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Novel methods in nuclear magnetic resonance (NMR) spectroscopy have recently been developed to investigate the binding properties of intermolecular complexes endowed with biomedical functions. Among these methods is the saturation transfer difference (STD), which enables the mapping of specific binding motifs of functional ligands. STD can efficiently uncover the specific and preferential binding sites of these ligands in their intermolecular complexes. This is particularly useful in the case of glycosaminoglycans (GAGs), a group of sulfated polysaccharides that play pivotal roles in various biological and pathological processes. The activity of GAGs is ultimately mediated through molecular interactions with key functional proteins, namely, GAG-binding proteins (GBPs). The quality of the GAG-GBP interactions depends on sulfation patterns, oligosaccharide length, and the composing monosaccharides of GAGs. Through STD NMR, information about the atoms of the GAG ligands involved in the complexes is provided. Here we highlight the latest achievements of the literature using STD NMR on GAG oligosaccharide-GBP complexes. Interestingly, most of the GBPs studied so far by STD NMR belong to one of the three major classes: coagulation factors, growth factors, or chemokine/cytokines. Unveiling the structural requirements of GAG ligands in bindings with their protein partners is a crucial step to understand the biochemical and medical actions of GAGs. This process is also a requirement in GAG-based drug discovery and development.

Keywords

saturation transfer difference nuclear magnetic resonance glycosaminoglycans carbohydrate-protein interactions

Introduction

In the past two decades, a variety of analytical techniques have been developed for explicit characterization of the interactions between proteins and their ligands. Among these are mass spectrometry,¹ surface plasmon resonance spectroscopy,² microarray,³ and nuclear magnetic resonance (NMR) spectroscopy.⁴ NMR has emerged as a powerful technique capable of uncovering a great amount of details regarding protein-ligand complexes in solution. This analytical technique can study a large variety of proteins because of the assessment of an impressive range of binding affinities, going from 10⁻⁹ to 10⁻³ molar.⁴

With respect to the diversity of NMR experiments that can be conducted to investigate properties of intermolecular complexes in solution, three individual goals can be defined. The first would be the understanding of the specific amino acid residues involved in the protein-ligand interactions. The second would be the mapping of atoms of the binding ligand(s) involved in the interactions. The third would be the bound conformations of the ligand necessary for the proper and best interactions and how these conformations change from the free (unbound) status to the bound status. Regardless of the goal wanted in research, NMR spectroscopy can surely provide methods capable of overviewing all of these three aspects of the protein-ligand complexes. The first goal can be addressed by chemical shift perturbation (CSP), the second can be addressed by saturation transfer difference (STD), and the third can be addressed by the transferred nuclear Overhauser effect (trNOE).^5,6

STD

The assessment of the ligand properties necessary for the specific interactions with their protein partners is crucial in any science concerned with the functionality of these proteins, including those of medical interest. As pointed above, the NMR STD method can properly address the questions related to how the ligands bind to their protein partners during intermolecular complex formation. As presented by Mayer and Meyer,⁷ the NMR spectrum of the saturated state (on-resonance) in interaction with its protein partner is compared with the NMR spectrum of the unsaturated state (off-resonance) of the unbound ligands in solution ( Fig. 1A ). This experiment is conducted on a single sample in which both the on-resonance and off-resonance spectra are acquired and compared. The STD method relies on the specific magnetization of certain protein chemical groups (usually nearby the binding site) and transfer, through spatial contacts, to the ligand ( Fig. 1B ). The resulting STD NMR spectrum should provide signals with decreased intensities derived from the contacts with the protein ( Fig. 1B ). By subtracting the off-resonance spectrum from the on-resonance spectrum, residual NMR signals provide the resultant intensities and the chemical shifts specifically related to the chemical groups of the ligand involved in the interaction ( Fig. 1C ). Besides the mapping of the atoms of the ligands involved in such interactions (through chemical shift notation), STD is also capable of qualitatively estimating the proximity of the contacts through the intensities of the resultant STD signals. For instance, protons within the ligand that are near the protein can receive more magnetization from the saturated protein through the cross-polarization phenomenon resembling the internuclear proximity seen in the NOE mechanism.⁷

Figure 1.

The overall saturation transfer difference (STD) nuclear magnetic resonance (NMR) mechanism is illustrated. (A) The reference NMR spectrum of the ligand is recorded in solution without saturation on the protein partner (saturation off). (B) The NMR spectrum of the ligand is recorded with saturation on the protein partner (saturation on). Ligand atoms in near proximity experience a loss in intensity when the saturation occurs, resulting in a spectrum with less signal intensities as compared with the reference spectrum. (C) The spectrum with saturation on is then subtracted from the reference spectrum with saturation off, leaving the NMR signal intensities, which are relative to the STD.

Through STD NMR, dissociation constants of the complexes (K_D) can be determined.⁸ This experiment is typically performed with a sample prepared with ~5- to 100-fold molar excess of ligand in comparison with the protein concentration in solvent. This ensures that all observed signals are strictly from the ligand under investigation. Therefore, it is important to perform an on-resonance control experiment of the ligand being observed in solution without the protein. This ensures that no signals from the ligand are being observed from the selective pulse designed for the protein. In case signals from the protein are still present from the selective magnetization, these unwanted signals from the protein without the bound ligand can be filtered out from the STD spectra. This method is called saturation transfer double-difference (STDD).^9,10 Although primarily conducted as a one-dimensional (1D) NMR experiment, STD can also be applied in two-dimensional (2D) NMR experiments. An example are the studies involving NMR in the heteronuclear single-quantum coherence (HSQC) spectrum¹¹ in events in which signal overlap of the complexed ligand is too difficult for interpretation through the usual 1D techniques.¹²

The application of STD is subject to several experimental conditions, besides the concentration requirements as described above. These conditions must be carefully considered during the experiment so that accurate and well-resolved information can be acquired from the STD NMR technique. Among them is molecular weight (MW). STD signals can be limited by the MW of the molecule with which the ligand is interacting. Medium-sized or small macromolecule (<15 kDa) will not be subject to the intense ¹H-¹H cross polarization seen for larger complexes.^13,14 The magnetization source leading to spin diffusion is the selective saturation applied to the protein in the on-resonance spectrum. As described above, this magnetization is intended to only excite the protein involved in the complex. Thankfully, in the case of carbohydrates, the frequently irradiated regions of the proteins do not overlap with the carbohydrate signals.¹⁵ Because many binding proteins show their binding sites composed of lysine and arginine residues, the methylene protons of these amino acids can be chosen as targets for specific magnetization.¹⁶ The length of this magnetic excitation is also crucial to the mechanism of STD. The excitement must be long enough to allow proper magnetization of the ligand from the initially radiated protein in the bound state. To assess this phenomenon, signal intensity buildup curves should be performed often to determine the optimal length of saturation time.¹⁷ The preparation of the samples to be analyzed by STD NMR is also routinely performed using buffered solutions in which the salt concentrations can mimic the physiological conditions.¹⁸ Temperature may also play a key role in the quality of the STD-derived information. Although frequently performed at room temperature (25 °C), the use of lower temperatures can be explored to increase resolution.^15,19

STD was originally developed for use in screening of potential pharmaceutical agents from libraries of small MW compounds. Currently, STD NMR has been applied in a more diverse set of investigations. Recent examples are studies in which multiple ligands exist for interactions with functional biomacromolecules. More specifically, STD has been applied in the research of nucleic acids²⁰ and peptides²¹ and, in a later stage, optimized for carbohydrate studies.²² Recent analytical advancements have also greatly increased the types of binding targets subjected to scientific investigations. Whole cells,²³ viruses,²⁴ and G protein–coupled receptors (GPCRs)²⁵ are among these recent new binding targets successfully studied nowadays through STD NMR. Conclusions from these experiments are viable for therapeutic applications. The use of multiple ligands in solution allows for the further mapping of the specific binding epitopes of structurally similar ligands under the same screening. This information is exceptionally relevant in investigations of structurally complex polysaccharides that participate in a wide range of biomedical functions. Example of these polysaccharides endowed of numerous biomedical actions are the glycosaminoglycans (GAGs). GAGs are vastly exploited in the market and research as both pharmaceutical and nutraceutical ingredients.

GAGs and GAG-Binding Proteins

GAGs are a structurally diverse group of complex linear polysaccharides that are often highly sulfated and of high MW.^5,26 GAGs are expressed as heterogeneous chains composed of disaccharide building blocks. These building blocks are in turn composed of alternating units of one hexosamine (N-acetylglucosamine [GlcNAc] or N-acetylgalactosamine [GalNAc]) and one hexuronic acid (glucuronic acid [GlcA] or iduronic acid [IdoA]) or a galactose (Gal) unit.^5,26 Depending on the composition, GAGs fall within one of the four major specific families: heparin (Hp)/heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and keratan sulfate (KS). All of these can vary greatly in terms of N- and O-sulfation patterns, except HA, which is naturally not sulfated.^26,27 The structures of these four groups are depicted in Figure 2.

Figure 2.

The structural representation of the four major glycosaminoglycan (GAG) families is provided. (A) Representative repeating disaccharide unit of heparan sulfate (HS) and heparin (Hp) structures. Both are composed of alternating 4-linked uronic acid and 4-linked α-glucosamine (GlcNAc) units. HS has β-D-glucuronic acid (GlcA) as its major uronic acid type, whereas Hp has α-L- iduronic acid (IdoA). HS is frequently N-sulfated at the GlcNAc unit, with less extension of N-acetylation and just rare amounts of N-free (just the amino NH₂ group), whereas Hp is predominantly composed of 2-sulfated IdoA together with N,6-di-sulfated GlcNAc units. Although the 3-O-sulfation at the GlcNAc unit occurs more often at Hp than HS, it still happens but rarely. (B) Representative repeating disaccharide unit of chondroitin sulfate (CS) and dermatan sulfate (DS) structures. CSs are composed of alternating 4-linked β-D-GlcA and 3-linked N-acetyl-α-D-galactosamine (GalNAc) units. CS-A is mostly 4-sulfated at the GalNAc units, whereas CS-C is predominantly 6-sulfated. CS-B, widely known as DS, has α-L-iduronic acid (IdoA) units rather than β-GlcA. The IdoA units in DS may contain 2-sulfation, whereas the GalNAc units are mostly 4-sulfated. (C) Representative repeating disaccharide unit of keratan sulfate (KS) structure. It is composed of alternating 3-linked β-galactose (Gal) and 4-linked α-GlcNAc units. KS can bear sulfation at the 6-position of each unit, although sulfation at GlcNAc occurs more often. (D) Representative repeating disaccharide unit of hyaluronic acid (HA). It is composed of alternating 4-linked β-D-GlcA and 3-linked β-D-GlcNAc units.

GAGs are highly evolutionarily conserved across animalia as a key component of the extracellular matrix (ECM). These molecules act as binders and regulators of a variety of key proteins involved in numerous physiological and pathological events.^28–33 Because GAGs show high degrees of structural diversity, they can interact and control multiple proteins. These proteins are colloquially named GAG-binding proteins (GBPs). This structural complexity depends on the microheterogeneity of the oligosaccharide sequences seen in their polymeric chains. Structural specificity is a key determinant for protein interactions.³² Hence, the discovery of binding motifs or functional epitopes in GAG molecules is a priority.

The GBPs are key players in the coagulation system,^34,35 inflammatory response,³⁶ tumorigenesis,³⁷ tissue development, repair and fibrosis,³⁸ neurologic functions,³⁹and virus infections.^40,41 Although hundreds of GBPs have been identified⁴² and are currently available for biomedical and physicochemical studies, those who have been investigated by STD NMR lately are in fact the minority. Interestingly, over the past decade, STD NMR studies of GAGs have been primarily focused on three major groups of GBPs: coagulation factors, fibroblast growth factors (FGFs), and cytokines/chemokines. A few other reports may exist but can still be generalized as exceptions.^43,44 In all three of these GBP groups, the binding appears to be reliant on variable features within GAGs. These are chain length, specific sulfation pattern and planar 3D arrangement of the sulfate groups, and the monosaccharide composition.

Because the GAG structures are often expressed heterogeneously in nature, a common theme within the STD studies of the three GBPs is the exploitation of synthetically produced GAG-related defined oligosaccharides. The production of structurally defined oligosaccharides in terms of size and sulfation patterns assist the structure-activity relationship to define the proper or best GAG ligand or motif responsible for GBP interaction and regulation. This is usually accomplished through the use of oligosaccharides with lengths of four (tetrasaccharide), six (hexasaccharide), or eight (octasaccharide) units. These ligands enable more robust conclusions regarding the binding contributions of the individual monosaccharides and their sulfation patterns. Identification of the individual units within longer oligosaccharides becomes much more difficult primarily because of the chemical shift overlap (internal monosaccharides with nearly identical physicochemical properties). On the other hand, smaller oligosaccharides are not always the functional epitopes, although key conclusions can be reached using these short fragments.⁴⁵ Below, we highlight the main findings obtained so far by STD NMR studies of GAGs in interactions with the three major GBPs.

Coagulation Factors

Antithrombin

The most commonly exploited medical application of GAGs is the anticoagulant activity that is exhibited by Hp ( Fig. 2A ). This function relies on binding and subsequent activation of antithrombin (AT), leading to downstream effects within the coagulation pathway, ultimately inhibiting factors Xa and IIa. The Hp-AT binding event is primarily mediated through a pentasaccharide sequence, as pictured in Figure 3. This structure was initially described through x-ray crystallography, where a marked conformational change was observed in the 3D structure of AT as well.⁴⁶ This binding event is dependent on the negatively charged sulfate groups present in the pentasaccharide sequence. In particular, the 3-O-sulfation on the middle GlcNS3S6S unit and the 2-O-sulfation of the IdoA residue play crucial roles.

Figure 3.

The antithrombin-binding heparin sequence is composed of the following pentasaccharide structure: [→4)-D-GlcNAc/GlcNS6S-α-(1→4)-D-GlcA-β-(1→4)-D-GlcNS3S6S-α-(1→4)-L-IdoA2S-α-(1→4)-D-GlcNS6S-α-(1→].

Stancanelli et al.⁴⁷ sought to further elucidate the importance of the pentasaccharide motif ( Fig. 3 ). To this end, the investigators used two different synthetic Hp hexasaccharides containing the pentasaccharide sequence. A modification was made introducing the addition of a GlcA on the reducing end of the oligosaccharide.⁴⁷ The two hexasaccharide structures differ only in the sulfation present on the IdoA. One contains the sulfation on the second position of IdoA, whereas the other lacks sulfation on this position. To address how these changes to the core pentasaccharide affect the binding interactions with AT, STD experiments were performed on both hexasaccharides. Predictably, it was observed that the additional reducing end GlcA added to the pentasaccharide sequence lacks a strong interaction with AT. This is observed through the lack of intensity of the resulting STD signal. Comparing the STD spectra of both GAG ligands, it was observed that the hexasaccharide residues necessary for the AT interaction are very similar between the two compounds. This indicates that the presence of 2-O-sulfation on IdoA is not a crucial feature to the binding mechanism of the pentasaccharide to AT. Figure 4 illustrates these findings, showing the interacting intensities of both Hp hexasaccharides.

Figure 4.

The hexasaccharides used in saturation transfer difference (STD) studies in the interaction with antithrombin (AT) are pictured. Both oligomers contain the AT-binding heparin pentasaccharide but with slight variation on the sulfation pattern as well as an addition of the glucuronic acid present at the reducing end. The hexasaccharide at (A) contains the 2-sulfation at the iduronic acid (IdoA) residue, whereas the one at (B) is not sulfated at this position of the IdoA motif (highlighted in the red box). Gray markers correspond to STD signal intensities and are displayed relative to the maximum intensity observed for H of IdoA after selective magnetization of AT. Adapted with permission from ref. 47.

In further work, Guerrini et al.⁴⁸ identified an increase in binding magnitude with the addition of sulfation on the 3-O-position of the GlcNS3S6S unit in the pentasaccharide sequence.⁴⁸ This work has validated the contribution of the rare and functional 3-O-sulfation site of the pentasaccharide for the proper interaction with AT. Viskov et al.⁴⁹ performed STD of a decasaccharide consisting of two contiguous pentasaccharide moieties as described earlier. The STD spectrum has revealed signals across the entire decasaccharide. However, the most intense signals were observed in the nonreducing end pentasaccharide sequence, suggesting some preference for one specific side of the decasaccharide.⁴⁹

FGFs

FGF2 and FGF10

FGFs are another family of GBPs that has been widely investigated using STD NMR technology. FGFs are key signaling proteins involved in multiple biological processes. Examples of these events are regeneration of both hard and soft tissues across multiple organ systems.^50–52 These regulatory functions of FGFs have been exploited for therapeutic applications toward certain diseases such as Parkinson’s disease and diabetes.⁵³ The 23 currently known FGFs exhibit a conserved amino acid sequence.⁵³ A few of them display the lack of binding to GAGs, particularly to Hp and CS.⁵⁴ Because this relationship is so extensive, STD is a powerful tool to elucidate whether the structure-activity relationship between GAGs and this GBP family is a conserved mechanism or not.

The work of Yu et al.¹⁶ provided excellent information about the FGF interactions. In their report, the authors compared two members of the FGF family, FGF2 and FGF10, in intermolecular complexes with a small library of three synthetic Hp octasaccharides. These Hp octasaccharides differ solely on sulfation patterns.¹⁶ Regarding the Hp oligosaccharide structures, it is crucial to point out that each disaccharide unit is disulfated. However, only octasaccharide 2 contains a disulfated monosaccharide unit, GlcNS6S. Both octasaccharides 1 and 3 are sulfated on the C2 position of IdoA but with distinct sulfations at the hexosamine. Octasaccharide 1 bears the GlcNAc6S unit, whereas octasaccharide 3 bears the GlcNS unit.¹⁶

FGF2 and FGF10 show markedly different patterns in the STD approach. In FGF2 interactions, all three octasaccharides display a transfer of saturation, indicating binding between the protein and ligands. However, only octasaccharide 3 displayed discrete intensities that are indicative of protons crucial to a specific binding interface. To further highlight the specificity of interaction with octasaccharide 3 as compared with the other two Hp oligosaccharides, all three fragments were immersed in the same solution for the STD study. A similar STD on-resonance spectrum of octasaccharide 3 alone was observed. This suggests selectivity of octasaccharide 3 for interaction with FGF2 as compared with the other two octasaccharides. This experiment has proved a preferential affinity toward the Hp fragment containing the GlcNS unit. When viewing the proton STD signals involved in this specific event, it is apparent that only the innermost disaccharide units within the oligosaccharide are involved in the binding. This is supported by the expected lack of signals from both reducing and nonreducing ends of the oligosaccharide chain. The STD results indicate that the Hp ligand binds to the FGF2 preferably by its internal middle region. Taking all data together,⁵⁴ it becomes apparent that the GAG binding to FGF2 not only requires a specific spatial arrangement of sulfation present but is also selective toward a residue during the binding ( Fig. 5 ).

Figure 5.

Nuclear magnetic resonance assignments of the saturation transfer difference (STD) spectrum obtained from the complex of a heparin (Hp) octasaccharide and fibroblast growth factor 2 (FGF2). (A) Reference spectrum recorded in the presence of FGF2 but without saturation on the protein with magnetization. (B) Structure of the Hp octasaccharide used in the analysis. This ligand is composed of alternating IdoA2S and GlcNS units. The internal tetrasaccharide highlighted by the shadowed box indicates the binding motif recognized by the STD experiment. (C) The gray dots on the structural units indicate the signals assigned in the STD spectrum. Adapted with permission from ref. 16.

Conclusions yielded from STD NMR studies of FGF10 and the same Hp oligosaccharides proved a much more promiscuous binding event in comparison with the FGF2 study. Of the three used Hp fragments, both octasaccharides 2 and 3 displayed preferential NMR signals alone and of similar intensities when co-incubated in solution. Octasaccharide 2 displayed signals from both monosaccharide units; however, STD signals were mostly pronounced from the GlcNS6S units. This was expected because of the presence of sulfation at only this unit. The resulting STD spectra of octasaccharide 3 with FGF10 displayed similar signals observed in the STD study with FGF2. In both Hp octasaccharides, the signals observed are localized to the innermost residues of their chains. Negligible intensity of reducing and nonreducing ends was seen. This set of results has pointed again toward the fact that GAG oligosaccharides prefer to interact with the two forms of FGFs through the middle of their oligosaccharide chain.¹⁶

Cytokines and Chemokines

Another application of STD in studies of GAGs is to understand their regulatory mechanisms of action over chemokines and cytokines. Regulation of these signaling proteins, mediated by GAG interactions, is crucial for the prevention and mediation of serious autoimmune disorders and inflammatory issues. In comparison with coagulation and fibroblast growth factors, STD-derived information related to chemokines and cytokines has been much more heavily explored lately. This is likely because of the functional and structural diversity of these GBPs themselves. This structural diversity has assisted the information generation about the GAG-binding aspects of these proteins. Motifs found in GAG oligosaccharides are required to form the functional GAG-chemokine/cytokine complexes, and thus, STD NMR studies are key to reveal these findings. The GAG binding is a requirement step for the functions of chemokines/cytokines. Upon interaction, GAG will induce chemokine/cytokine oligomerization. The oligomerized chemokines/cytokines are consequently activated and hold to the surface of vessels in a much tighter fashion so that they can exert their signaling roles to the migration of cells.

CXCL12α

Laguri et al.⁵⁵ used a synthetic ¹³C-labeled HS ( Fig. 2A ) octasaccharide with sulfation present on the C6 and N-positions of the hexosamine (GlcNS6S) in binding studies with the GBP stromal cell-derived factor-1 alpha (CXCL12α). CXCL12α is a splice variant of CXCL12, a chemokine with involvement across multiple processes. Example of these processes are embryogenesis, angiogenesis, and inflammation. CXCL12α is expressed in a diverse set of cell types.⁵⁶ The importance of GAG binding to the function of this protein function has been previously described in which GAG binding is responsible to sequestrate and hold these proteins at the vessel surface for proper action.⁵⁷ This functional relationship has been further explored for therapeutic purposes. Examples of these applications are the inhibition of ECM GAGs required for cell migration during the development of the hepatocellular carcinoma.⁵⁸

STD NMR studies of the CXCL12α-HS oligosaccharide complex provided interesting conclusions regarding the GAG moieties involved in binding of CXCL12α. Figure 6 illustrates the major findings from the GAG ligand viewpoint. Basically, during the analysis, the resultant NMR spectra displayed a loss of signals from residues not present in the innermost units likely because of the nature of the binding being again focused on the center of the oligosaccharide chain.⁵⁵ This occurred in a similar fashion to the results obtained by Yu et al.⁵⁴ with FGFs ( Fig. 5 ), where GAGs show their outermost monosaccharides not directly involved in the binding process. In addition to the relevant information observed in proton difference spectra, Laguri et al.⁵⁵ also observed that the signal intensity changes considerably in ¹³C-¹H correlation spectra generated with the synthetic ¹³C isotopically labeled Hp ligand used in the work.⁵⁵

Figure 6.

The heparan sulfate (HS) octasaccharide containing the GlcNS6S motif used in the saturation transfer difference (STD) study in the interaction with CXCL12 is displayed. STD intensities at appropriate protons are shown qualitatively through the gray markers present. The intensities are derived from the STD spectra of the excess HS octasaccharide in interaction with CXCL12α in solution after selective magnetization of the chemokine. Adapted with permission from ref. 55, 2011 American Chemical Society.

Midkine

Using CS oligosaccharides, Solera et al.⁵⁹ applied STD to investigate their binding with the cytokine Midkine (MK).⁵⁹ MK is a GBP that has great binding affinity to Hp ( Fig. 2A ) and CS ( Fig. 2B ).⁶⁰ MK is expressed primarily during inflammation, where it functions to drive the migratory pathways of inflammatory cells.⁶¹ This expression is tightly controlled because this cytokine is associated with multiple disease states in which inhibition can lead to autoimmune disorders.⁶² Overexpression of MK has a relationship with tumorigenesis.⁶³ Hence, it is important to uncover the molecular details where GAGs bind to MK so that potential interventions can be proposed to control these malignant events.

In their STD studies, Solera et al.⁵⁹ employed two synthetic CS tetrasaccharides ( Fig. 2B ) differing in sulfation pattern and backbone. These CS show the following structures: GalNAc4S6S-GlcA-GalNAc4S6S-GlcA ( Fig. 7A ) and IdoA2S4S-GalNAc4S6S-IdoA2S-GalNAc4S6S ( Fig. 7B ). As seen, they differ in sulfation patterns present on the uronic acid units. The presence of GlcA has been exchanged by IdoA in the second structure on the nonreducing end. Consequently, the order of the monosaccharides within the tetrasaccharide units has changed. In the interaction with MK, the authors observed that in both resultant complexes, magnetization transfer occurs across the entire oligosaccharides. However, the highest intensity occurs in the reducing ends of both CS tetrasaccharides, with some bindings also occurring on the internal region of the second CS ligand.⁵⁹ The results of this investigation are depicted in Figure 7 .

Figure 7.

Structures of chondroitin sulfate (CS) tetrasaccharides used in the interaction with midkine. The CS tetrasaccharide at (A) contains a GalNAc4S6S unit, whereas the one at (B) contains an IdoA2S4S as opposed to the glucuronic acid (GlcA) on the nonreducing end but with GalNac4S6S residues and GlcA2S also present. Gray markers indicate saturation transfer difference–derived intensities at specific protons. Adapted with permission from ref. 59.

From Figure 7 , significant STD effects are noted on both planes of the CS tetrasaccharides. This apparent double interaction is due to the binding of both domains within MK, which displays a hinge action required for proper function.⁶⁴ Regarding the structural GAG binding mechanism of MK, two independent domains fold and interact with one GAG, as seen through NMR studies of MK in interactions with Hp⁶⁵ and through simulations of molecular dynamics.⁵⁹ As opposed to the previous results from the STD analysis of GAGs in the interactions with FGFs¹⁶ and the CXCL12α,⁵⁵ for which the binding predominates in the middle of the GAG oligosaccharide chain, the binding of GAG oligosaccharides with MK prefers the reducing ends.⁵⁹ This comparative analysis exemplifies the specificity of interactions regarding the GBPs and GAGs.

CCL5

CCL5, colloquially known as regulated on the activation of normal T-cell expressed and secreted (RANTES), is, like most chemokines, actively involved in cell migration during the events of inflammation. CCL5 presents binding properties with GPCRs and ECM GAGs, where the interactions with GAGs are crucial for its biological activity in vivo.⁶⁶ Regulation and expression of CCL5 have become a topic of discussion recently because of its functions in multiple disease states such as liver fibrosis,⁶⁷ atherosclerosis,⁶⁸ and progression of pancreatic cancer.⁶⁹ Disruption of cellular binding that leads to these issues through GAG competition is currently under exploration as a possible route to mitigate CCL5’s proinflammatory activity. This has been observed in the work of Singh et al.,⁷⁰ in which Hp ( Fig. 2A ) tetrasaccharides bind to CCL5 on the CCR1 (CCL5 receptor) binding site. This binding results in an inhibitory effect by the Hp tetrasaccharide. This process was reported to be dependent on sulfation pattern and pH.⁷⁰

In the work of Deshauer et al.,⁷¹ two CS hexasaccharides extracted from natural sources and with different sulfation patterns at the C4 and C6 positions ( Fig. 2B ) were studied in interactions with CCL5. Using a fully 4-sulfated CS hexasaccharide, the STD method has proved to be a powerful tool to determine which protons from both GlcA and GalNAc units, and not only mostly from the sulfate unit, participate in the binding interface with CCL5. In addition to the standard STD experiment in which the GBP is first magnetized, the inverse of the STD experiment was performed in which the ligand itself is selectively set up to be magnetized in the experiment. Through this case of inverse STD, isotopically labeled CCL5 in molar excess concentration in solution was investigated through the ¹H-¹⁵N HSQC spectrum (2D HSQC STD NMR). The amino acids mapped were in concordance with the NMR observations based on CSP used to diagnose the GAG binding site on CCL5 in the same work. The technology of inverse STD and CSP can be applied to any GBP in interaction studies with any GAG in which amino acid residues of the GBP have already been properly assigned.

Interleukin-10

Interleukin-10 (IL10) is a tightly regulated pleiotropic cytokine that functions in both pro- and anti-inflammatory processes. IL10 facilitates the migration of cells, where its primary function is a downstream effector in response to pathogens.⁷² Multiple disease states are associated with IL10 when this cytokine is dysregulated. Among these diseases are autoimmune disorders such as inflammatory bowel disease,⁷³ viral pathogenesis,⁷⁴ and tumorigenesis.⁷⁵ As for all GBPs, it is important to distinguish how GAGs can play a role in the binding of IL10. Künze et al.¹⁸ employed a suite of various GAGs in binding studies with IL10, including CS, DS, HA, and Hp ( Fig. 2 ).

Künze et al.¹⁸ successfully carried out standard 1D STD and pseudo-2D STD experiments of their GAG ligands in complex with IL10, providing particularly robust conclusions about the binding interface. The 1D STD experiments indicated that most of the protons necessary for interaction are nearby sulfate groups. This conclusion is supported by the removal of sulfate groups and a consequent higher dissociation constant demonstrated by the reduction of the STD-related signal intensity when a comparative approach is made between a trisulfated CS disaccharide versus two different disulfated CS disaccharides. The CS disulfated disaccharides showed comparable signal intensity to each other but were markedly less intense than the trisulfated counterpart. The use of the HSQC experiment in this case has allowed more concise conclusions about the binding protons, especially when there is significant signal overlap in the spectrum.¹⁸

As previously mentioned, GAGs are biologically synthesized as long-chain polysaccharides. Hence, it is crucial to understand how chain length can affect the strength of the consequent interactions with GBPs. Interestingly, it has been observed that binding affinity is directly affected by the length of the GAG chain.^33,76 To explore this concept, Künze et al.¹⁹ capitalized the usage of various Hp ( Fig. 2A ) oligosaccharides with multiple chain lengths. These Hp oligosaccharides vary within degrees of polymerization 2 (dp 2, or disaccharide) to dp10 (decasaccharide).¹⁹ The authors found that there is clear evidence of positive cooperativity through STD analysis, which can be increased with the length of the GAG oligosaccharide at lower concentrations.

Leukocyte Common Antigen-Related

Leukocyte common antigen-related (LAR) is a receptor-like protein-tyrosine-phosphatase involved in numerous biological processes.⁷⁷ Some of these events are controlled by interactions with ECM GAGs. Binding to CS proteoglycans (CSPGs) is an example in which CSPGs function to inhibit axon growth and elongation.⁷⁸ This is in stark contrast to HS proteoglycans, which invoke an opposite response in which interactions with LAR, axon guidance, and proliferation are promoted.^79,80 Because of this complex and antagonistic relationship between LAR and GAGs, it is important to determine the structural differences that mediate binding and differentiation of downstream effects.

Using the known binding epitope of the Hp pentasaccharide sequence of high affinity for AT ( Fig. 3 ), Gao et al.⁸¹ sought to determine the interaction of this epitope with LAR associated with the two immunoglobulin domains (Ig12). Because of issues arising from lower signal intensities as well as signal interference from LAR-Ig12, a 30:1 ratio of Hp pentasaccharide:LAR-Ig12 was made, and the complex was submitted to STDD experiments. By effectively removing the residual signals coming from the GBP, the investigators of this work were able to generate clean information covering only protons from the GAG ligand. The results of STDD from this reference have displayed a trend of signals arising from the nonreducing end of the pentasaccharide with no clear indication of transfer happening to the reducing end of the ligand. Hence, the binding preference has been demonstrated to be driven by a conjunction of balanced sulfation pattern/content and sugar composition within the GAG oligosaccharide. This conclusion comes from the fact that the nonreducing end of the Hp pentasaccharide is less sulfated than the reducing-end region containing the 2-O-sulfated IdoA unit ( Fig. 3 ). The preferential binding of the Hp pentasaccharide with LAR-Ig1-2 at the nonreducing end as opposed to the middle of the GAG chain in interactions with FGFs¹⁶ and with CXCL12α⁵⁵ highlights again the diversity and specificity of the GAG-GBP interactions.

Closing Remarks

STD is a very powerful NMR technique used to investigate at the atomic level the contacts of a ligand, such as GAG oligosaccharides, in interactions with proteins. Through the experimental results revisited here, the potential of STD has been proven very useful to distinguish between ligands in a mixture of GAG oligosaccharides and to understand the minimal structural features, such as sulfation patterns/contents, monosaccharide composition, chain length, and local binding preferences. All of these features are necessary and work altogether for proper complex formation with the GBPs. Among all structural biology–related studies that are recently being generated in this current glycosaminoglycanomics era, STD seems to play a crucial role in deciphering among the best binding moieties of GAG ligands required for the proper interaction with GBPs. Interestingly, the three major GBP groups mostly studied so far by STD are the coagulation factors, FGFs, and chemokines/cytokines. The STD NMR studies using these GBPs have shown that a diverse and distinguished set of GAG structures is required for adequate interactions. This observation strongly supports the fact that GAG-GBP complexes are highly specific. Understanding the structures of GAG epitopes necessary for GBP interactions is a requirement in drug discovery and development in which both biological macromolecules (GAGs and GBPs) must act cooperatively.

Footnotes

Acknowledgements

We are grateful to Christine Diedrich (SLAS Technology editorial manager) and Joe Olechno (SLAS Technology associate editor) as well as Drs. Parastoo Azadi and Christian Heiss (guest editors of the special issue “Carbohydrate Structure Analysis: Methods and Applications” in SLAS Technology) for their kind invitation to have one article of our group in their special issue.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received financial support for the research, authorship, and/or publication of this article: Financial support is received from the University of Mississippi and the American Association of Colleges of Pharmacy 2019 New Investigator Award. Copyrights and licenses were properly obtained for all figures reused or modified from the literature.

References

Ishii

Noda

Uchiyama

Mass Spectrometric Analysis of Protein–Ligand Interactions. Biophys. Physicobiol. 2016, 13, 87–95.

Frostell

Vinterbäck

Sjöbom

Protein–Ligand Interactions Using SPR Systems. In: Williams

Daviter

, Eds.; Protein-Ligand Interactions: Methods and Applications; Humana Press: Totowa, NJ, 2013, p 139–165.

Vegas

A. J.

Fuller

J. H.

Koehler

A. N.

Small-Molecule Microarrays as Tools in Ligand Discovery. Chem. Soc. Rev. 2018, 37, 1385–1394.

Cala

Guillière

Krimm

NMR-Based Analysis of Protein–Ligand Interactions. Anal. Bioanal. Chem. 2014, 406, 943–956.

Pomin

V. H.

Wang

Glycosaminoglycan-Protein Interactions by Nuclear Magnetic Resonance (NMR) Spectroscopy. Molecules 2018, 23, 2314

Pomin

V. H.

Biological Findings from the Recent NMR-Based Studies of Glycosaminoglycan–Protein Interactions. Glycobiology 2014, 24, 991–1003.

Mayer

Meyer

Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy. Angew. Chem. Int. Ed. 1999, 38, 1784–1788.

Viegas

Manso

Nobrega

F. L.

; et al. Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding. J. Chem. Educ. 2011, 88, 990–994.

Bergeron

S. J.

Henry

I. D.

Santini

R. E.

; et al. Saturation Transfer Double-Difference NMR Spectroscopy Using a Dual Solenoid Microcoil Difference Probe. Magn Reson Chem. 2008, 46, 925–929.

10.

Shirzadi

Simpson

M. J.

; et al. Application of Saturation Transfer Double Difference NMR to Elucidate the Mechanistic Interactions of Pesticides with Humic Acid. Environ. Sci. Technol. 2008, 42, 1084–1090.

11.

Megy

Bertho

Gharbi-Benarous

; et al. STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein β-Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the β-TrCP Protein. J. Biol. Chem. 2005, 280, 29107–29116.

12.

Bhunia

Bhattacharjya

Chatterjee

Applications of Saturation Transfer Difference NMR in Biological Systems. Drug Discov. Today. 2012, 17, 505–513.

13.

Aretz

Rademacher

Ranking Hits from Saturation Transfer Difference Nuclear Magnetic Resonance–Based Fragment Screening. Front. Chem. 2019, 7, 215.

14.

Angulo

Nieto

P. M.

STD-NMR: Application to Transient Interactions between Biomolecules—A Quantitative Approach. Eur. Biophys J. 2011, 40, 1357–1369.

15.

Teilum

Kunze

M. B. A.

Erlendsson

; et al. (S)Pinning Down Protein Interactions by NMR. Protein Sci. 2017, 26, 436–451.

16.

Roy

Arevalo

; et al. Characterization of Heparin–Protein Interaction by Saturation Transfer Difference (STD) NMR. Anal. Bioanal. Chem. 2014, 406, 3079–3089.

17.

Groves

Kövér

K. E.

André

; et al. Temperature Dependence of Ligand–Protein Complex Formation as Reflected by Saturation Transfer Difference NMR Experiments. Magn. Reson. Chem. 2007, 45, 745–748.

18.

Künze

Köhling

Vogel

; et al. Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy. J. Biol. Chem. 2016, 291, 3100–3113.

19.

Künze

Gehrcke

J.-P.

Pisabarro

M. T.

; et al. NMR Characterization of the Binding Properties and Conformation of Glycosaminoglycans Interacting with Interleukin-10. Glycobiology. 2014, 24, 1036–1049.

20.

Harris

K. A.

Shekhtman

Agris

P. F.

Specific RNA-Protein Interactions Detected with Saturation Transfer Difference NMR. RNA Biol. 2013, 10, 1307–1311.

21.

Wagstaff

J. L.

Vallath

Marshall

J. F.

; et al. Two-Dimensional Heteronuclear Saturation Transfer Difference NMR Reveals Detailed Integrin αvβ6 Protein–Peptide Interactions. Chem. Commun. 2010, 46, 7533–7535.

22.

Mayer

Meyer

Group Epitope Mapping by Saturation Transfer Difference NMR to Identify Segments of a Ligand in Direct Contact with a Protein Receptor. J. Am. Chem. Soc. 2001, 123, 6108–6117.

23.

Airoldi

Giovannardi

La Ferla

; et al. Saturation Transfer Difference NMR Experiments of Membrane Proteins in Living Cells under HR-MAS Conditions: The Interaction of the SGLT1 Co-Transporter with Its Ligands. Chem. Eur. J. 2011, 17, 13395–13399.

24.

Rademacher

Krishna

N. R.

Palcic

; et al. NMR Experiments Reveal the Molecular Basis of Receptor Recognition by a Calicivirus. J. Am. Chem. Soc. 2008, 130, 3669–3675.

25.

Pereira

Pfeifer

T. A.

Grigliatti

T. A.

; et al. Functional Cell-Based Screening and Saturation Transfer Double-Difference NMR Have Identified Haplosamate A as a Cannabinoid Receptor Agonist. Chem. Biol. 2009, 4, 139–144.

26.

Pomin

V. H.

Mulloy

Glycosaminoglycans and Proteoglycans. Pharmaceuticals (Basel) 2018, 11, 27.

27.

Almond

Hyaluronan. Cell. Mol. Life Sci. 2007, 64, 1591–1596.

28.

Yamada

Sugahara

Ozbek

Evolution of Glycosaminoglycans: Comparative Biochemical Study. Commun. Integr. Biol. 2011, 4, 150–158.

29.

Pomin

V. H.

A Dilemma in the Glycosaminoglycan-Based Therapy: Synthetic or Naturally Unique Molecules?

Med. Res. Rev. 2015, 35, 1195–1219.

30.

Hileman

R. E.

Fromm

J. R.

Weiler

J. M.

; et al. Glycosaminoglycan-Protein Interactions: Definition of Consensus Sites in Glycosaminoglycan Binding Proteins. BioEssays 1998, 20, 156–167.

31.

Imberty

Lortat-Jacob

Pérez

Structural View of Glycosaminoglycan–Protein Interactions. Carb. Res. 2007, 342, 430–439.

32.

Spillmann

Lindahl

Glycosaminoglycan-Protein Interactions: A Question of Specificity. Curr. Opin. Struct. Biol. 1994, 4, 677–682.

33.

Gandhi

N. S.

Mancera

R. L.

The Structure of Glycosaminoglycans and Their Interactions with Proteins. Chem. Biol. Drug Design. 2008, 72, 455–482.

34.

Kaiser

Harenberg

Hoppensteadt

; et al. Effects of a Heparin-Binding Protein on Blood Coagulation and Platelet Function. Semin. Thromb. Hemost. 2001, 27, 495–502.

35.

Tollefsen

D. M.

Pestka

C. A.

Monafo

W. J.

Activation of Heparin Cofactor II by Dermatan Sulfate. J. Biol. Chem., 1983, 258, 6713–6716.

36.

Pomin

V. H.

Sulfated Glycans in Inflammation. Eur. J. Med. Chem. 2015, 92, 353–369.

37.

Yip

G. W.

Smollich

Götte

Therapeutic Value of Glycosaminoglycans in Cancer. Mol. Cancer Therap. 2006, 5, 2139–2148.

38.

Ghatak

Maytin

E. V.

Mack

J. A.

; et al. Roles of Proteoglycans and Glycosaminoglycans in Wound Healing and Fibrosis. Int. J. Cell. Biol. 2015, 2015, 834893–834893.

39.

Hayes

A. J.

Melrose

Glycans and Glycosaminoglycans in Neurobiology: Key Regulators of Neuronal Cell Function and Fate. Biochem. J. 2018, 475, 2511–2545.

40.

Aquino

R. S.

Park

P. W.

Glycosaminoglycans and Infection. Front. Biosci. 2016, 21, 1260–1277.

41.

Jinno

Park

P. W.

Role of Glycosaminoglycans in Infectious Disease. Methods Mol. Biol. 2015, 1229, 567–585.

42.

Esko

Linhardt

R. J.

Proteins That Bind Sulfated Glycosaminoglycans. In: Varki

Cummings

Esko

; et al., Eds.; Essentials of Glycobiology, 3rd ed.; Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY, 2017.

43.

Singh

Gitti

R. K.

Diouf

; et al. Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x is Identified as a Minimal Chondroitin Sulfate A-Binding Region. J. Biol. Chem. 2010, 285, 24855–24862.

44.

Muñoz-García

J. C.

Chabrol

Vivès

R. R.

; et al. Langerin-Heparin Interaction: Two Binding Sites for Small and Large Ligands as Revealed by a Combination of NMR Spectroscopy and Cross-Linking Mapping Experiments. J. Am. Chem. Soc. 2015, 137, 4100–4110.

45.

García-Jiménez

M. J.

Gil-Caballero

Canales

Á.

; et al. Interactions between a Heparin Trisaccharide Library and FGF-1 Analyzed by NMR Methods. Int. J. Mol. Sci. 2017, 18, pii: E1293.

46.

Jin

Abrahams

J. P.

Skinner

; et al. The Anticoagulant Activation of Antithrombin by Heparin. Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 14683–14688.

47.

Stancanelli

Elli

Hsieh

P. H.

; et al. Recognition and Conformational Properties of an Alternative Antithrombin Binding Sequence Obtained by Chemoenzymatic Synthesis. ChemBioChem 2018, 19, 1178–1188.

48.

Guerrini

Elli

Mourier

; et al. An Unusual Antithrombin-Binding Heparin Octasaccharide with an Additional 3-O-sulfated Glucosamine in the Active Pentasaccharide Sequence. Biochem. J. 2012, 449, 343–351.

49.

Viskov

Elli

Urso

; et al. Heparin Dodecasaccharide Containing Two Antithrombin-Binding Pentasaccharides: Structural Features and Biological Properties. J. Biol. Chem. 2013, 288, 25895–25907.

50.

Hankemeier

Keus

Zeichen

Modulation of Proliferation and Differentiation of Human Bone Marrow Stromal Cells by Fibroblast Growth Factor 2: Potential Implications for Tissue Engineering of Tendons and Ligaments. Tissue Eng. 2005, 11, 41–49.

51.

Delgado-Rivera

Harris

S. L.

Ahmed

; et al. Increased FGF-2 Secretion and Ability to Support Neurite Outgrowth by Astrocytes Cultured on Polyamide Nanofibrillar Matrices. Matrix Biol. 2009, 28, 137–147.

52.

Rophael

J. A.

Craft

R. O.

Palmer

J. A.

Angiogenic Growth Factor Synergism in a Murine Tissue Engineering Model of Angiogenesis and Adipogenesis. Am. J. Pathol. 2007, 171, 2048–2057.

53.

Yun

Y.-R.

Won

J. E.

Jeon

; et al. Fibroblast Growth Factors: Biology, Function, and Application for Tissue Regeneration. J. Tissue Eng. 2010, 2010, 218142–218142.

54.

Asada

Shinomiya

Suzuki

; et al. Glycosaminoglycan Affinity of the Complete Fibroblast Growth Factor Family. Biochim. Biophys. Acta. Gen. Sub. 2009, 1790, 40–48.

55.

Laguri

Sapay

Simorre

J.-P.

; et al. 13C-Labeled Heparan Sulfate Analogue as a Tool To Study Protein/Heparan Sulfate Interactions by NMR Spectroscopy: Application to the CXCL12α Chemokine. J. Am. Chem. Soc. 2011, 133, 9642–9645.

56.

Janssens

Struyf

Proost

The Unique Structural and Functional Features of CXCL12. Cell. Mol. Immunol. 2018, 15, 299–311.

57.

Murphy

J. W.

Cho

Sachpatzidis

Structural and Functional Basis of CXCL12 (Stromal Cell-Derived Factor-1 Alpha) Binding to Heparin. J. Biol. Chem. 2007, 282, 10018–10027.

58.

Friand

Haddad

Papy-Garcia

; et al. Glycosaminoglycan Mimetics Inhibit SDF-1/CXCL12-Mediated Migration and Invasion of Human Hepatoma Cells. Glycobiology. 2009, 19, 1511–1524.

59.

Solera

Macchione

Maza

; et al. Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine. Chem. Eur. J. 2016, 22, 2356–2369.

60.

Zou

Muramatsu

Ikematsu

; et al. A Heparin-Binding Growth Factor, Midkine, Binds to a Chondroitin Sulfate Proteoglycan, PG-M/Versican. Eur. J. Biochem. 2000, 267, 4046–4053.

61.

Kadomatsu

Kishida

Tsubota

The Heparin-Binding Growth Factor Midkine: The Biological Activities and Candidate Receptors. J. Biochem. 2013, 153, 511–521.

62.

Wang

Takeuchi

Sonobe

; et al. Inhibition of Midkine Alleviates Experimental Autoimmune Encephalomyelitis Through the Expansion of Regulatory T Cell Population. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3915–3920.

63.

Kadomatsu

Muramatsu

Midkine and Pleiotrophin in Neural Development and Cancer. Cancer Lett. 2004, 204, 127–143.

64.

Lim

Yao

Graf

; et al. Structure–Function Analysis of Full-Length Midkine Reveals Novel Residues Important for Heparin Binding and Zebrafish Embryogenesis. Biochem. J. 2013, 451, 407–415.

65.

Iwasaki

Nagata

Hatanaka

; et al. Solution Structure of Midkine, a New Heparin-Binding Growth Factor. EMBO J. 1997, 16, 6936–6946.

66.

Proudfoot

A. E. I.

Handel

T. M.

Johnson

; et al. Glycosaminoglycan Binding and Oligomerization Are Essential for the In Vivo Activity of Certain Chemokines. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1885–1890.

67.

Seki

De Minicis

Gwak

G.-Y.

; et al. CCR1 and CCR5 Promote Hepatic Fibrosis in Mice. J. Clin. Invest. 2009, 119, 1858–1870.

68.

Koenen

R. R.

von Hundelshausen

Nesmelova

I. V.

; et al. Disrupting Functional Interactions between Platelet Chemokines Inhibits Atherosclerosis in Hyperlipidemic Mice. Nat. Med. 2009, 15, 97–103.

69.

Singh

S. K.

Mishra

M. K.

Eltoum

I.-E.A.

; et al. CCR5/CCL5 Axis Interaction Promotes Migratory and Invasiveness of Pancreatic Cancer Cells. Sci. Rep. 2018, 8, 1323–1323.

70.

Singh

Kett

W. C.

Severin

I. C.

; et al.The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH. J. Biol. Chem. 2015, 290, 15421–15436.

71.

Deshauer

Morgan

A. M.

Ryan

E. O.

; et al. Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands. Structure 2015, 23, 1066–1077.

72.

Couper

K. N.

Blount

D. G.

Riley

E. M.

IL-10: The Master Regulator of Immunity to Infection. J. Immunol. 2008, 180, 5771–5777.

73.

Schmulson

Pulido-London

Rodriguez

Lower Serum IL-10 Is an Independent Predictor of IBS among Volunteers in Mexico. Am. J. Gastroenterol. 2012, 107, 747–753.

74.

Yamamoto-Tabata

McDonagh

Chang

H.-T.

; et al. Human Cytomegalovirus Interleukin-10 Downregulates Metalloproteinase Activity and Impairs Endothelial Cell Migration and Placental Cytotrophoblast Invasiveness In Vitro. J. Virol. 2004, 78, 2831–2840.

75.

Lane

Matte

Garde-Granger

; et al. Ascites IL-10 Promotes Ovarian Cancer Cell Migration. Cancer Microenviron. 2018, 11, 115–124.

76.

Stopschinski

B. E.

Holmes

B. B.

Miller

G. M.

; et al. Specific Glycosaminoglycan Chain Length and Sulfation Patterns are Required for Cell Uptake of Tau Versus α-Synuclein and β-Amyloid Aggregates. J. Biol. Chem. 2018, 293, 10826–10840.

77.

Charles

M. L.

Robert

A. M.

The Leukocyte Common Antigen-Related Protein LAR: Candidate PTP for Inhibitory Targeting. Curr. Top. Med. Chem. 2003, 3, 809–819.

78.

Fisher

Xing

Dill

; et al. Leukocyte Common Antigen-Related Phosphatase Is a Functional Receptor for Chondroitin Sulfate Proteoglycan Axon Growth Inhibitors. J. Neurosci. 2011, 31, 14051–14066.

79.

Fox

A. N.

Zinn

The Heparan Sulfate Proteoglycan Syndecan Is an In Vivo Ligand for the Drosophila LAR Receptor Tyrosine Phosphatase. Curr. Biol. 2005, 15, 1701–1711.

80.

Poe

A. R.

Tang

Wang

; et al. Dendritic Space-Filling Requires a Neuronal Type-Specific Extracellular Permissive Signal in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, E8062–E8071.

81.

Gao

Yang

J.-Y.

Moremen

K. W.

; et al. Structural Characterization of a Heparan Sulfate Pentamer Interacting with LAR-Ig1-2. Biochem. 2018, 57, 2189–2199.

Saturation Transfer Difference in Characterization of Glycosaminoglycan-Protein Interactions

Abstract

Keywords

Introduction

STD

GAGs and GAG-Binding Proteins

Coagulation Factors

Antithrombin

FGFs

FGF2 and FGF10

Cytokines and Chemokines

CXCL12α

Midkine

CCL5

Interleukin-10

Leukocyte Common Antigen-Related

Closing Remarks

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References