Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN.

Due to their ability to recognize carbohydrate structures, lectins emerged as potential receptors for bacterial lipopolysaccharides (LPS). Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. We contributed to fill this gap by unveiling the molecular basis of the interaction between the lipooligosaccharide of Escherichia coli and the dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN). Specifically, a combination of different techniques, including fluorescence microscopy, surface plasmon resonance, NMR spectroscopy, and computational studies, demonstrated that DC-SIGN binds to the purified deacylated R1 lipooligosaccharide mainly through the recognition of its outer core pentasaccharide, which acts as a crosslinker between two different tetrameric units of DC-SIGN. Our results contribute to a better understanding of DC-SIGN-LPS interaction and may support the development of pharmacological and immunostimulatory strategies for bacterial infections, prevention, and therapy.

Staphylococcus aureus sacculus mediates activities of M23 hydrolases.

Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently.

The unique 3D arrangement of macrophage galactose lectin enables Escherichia coli lipopolysaccharide recognition through two distinct interfaces.

Lipopolysaccharides are a hallmark of gram-negative bacteria, and their presence at the cell surface is key for bacterial integrity. As surface-exposed components, they are recognized by immunity C-type lectin receptors present on antigen-presenting cells. Human macrophage galactose lectin binds Escherichia coli surface that presents a specific glycan motif. Nevertheless, this high-affinity interaction occurs regardless of the integrity of its canonical calcium-dependent glycan-binding site. NMR of macrophage galactose-type lectin (MGL) carbohydrate recognition domain and complete extracellular domain revealed a glycan-binding site opposite to the canonical site. A model of trimeric macrophage galactose lectin was determined based on a combination of small-angle X-ray scattering and AlphaFold. A disulfide bond positions the carbohydrate recognition domain perpendicular to the coiled-coil domain. This unique configuration for a C-type lectin orients the six glycan sites of MGL in an ideal position to bind lipopolysaccharides at the bacterial surface with high avidity.

Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.

Preparation and Characterization of Heparan Sulfate-Derived Oligosaccharides to Investigate Protein-GAG Interaction and HS Biosynthesis Enzyme Activity.

Heparan sulfate chains are complex and structurally diverse polysaccharides that interact with a large number of proteins, thereby regulating a vast array of biological functions. Understanding this activity requires obtaining oligosaccharides of defined structures. Here we describe methods for isolating, engineering, and characterizing heparan sulfate-derived oligosaccharides and approaches based on high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and bio-layer interferometry (BLI) to study their structures, modifications, and interactions.

The lipopolysaccharide-transporter complex LptB2FG also displays adenylate kinase activity in vitro dependent on the binding partners LptC/LptA.

Lipopolysaccharide (LPS) is an essential glycolipid that covers the surface of gram-negative bacteria. The transport of LPS involves a dedicated seven-protein transporter system called the lipopolysaccharide transport system (Lpt) machinery that physically spans the entire cell envelope. The LptB2FG complex is an ABC transporter that hydrolyzes ATP to extract LPS from the inner membrane for transport to the outer membrane. Here, we extracted LptB2FG directly from the inner membrane with its original lipid environment using styrene-maleic acid polymers. We found that styrene-maleic acid polymers-LptB2FG in nanodiscs display not only ATPase activity but also a previously uncharacterized adenylate kinase (AK) activity, as it catalyzed phosphotransfer between two ADP molecules to generate ATP and AMP. The ATPase and AK activities of LptB2FG were both stimulated by the interaction on the periplasmic side with the periplasmic LPS transport proteins LptC and LptA and inhibited by the presence of the LptC transmembrane helix. We determined that the isolated ATPase module (LptB) had weak AK activity in the absence of transmembrane proteins LptF and LptG, and one mutation in LptB that weakens its affinity for ADP led to AK activity similar to that of fully assembled complex. Thus, we conclude that LptB2FG is capable of producing ATP from ADP, depending on the assembly of the Lpt bridge, and that this AK activity might be important to ensure efficient LPS transport in the fully assembled Lpt system.

Deciphering the structural attributes of protein-heparan sulfate interactions using chemo-enzymatic approaches and NMR spectroscopy.

Heparan sulfates (HS) is a polysaccharide found at the cell surface, where it mediates interactions with hundreds of proteins and regulates major pathophysiological processes. HS is highly heterogeneous and structurally complex and examples that define their structure-activity relationships remain limited. Here, in order to characterize a protein-HS interface and define the corresponding saccharide-binding domain, we present a chemo-enzymatic approach that generates 13C-labeled HS-based oligosaccharide structures. Nuclear magnetic resonance (NMR) spectroscopy, which efficiently discriminates between important or redundant chemical groups in the oligosaccharides, is employed to characterize these molecules alone and in interaction with proteins. Using chemokines as model system, docking based on NMR data on both proteins and oligosaccharides enable the identification of the structural determinant involved in the complex. This study shows that both the position of the sulfo groups along the chain and their mode of presentation, rather than their overall number, are key determinant and further points out the usefulness of these 13C-labeled oligosaccharides in obtaining detailed structural information on HS-protein complexes.

SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli.

Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.IMPORTANCEEscherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.

Defining the function of OmpA in the Rcs stress response.

OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of ß-barrel proteins for several decades. In Escherichia coli, OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal ß-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results suggest a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response.

Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC-LptA Interaction and Decreasing LptA Stability.

The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal ß-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC-LptA and LptA-LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC-LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.

Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa.

Peptidoglycan (PG) is an essential component of the bacterial cell wall and is assembled from a lipid II precursor by glycosyltransferase and transpeptidase reactions catalyzed in particular by bifunctional class A penicillin-binding proteins (aPBPs). In the major clinical pathogen Pseudomonas aeruginosa, PBP1B is anchored within the cytoplasmic membrane but regulated by a bespoke outer membrane-localized lipoprotein known as LpoP. Here, we report the structure of LpoP, showing an extended N-terminal, flexible tether followed by a well-ordered C-terminal tandem-tetratricopeptide repeat domain. We show that LpoP stimulates both PBP1B transpeptidase and glycosyltransferase activities in vitro and interacts directly via its C terminus globular domain with the central UB2H domain of PBP1B. Contrary to the situation in E. coli, P. aeruginosa CpoB does not regulate PBP1B/LpoP in vitro. We propose a mechanism that helps to underscore similarities and differences in class A PBP activation across Gram-negative bacteria.

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide.

Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOSR1 mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.

Solid State NMR Studies of Intact Lipopolysaccharide Endotoxin.

Lipopolysaccharides (LPS) are complex glycolipids forming the outside layer of Gram-negative bacteria. Their hydrophobic and heterogeneous nature greatly hampers their structural study in an environment similar to the bacterial surface. We have studied LPS purified from E. coli and pathogenic P. aeruginosa with long O-antigen polysaccharides assembled in solution as vesicles or elongated micelles. Solid-state NMR with magic-angle spinning permitted the identification of NMR signals arising from regions with different flexibilities in the LPS, from the lipid components to the O-antigen polysaccharides. Atomic scale data on the LPS enabled the study of the interaction of gentamicin antibiotic bound to P. aeruginosa LPS, for which we could confirm that a specific oligosaccharide is involved in the antibiotic binding. The possibility to study LPS alone and bound to a ligand when it is assembled in membrane-like structures opens great prospects for the investigation of proteins and antibiotics that specifically target such an important molecule at the surface of Gram-negative bacteria.

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus.

Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to ß-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of ß-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to ß-lactams in S. aureus.

Interaction of lipopolysaccharides at intermolecular sites of the periplasmic Lpt transport assembly.

Transport of lipopolysaccharides (LPS) to the surface of the outer membrane is essential for viability of Gram-negative bacteria. Periplasmic LptC and LptA proteins of the LPS transport system (Lpt) are responsible for LPS transfer between the Lpt inner and outer membrane complexes. Here, using a monomeric E. coli LptA mutant, we first show in vivo that a stable LptA oligomeric form is not strictly essential for bacteria. The LptC-LptA complex was characterized by a combination of SAXS and NMR methods and a low resolution model of the complex was determined. We were then able to observe interaction of LPS with LptC, the monomeric LptA mutant as well as with the LptC-LptA complex. A LptC-LPS complex was built based on NMR data in which the lipid moiety of the LPS is buried at the interface of the two ß-jellyrolls of the LptC dimer. The selectivity of LPS for this intermolecular surface and the observation of such cavities at homo- or heteromolecular interfaces in LptC and LptA suggests that intermolecular sites are essential for binding LPS during its transport.

Heparan sulfate differentially controls CXCL12α- and CXCL12γ-mediated cell migration through differential presentation to their receptor CXCR4.

Chemokines stimulate signals in cells by binding to G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. These chemoattractant cytokines also interact with heparan sulfate (HS), which provides positional information within tissues in the form of haptotactic gradients along which cells can migrate directionally. To investigate the mechanism by which HS modulates chemokine functions, we used the CXC chemokine CXCL12, which exists in different isoforms that all signal through CXCR4 but have distinct HS-binding domains. In experiments with both cell-associated and solubilized CXCR4, we found that although CXCL12γ bound to CXCR4 with a higher affinity than did CXCL12α, CXCL12γ displayed reduced signaling and chemotactic activities. These properties were caused by the specific carboxyl-terminal region of CXCL12γ, which, by interacting with CXCR4 sulfotyrosines, mediated high-affinity, but nonproductive, binding to CXCR4. HS prevented CXCL12γ from interacting with the CXCR4 sulfotyrosines, thereby functionally presenting the chemokine to its receptor such that its activity was similar to that of CXCL12α. HS had no effects on the binding of CXCL12α to CXCR4 or its biological activity, suggesting that this polysaccharide controls CXCL12 in an isoform-specific manner. These data suggest that the HS-dependent regulation of chemokine functions extends beyond the simple process of immobilization and directly modulates receptor ligation and activation.

Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ.

Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm.

In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of ß-lactam antibiotics is bypassed by L,D-transpeptidases that form unusual 3 → 3 peptidoglycan cross-links. ß-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium L,D-transpeptidase Ldt(fm) by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt(fm) that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt(fm) active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with ß-lactams.

Hydration water mobility is enhanced around tau amyloid fibers.

The paired helical filaments (PHF) formed by the intrinsically disordered human protein tau are one of the pathological hallmarks of Alzheimer disease. PHF are fibers of amyloid nature that are composed of a rigid core and an unstructured fuzzy coat. The mechanisms of fiber formation, in particular the role that hydration water might play, remain poorly understood. We combined protein deuteration, neutron scattering, and all-atom molecular dynamics simulations to study the dynamics of hydration water at the surface of fibers formed by the full-length human protein htau40. In comparison with monomeric tau, hydration water on the surface of tau fibers is more mobile, as evidenced by an increased fraction of translationally diffusing water molecules, a higher diffusion coefficient, and increased mean-squared displacements in neutron scattering experiments. Fibers formed by the hexapeptide (306)VQIVYK(311) were taken as a model for the tau fiber core and studied by molecular dynamics simulations, revealing that hydration water dynamics around the core domain is significantly reduced after fiber formation. Thus, an increase in water dynamics around the fuzzy coat is proposed to be at the origin of the experimentally observed increase in hydration water dynamics around the entire tau fiber. The observed increase in hydration water dynamics is suggested to promote fiber formation through entropic effects. Detection of the enhanced hydration water mobility around tau fibers is conjectured to potentially contribute to the early diagnosis of Alzheimer patients by diffusion MRI.

C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-O-sulfated heparan sulfate domains.

Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins and, as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed a chemically defined 13C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C5-epimerase and 2-O-sulfotransferase, which catalyze the production of iduronic acid and its 2-O-sulfation, respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C5-epimerase and 2-O-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface plasmon resonance experiments.