Downstream Products are Potent Inhibitors of the Heparan Sulfate 2-O-Sulfotransferase

Thieker, David F.; Xu, Yongmei; Chapla, Digantkumar; Nora, Chelsea; Qiu, Hong; Felix, T Wieland; Wang, Lianchun; Moremen, Kelley W.; Liu, Jian; Woods, Robert J.

doi:10.1038/s41598-018-29602-4

Cited by 11 publications

(14 citation statements)

References 73 publications

(79 reference statements)

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“…These data are in line with the described stepwise mechanism underlying HS biosynthesis, further supporting that epimerization and 2- O -sulfation reactions usually take place prior to 6- O -sulfation ( Figure 1 ). More recently, it was uncovered that 6- O -sulfated HS oligosaccharides were able to bind to 2OST1, with higher affinity than 2OST1 direct substrates, and inhibit its catalytic activity ( 42 ). In light of these results, it was hypothesized that enzymes involved in the HS biosynthetic pathway might be temporarily and/or spatially separated from the intermediate substrates produced downstream on the Golgi.…”

Section: Hs Biosynthesis and Modificationmentioning

confidence: 99%

Heparan Sulfate Biosynthesis and Sulfation Profiles as Modulators of Cancer Signalling and Progression

et al. 2021

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Heparan Sulfate Proteoglycans (HSPGs) are important cell surface and Extracellular Matrix (ECM) maestros involved in the orchestration of multiple cellular events in physiology and pathology. These glycoconjugates bind to various bioactive proteins via their Heparan Sulfate (HS) chains, but also through the protein backbone, and function as scaffolds for protein-protein interactions, modulating extracellular ligand gradients, cell signalling networks and cell-cell/cell-ECM interactions. The structural features of HS chains, including length and sulfation patterns, are crucial for the biological roles displayed by HSPGs, as these features determine HS chains binding affinities and selectivity. The large HS structural diversity results from a tightly controlled biosynthetic pathway that is differently regulated in different organs, stages of development and pathologies, including cancer. This review addresses the regulatory mechanisms underlying HS biosynthesis, with a particular focus on the catalytic activity of the enzymes responsible for HS glycan sequences and sulfation motifs, namely D-Glucuronyl C5-Epimerase, N- and O-Sulfotransferases. Moreover, we provide insights on the impact of different HS structural epitopes over HSPG-protein interactions and cell signalling, as well as on the effects of deregulated expression of HS modifying enzymes in the development and progression of cancer. Finally, we discuss the clinical potential of HS biosynthetic enzymes as novel targets for therapy, and highlight the importance of developing new HS-based tools for better patients’ stratification and cancer treatment.

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Section: Hs Biosynthesis and Modificationmentioning

confidence: 99%

Heparan Sulfate Biosynthesis and Sulfation Profiles as Modulators of Cancer Signalling and Progression

et al. 2021

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“…As noted above, knowledge of the effective concentration of immobilized DNA was critical to data interpretation. For ForteBio High Precision Streptavidin (SAX) tips, we determined this value was most frequently~2 × 10 −9 M. Thus, for a reaction to be in the equilibrium binding regime, its K d value must be >10 −8 M. The binding affinities measured by Theiker et al 19 for a sulfotransferase binding to hexasaccharides ranged from 10 −8 to 10 −7 M. These experiments used slightly different Forte-Bio streptavidin tips than the current work (SA rather than SAX), but if the tips have comparable numbers of binding sites, these reactions should have been in the equilibrium binding regime. In addition, we obtained a reasonable estimate of K d for an experiment in the intermediate binding regime using Equation (2) for global fitting of multiple replicates.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, Thieker et al used BLI amplitudes to monitor carbohydrate-protein titrations and thereby determine binding affinities. 19 However, they did not have a well-established technique available by which to benchmark their measurements. In particular, one must establish whether the concentration of the immobilized substrate is above or below the K d of the binding reaction, because the two scenarios require different data fitting and interpretation.…”

Section: F I G U R Ementioning

confidence: 99%

“…Thus, we were motivated to determine whether the amplitude of the BLI plateau at equilibrium—which is reported to be a function of changes to the average optical thickness —could be used to monitor equilibrium titration experiments. Previously, Thieker et al used BLI amplitudes to monitor carbohydrate‐protein titrations and thereby determine binding affinities . However, they did not have a well‐established technique available by which to benchmark their measurements.…”

Section: Introductionmentioning

confidence: 99%

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The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules

et al. 2020

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Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein‐DNA binding affinities, nitrocellulose filter binding assays with 32P‐labeled DNA quantify Kd values from 10−12 to 10−8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio koff/kon determines Kd. However, for lactose repressor protein (LacI) and an engineered repressor protein (“LLhF”) binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below Kd (equilibrium binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10−9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10−8 M should reliably be in the equilibrium binding regime. We also observed effects from multi‐valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein‐DNA binding and to assess the affinity of fructose‐1‐kinase for the DNA‐LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.

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“…A substrate specificity analysis showed that the 2-OST acceptor residues must be flanked by two GlcNS, consistently with the observation that N- sulfo groups mediated many contacts with several amino acids of the enzyme [ 187 ]. Oligosaccharides containing a 6- O -sulfate group also bound strongly to the 2-OST active site, but the 6- O -sulfate moiety appeared to occupy the PAPS binding site and thus inhibited the 2- O sulfation reaction [ 188 ]. Altogether, these data suggest 2- O -sulfation occurs prior to 6- O -sulfation to generate IdoA2S-GlcNS6S sequences and that the 2-OST exhibits multiple structural features to recognize its substrate sulfation pattern.…”

Section: Heparan Sulfate Maturationmentioning

confidence: 99%

Heparan Sulfate Proteoglycans Biosynthesis and Post Synthesis Mechanisms Combine Few Enzymes and Few Core Proteins to Generate Extensive Structural and Functional Diversity

et al. 2020

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Glycosylation is a common and widespread post-translational modification that affects a large majority of proteins. Of these, a small minority, about 20, are specifically modified by the addition of heparan sulfate, a linear polysaccharide from the glycosaminoglycan family. The resulting molecules, heparan sulfate proteoglycans, nevertheless play a fundamental role in most biological functions by interacting with a myriad of proteins. This large functional repertoire stems from the ubiquitous presence of these molecules within the tissue and a tremendous structural variety of the heparan sulfate chains, generated through both biosynthesis and post synthesis mechanisms. The present review focusses on how proteoglycans are “gagosylated” and acquire structural complexity through the concerted action of Golgi-localized biosynthesis enzymes and extracellular modifying enzymes. It examines, in particular, the possibility that these enzymes form complexes of different modes of organization, leading to the synthesis of various oligosaccharide sequences.

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Downstream Products are Potent Inhibitors of the Heparan Sulfate 2-O-Sulfotransferase

Cited by 11 publications

References 73 publications

Heparan Sulfate Biosynthesis and Sulfation Profiles as Modulators of Cancer Signalling and Progression

Heparan Sulfate Biosynthesis and Sulfation Profiles as Modulators of Cancer Signalling and Progression

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules

Heparan Sulfate Proteoglycans Biosynthesis and Post Synthesis Mechanisms Combine Few Enzymes and Few Core Proteins to Generate Extensive Structural and Functional Diversity

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