigenesis and tumor immunity.

Analysis of protein-heparin interactions using a portable SPR instrument

Optical biosensors such as those based on surface plasmon resonance (SPR) are a key analytical tool for understanding biomolecular interactions and function as well as the quantitative analysis of analytes in a wide variety of settings. The advent of portable SPR instruments enables analyses in the field. A critical step in method development is the passivation and functionalisation of the sensor surface. We describe the assembly of a surface of thiolated oleyl ethylene glycol/biotin oleyl ethylene glycol and its functionalisation with streptavidin and reducing end biotinylated heparin for a portable SPR instrument. Such surfaces can be batch prepared and stored. Two examples of the analysis of heparin-binding proteins are presented. The binding of fibroblast growth factor 2 and competition for the binding of a heparan sulfate sulfotransferase by a library of selectively modified heparins and suramin, which identify the selectivity of the enzyme for sulfated structures in the polysaccharide and demonstrate suramin as a competitor for the enzyme’s sugar acceptor site. Heparin functionalised surfaces should have a wide applicability, since this polysaccharide is a close structural analogue of the host cell surface polysaccharide, heparan sulfate, a receptor for many endogenous proteins and viruses.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3′-phosphate-5′-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3′,5′-diphosphate) as a side product. We present a europium(iii)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Histochemical Analysis of Heparan Sulfate 3-O-sulfotransferase Expression in Mouse Brain.

In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify the cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with non-radioactive nucleotides.

Structural Determinants of Substrate Recognition and Catalysis by Heparan Sulfate Sulfotransferases.

Heparan sulfate (HS) and heparin contain imprinted "sulfation codes", which dictate their diverse physiological and pathological functions. A group of orchestrated biosynthetic enzymes cooperate in polymerizing and modifying HS chains. The biotechnological development of enzymes that can recreate this sulfation pattern on synthetic heparin is challenging, primarily due to the paucity of quantitative data for sulfotransferase enzymes. Herein, we identified critical structural characteristics that determine substrate specificity and shed light on the catalytic mechanism of sugar sulfation of two HS sulfotransferases, 2-O-sulfotransferase (HS2ST) and 6-O-sulfotransferase (HS6ST). Two sets of molecular clamps in HS2ST recognize appropriate substrates; these clamps flank the acceptor binding site on opposite sides. The hexuronic epimers, and not their puckers, have a critical influence on HS2ST selectivity. In contrast, HS6ST recognizes a broader range of substrates. This promiscuity is granted by a conserved tryptophan residue, W210, that positions the acceptor within the active site for catalysis by means of strong electrostatic interactions. Lysines K131 and K132 act in concert with a second tryptophan, W153, shedding water molecules from within the active site, thus providing HS6ST with a binding preference toward 2-O-sulfated substrates. QM/MM calculations provided valuable mechanistic insights into the catalytic process, identifying that the sulfation of both HS2ST and HS6ST follows a SN2-like mechanism. When they are taken together, our findings reveal the molecular basis of how these enzymes recognize different substrates and catalyze sugar sulfation, enabling the generation of enzymes that could create specific heparin epitopes.

Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment, and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR/Cas9 screens with a small molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting heparan sulfate formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of heparan sulfate-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure, and serves as a master regulator of the extracellular matrix.

Prognostic significance of hedgehog signaling network-related gene expression in breast cancer patients.

Breast cancer continues to be a serious public health problem. The role of the hedgehog pathway in normal development of the mammary gland as well as in carcinogenesis and progression of breast cancer is the subject of intense investigation, revealing functional interactions with cell surface heparan sulfate. Nevertheless, its influence on breast cancer prognosis, and its relation to specific sulfation motifs in heparan sulfate have only been poorly studied in large patient cohorts. Using the public database KMplotter that includes gene expression and survival data of 3951 patients, we found that the higher expression of SHH, HHAT, PTCH1, GLI1, GLI2, and GLI3 positively influences breast cancer prognosis. Stratifying patients according to the expression of hormone receptors, histological grade, lymph node metastasis, and systemic therapy, we observed that GLI1, GLI2, and GLI3 expression, as well as co-expression of SHH and ELP1 were associated with worse relapse-free survival in patients with HER2-positive tumors. Moreover, GLI1 expression in progesterone receptor-negative tumors and GLI3 expression in grade 3 tumors correlated with poor prognosis. SHH,in a panel of cell lines representing different breast cancer subtypes, and HHAT, PTCH1, GLI1, GLI2, and GLI3 were mostly expressed in cell lines classified as HER2-positive and basal-like. Expression of SHH, HHAT, GLI2, and GLI3 was differentially affected by overexpression of the heparan sulfate sulfotransferases HS2ST1 and HS3ST2 in vitro. Althoughhigh HS2ST1 expression was associated with poor prognosis in KMplotter analysis, high levels of HS3ST2 were associated with a good prognosis, except for ER-positive breast cancer. We suggest the GLI transcription factors as possible markers for the diagnosis, treatment, and prognosis of breast cancer especially in HER2-positive tumors, but also in progesterone receptor-negative and grade-3 tumors. The pathway interaction and prognostic impact of specific heparan sulfate sulfotransferases provide novel perspectives regarding a therapeutical targeting of the hedgehog pathway in breast cancer.

The Heparan Sulfate Sulfotransferases HS2ST1 and HS3ST2 Are Novel Regulators of Breast Cancer Stem-Cell Properties

Heparan sulfate (HS) is a glycosaminoglycan found mainly in its protein-conjugated form at the cell surface and the extracellular matrix. Its high sulfation degree mediates functional interactions with positively charged amino acids in proteins. 2-O sulfation of iduronic acid and 3-O sulfation of glucosamine in HS are mediated by the sulfotransferases HS2ST and HS3ST, respectively, which are dysregulated in several cancers. Both sulfotransferases regulate breast cancer cell viability and invasion, but their role in cancer stem cells (CSCs) is unknown. Breast CSCs express characteristic markers such as CD44+/CD24−/low, CD133 and ALDH1 and are involved in tumor initiation, formation, and recurrence. We studied the influence of HS2ST1 and HS3ST2 overexpression on the CSC phenotype in breast cancer cell lines representative of the triple-negative (MDA-MB-231) and hormone-receptor positive subtype (MCF-7). The CD44+/CD24−/low phenotype was significantly reduced in MDA-MB-231 cells after overexpression of both enzymes, remaining unaltered in MCF-7 cells. ALDH1 activity was increased after HS2ST1 and HS3ST2 overexpression in MDA-MB-231 cells and reduced after HS2ST1 overexpression in MCF-7 cells. Colony and spheroid formation were increased after HS2ST1 and HS3ST2 overexpression in MCF-7 cells. Moreover, MDA-MB-231 cells overexpressing HS2ST1 formed more colonies and could not generate spheres. The phenotypic changes were associated with complex changes in the expression of the stemness-associated notch and Wnt-signaling pathways constituents, syndecans, heparanase and Sulf1. The results improve our understanding of breast CSC function and mark a subtype-specific impact of HS modifications on the CSC phenotype of triple-negative and hormone receptor positive breast cancer model cell lines.

Changes in heparan sulfate sulfotransferases and cell-surface heparan sulfate during SKM-1 cells granulocytic differentiation and A549 cells epithelial-mesenchymal transition

Heparan sulfate (HS) with various sulfation patterns is one of important modulators of cancer cell fate through interacting with numerous growth factors. Here we found HS 2-O sulfotransferase 1 (HS2ST1) was downregulated at both mRNA and protein levels during granulocytic differentiation of SKM-1 leukemia cells and also HS (glucosamine) 3-O sulfotransferase 3A (HS3ST3A) in epithelial-mesenchymal transition (EMT) of A549 lung cancer cells, meanwhile, cell-surface HS recognized by anti-HS antibody was also changed in both cancer cell lines. Further, HS3ST3A was negatively correlated with the in vitro cell metastasis capability of A549 cells confirmed by RNA interference technology, wound-healing assay and in vitro Matrigel invasion assay. Together, specific HS sulfotransferases may serve as molecular markers and targets for cancer treatment.

Structure Based Substrate Specificity Analysis of Heparan Sulfate 6-O-Sulfotransferases.

Heparan sulfate (HS) is a sulfated polysaccharide exhibiting essential physiological functions. HS 6-O-sulfotransferase (6-OST) transfers a sulfo group to the 6-OH position of glucosamine units to confer a variety of HS biological activities. There are three different isoforms of 6-OST in the human genome. Here, we report crystal structures of the ternary complex of 6-OST with the sulfo donor analog 3'-phosphoadenosine 5'-phosphate and three different oligosaccharide substrates at 1.95 to 2.1 Å resolutions. Structural and mutational analyses reveal amino acid residues that contribute to catalysis and substrate recognition of 6-OST. Unexpectedly, the structures reveal 6-OST engages HS in a completely different orientation than other HS sulfotransferases and sheds light on the basic HS requirements for specificity. These findings also contribute structural information to understand mutations in human 6-OST isoform 1 associated with the human genetic disease idiopathic hypogonadotropic hypogonadism characterized by incomplete or lack of puberty.

Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans.

Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS.

The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer.

Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer. We show that 3-OST3A is epigenetically repressed in all breast cancer cell lines of a panel representative of distinct molecular subgroups, except in human epidermal growth factor receptor 2-positive (HER2+) sloan-kettering breast cancer (SKBR3) cells. Epigenetic mechanisms involved both DNA methylation and histone modifications, producing different repressive chromatin environments depending on the cell molecular signature. Gain and loss of function experiments by cDNA and siRNA transfection revealed profound effects of 3-OST3A expression on cell behavior including apoptosis, proliferation, response to trastuzumab in vitro and tumor growth in xenografted mice. 3-OST3A exerted dual activities acting as tumor-suppressor in lumA-michigan cancer foundation (MCF)-7 and triple negative-MD Anderson (MDA) metastatic breast (MB)-231 cells, or as an oncogenic factor in HER2+-SKBR3 cells. Mechanistically, fluorescence-resonance energy transfer-fluorescence-lifetime imaging microscopy experiments indicated that the effects of 3-OST3A in MCF-7 cells were mediated by altered interactions between HS and fibroblast growth factor-7 (FGF-7). Further, this interplay between HS and FGF-7 modulated downstream ERK, AKT and p38 cascades, suggesting that altering 3-O-sulfation affects FGFR2IIIb-mediated signaling. Corroborating our cellular data, a clinical study conducted in a cohort of breast cancer patients uncovered that, in HER2+ patients, high level expression of 3-OST3A in tumors was associated with reduced relapse-free survival. Our findings define 3-OST3A as a novel regulator of breast cancer pathogenicity, displaying tumor-suppressive or oncogenic activities in a cell- and tumor-dependent context, and demonstrate the clinical value of the HS-O-sulfotransferase 3-OST3A as a prognostic marker in HER2+ patients.

Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum).

Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain‐of‐function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position‐specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position‐specific, developmental‐stage‐specific, and heparan sulfate‐dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals.

Heparan sulfate 6-O-sulfotransferase 3 is involved in bone marrow mesenchymal stromal cell osteogenic differentiation‍.

The roles of sugar chains such as heparan sulfate (HS) in stem cell self-renewal and differentiation are poorly understood. HS is a sugar chain with linear sulfated polyanionic disaccharide repeating structures that interact with many proteins, including structural proteins in the extracellular matrix and growth factors and their receptors. Thus, unraveling the role of HS in stem cell self-renewal and differentiation could provide new insights and technical routes in clinical stem cell applications. Here, we purified rat bone marrow mesenchymal stromal cells (BMMSCs) by density gradient centrifugation, analyzed mesenchymal stromal cell surface stemness marker expression by flow cytometry, and identified the sulfotransferases responsible for sulfation ester modification of HS. An osteogenic differentiation model was established by chemical induction reagents and confirmed via alkaline phosphatase (ALP) activity detection and the expression of the osteogenic differentiation markers Runx2 and Ocn. The expression profiles of HS sulfotransferases in rat BMMSCs before and after osteogenic induction were detected by RT-PCR and Western blot. Cell spheroids were formed in both control and osteogenic culture systems when BMMSCs were grown to high confluence. We determined that this type of cell spheroid was a highly calcified nodule by histochemical staining. Among all the sulfotransferases examined, heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) mRNA and protein were upregulated in these calcified cell spheroids. HS6ST3 knockdown BMMSCs were established with RNA interference, and they had significantly lower ALP activity and decreased expression of the osteogenic differentiation markers Runx2 and Ocn. These findings suggest that HS6ST3 is involved in BMMSC differentiation, and new glycotherapeutic-based technologies could be developed in the future.

Histochemical analysis of heparan sulfate 3-O-sulfotransferase expression in mouse brain.

In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with nonradioactive nucleotides.

Overexpression of heparan sulfate 6-O-sulfotransferase-2 in colorectal cancer

The heparan sulfate sulfotransferase gene family catalyzes the transfer of sulfate groups to heparan sulfate and regulates various growth factor-receptor signaling pathways. However, the involvement of this gene family in cancer biology has not been elucidated. It was demonstrated that the heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2 (HS6ST2) gene is overexpressed in colorectal cancer (CRC) and its clinical significance in patients with CRC was investigated. The mRNA levels of HS6ST2 in clinical CRC samples and various cancer cell lines were assessed using a microarray analysis and quantitative RT-PCR, respectively. An immunohistochemical (IHC) analysis of the HS6ST2 protein was performed using 102 surgical specimens of CRC. The correlations between the HS6ST2 expression status and clinicopathological characteristics were then evaluated. HS6ST2 mRNA was significantly overexpressed by 37-fold in CRC samples compared to paired colonic mucosa. High levels of HS6ST2 mRNA expression were also observed in colorectal, esophageal and lung cancer cell lines. The IHC analysis demonstrated that HS6ST2 was expressed in the cytoplasmic region of CRC cells, but not in normal colonic mucosal cells. Positive staining for HS6ST2 was detected in 40 patients (39.2%). There was no significant association between the clinicopathological characteristics and HS6ST2 expression. However, positive staining for HS6ST2 was associated with a poor survival (P=0.074, log-rank test). In conclusion, HS6ST2 was found to be overexpressed in CRC and its expression tended to be a poor prognostic factor, although the correlation was not significant. These findings indicate that HS6ST2 may be a novel cancer-related marker that may provide insight into the glycobiology of CRC.

Drosophila Heparan Sulfate 6-O-Endosulfatase Sulf1 Facilitates Wingless (Wg) Protein Degradation

Heparan sulfate proteoglycans regulate various physiological and developmental processes through interactions with a number of protein ligands. Heparan sulfate (HS)-ligand binding depends on the amount and patterns of sulfate groups on HS, which are controlled by various HS sulfotransferases in the Golgi apparatus as well as extracellular 6-O-endosulfatases called "Sulfs." Sulfs are a family of secreted molecules that specifically remove 6-O-sulfate groups within the highly sulfated regions on HS. Vertebrate Sulfs promote Wnt signaling, whereas the only Drosophila homologue of Sulfs, Sulf1, negatively regulates Wingless (Wg) signaling. To understand the molecular mechanism for the negative regulation of Wg signaling by Sulf1, we studied the effects of Sulf1 on HS-Wg interaction and Wg stability. Sulf1 overexpression strongly inhibited the binding of Wg to Dally, a potential target heparan sulfate proteoglycan of Sulf1. This effect of Drosophila Sulf1 on the HS-Wg interaction is similar to that of vertebrate Sulfs. Using in vitro, in vivo, and ex vivo systems, we show that Sulf1 reduces extracellular Wg protein levels, at least partly by facilitating Wg degradation. In addition, expression of human Sulf1 in the Drosophila wing disc lowers the levels of extracellular Wg protein, as observed for Drosophila Sulf1. Our study demonstrates that vertebrate and Drosophila Sulfs have an intrinsically similar activity and that the function of Sulfs in the fate of Wnt/Wg ligands is context-dependent.

Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C(5)-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments.

Understanding the substrate specificity of the heparan sulfate sulfotransferases by an integrated biosynthetic and crystallographic approach

Heparan sulfates (HSs) have potential therapeutic value as anti-inflammatory and antimetastasis drugs, in addition to their current use as anticoagulants. Recent advances in chemoenzymatic synthesis of HS provide a way to conveniently produce homogenous HS with different biological properties. Crystal structures of sulfotransferases involved in this process are providing atomic detail of their substrate binding clefts and interactions with their HS substrates. In theory, the flexibility of this method can be increased by modifying the specificities of the sulfotransferases based on the structures, thereby producing a new array of products.

Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis

Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C(5)-epimerase (C(5)-epi) is a key enzyme in this pathway. C(5)-epi is known for being a two-way catalytic enzyme, displaying a "reversible" catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C(5)-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an "irreversible" catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C(5)-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.