Structural Determinants of Substrate Recognition and Catalysis by Heparan Sulfate Sulfotransferases
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PROVIDER: S-EPMC10550706 | biostudies-literature | 2021 Aug
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC5331487 | biostudies-literature
Project description:The biosynthesis of heparan sulfate (HS) involves an array of specialized sulfotransferases. Here, we present a study aimed at engineering the substrate specificity of different HS 3-O-sulfotransferase isoforms. Based on the crystal structures, we identified a pair of amino acid residues responsible for selecting the substrates. Mutations of these residues altered the substrate specificities. Our results demonstrate the feasibility of tailoring the specificity of sulfotransferases to modify HS with desired functions.
| S-EPMC2676843 | biostudies-literature
Project description: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.
| S-EPMC3711681 | biostudies-literature
Project description:Testosteronan, an unusual glycosaminoglycan (GAG) first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the GAG, heparan sulfate. Studies using (35) S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from (34) S-labeled 6-O-sulfo-N-sulfotestosteroran using (34) S-labeled PAPS because testosteronan was resistant to all of the tested GAG-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of (34) S into ∼14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ∼20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin (AT) III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to AT III.
| S-EPMC3819802 | biostudies-literature
Project description:The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.
| S-EPMC4105206 | biostudies-literature
Project description:The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.
| S-EPMC8341778 | biostudies-literature
Project description:Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.
| S-EPMC7212660 | biostudies-literature
Project description:The functions of heparan sulfate (HS) depend on the expression of structural domains that interact with protein partners. Glycosaminoglycans (GAGs) exhibit a high degree of polydispersity in their composition, chain length, sulfation, acetylation, and epimerization patterns. It is essential for the understanding of GAG biochemistry to produce detailed structural information as a function of spatial and temporal factors in biological systems. Toward this end, we developed a set of procedures to extract GAGs from various rat organ tissues and examined and compared HS expression levels using liquid chromatography/mass spectrometry. Here we demonstrate detailed variations in HS GAG chains as a function of organ location. These studies shed new light on the structural variation of GAG chains with respect to average length, disaccharide composition, and expression of low abundance structural epitopes, including unsubstituted amino groups and lyase-resistant oligosaccharides. The data show the presence of a disaccharide with an unsubstituted amino group that is endogenous and widely expressed in mammalian organ tissues.
| S-EPMC2673249 | biostudies-literature
Project description:Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family.
| S-EPMC4641920 | biostudies-literature
Project description: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.
| S-EPMC7546021 | biostudies-literature