Multitasking Human Lectin Galectin-3 Interacts with Sulfated

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The multitasking human lectin galectin-3 interacts with sulfated glycosaminoglycans and chondroitin sulfate-proteoglycans Melanie L Talaga, Ni Fan, Ashli L Fueri, Robert K Brown, Purnima Bandyopadhyay, and Tarun K. Dam Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00504 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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Biochemistry

The multitasking human lectin galectin-3 interacts with sulfated glycosaminoglycans and chondroitin sulfate-proteoglycans**

Melanie L Talaga1, Ni Fan1, Ashli L Fueri1, Robert K Brown1, Purnima Bandyopadhyay2, Tarun K Dam1,3,*

1

From the Laboratory of Mechanistic Glycobiology, Department of Chemistry, 2Department of

Biological Sciences, 3Life Science and Technology Institute, Michigan Technological University, Houghton, MI 49931

**Running title: Interaction of Galectin-3 with glycosaminoglycans

*To whom correspondence should be addressed: Dr. Tarun K. Dam, Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931,Telephone: (906) 487-2940; FAX: (906) 487-2061; [email protected]

**This work was supported by start-up fund (TKD, PB) and by Research Excellence Fund (TKD) provided by Michigan Technological University.

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Abbreviations and Textual Footnotes The abbreviations used are: CSA, chondroitin sulfate A; CSB, chondroitin sulfate B; CSC, chondroitin sulfate C; CSPG, chondroitin sulfate proteoglycan; ITC, isothermal titration calorimetry; BPG, bovine proteoglycan.


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Abstract Glycosaminoglycan (GAG)-binding proteins (GAGBPs) including growth factors, cytokines, morphogens and extracellular matrix proteins interact with both free GAGs and with those covalently linked to proteoglycans. Such interactions modulate a variety of cellular and extracellular events such as cell growth, metastasis, morphogenesis, neural development and inflammation. GAGBPs are structurally and evolutionarily unrelated proteins that typically recognize internal sequences of sulfated GAGs. GAGBPs are distinct from the other major group of glycan binding proteins, lectins. The multifunctional human galectin-3 (Gal-3) is a β-galactoside binding lectin that preferentially binds to N-acetyllactosamine moieties on glycoconjugates. Here we demonstrate through microcalorimetric and spectroscopic data that Gal-3 possesses the characteristics of a GAGBP. Gal-3 interacts with unmodified heparin, chondroitin sulfate-A (CSA), -B (CSB) and –C (CSC) as well as chondroitin sulfate proteoglycans (CSPGs). While heparin, CSA and CSC bind with micromolar affinity, the affinity of CSPGs is nanomolar. Significantly, CSA, CSC and a bovine CSPG were engaged in multivalent binding with Gal-3 and formed non-covalent crosslinked complex with the lectin. Binding of sulfated GAGs was completely abolished when Gal-3 was pre-incubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC and the bovine CSPG was reversed by β-lactose. Both observations strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Hill plot analysis of calorimetric data reveals that the binding of CSA, CSC and bovine CSPG to Gal-3 is associated with progressive negative cooperativity effects. Identification of Gal-3 as a GAGBP should help reveal new functions of Gal-3 mediated by GAGs and proteoglycans.


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Glycosaminoglycan (GAG)-binding proteins (GAGBPs) and lectins represent two major but distinct groups of glycan binding proteins. Unlike lectins, GAGBPs rarely share any common structural feature. While lectins typically bind to the terminal sugar residues of N- and O-glycans as well as glycolipids, GAGBPs interact with internal residues of sulfated GAG chains

1, 2

. The binding sites

of GAGBPs, which contain several basic amino acids, engage in moderate to high affinity monovalent interaction with a variety of sulfated GAGs. In contrast, low affinity single-site binding by lectins is enhanced by multivalency. The binding sites of lectins show higher stereospecificity and rarely require a patch of basic amino acids 1, 2.

GAGs including heparin/heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS or CSB) are linear polysaccharides made up of repeating disaccharide units [GlcNAc6Sα1-4GlcA/IdoA2S (for heparin/HS); GlcAβ1-3GalNAc4S (for CSA); IdoAα1-3GalNAc4S (for CSB); GlcAβ13GalNAc6S (for CSC)] and are often sulfated1, 3. CS and HS chains are distributed on the surface of virtually all cells and CS in particular is found throughout most extracellular matrices (ECM)4,5. CS bearing proteoglycans (CSPGs) and HS containing proteoglycans (HSPGs) are found as transmembrane receptors, in secretory granules and as ECM components. Binding of a variety of GAG-binding proteins (GAGBPs) to free GAGs or to the covalently-linked GAGs of proteoglycans is essential for many cellular events development/regeneration

12, 13

1, 2, 5-9

including metastasis

organogenesis14, wound healing

pathogens invasion 19, cell adhesion

20

3, 10

, morphogenesis11, neural

10, 15

, inflammation

16 17, 18

,

and coagulation 21. Growth factors, cytokines, morphogens,

ECM proteins, nuclear proteins and pathogen surface proteins are among the most notable GAGBPs. 4 ACS Paragon Plus Environment

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The multitasking β-galactoside-binding human lectin galectin-3 (Gal-3), through the interactions with its glycoprotein ligands, contributes to many of the biological events mentioned above. In particular, both GAGs and Gal-3 independently play important roles in cancer, inflammation and neural development

3, 22, 23 24 25 26, 27 28 29, 30

. However, direct interaction between GAGs and Gal-3

has not been clearly established by quantitative data.

Gal-3 is a bona fide lectin with no reported characteristics of a GAGBP. It recognizes neutral galactosyl moieties, primarily N-acetyllactosamine (LacNAc) residues, on glycoproteins. Most reported biological functions/interactions of Gal-3 have been shown to be via N- and O-linked glycans of glycoproteins. The integrity of three specific hydroxyl groups (4-OH, 6-OH of Gal and 3-OH of glucose/N-acetylglucosamine) of lactose/N-acetyllactosamine (LacNAc) moiety is crucial to be recognized by the glycan binding site of Gal-3

31, 32

. It is, therefore, a bit counterintuitive to

imagine that Gal-3 could bind to sulfated GAGs and proteoglycans through its glycan binding site.

In this communication, however, we present thermodynamic and spectroscopic data to show that Gal-3 interacts with unmodified sulfated GAGs (heparin, CSA, CSB and CSC) as well as CSPGs at physiological pH and ionic concentration. Interestingly, the present study also demonstrates that binding of Gal-3 to CSA, CSC and a CSPG leads to the formation of non-covalent cross-linked complexes. Such complexes are dissolved by lactose and sulfated GAGs. Our data strongly suggest that sulfated GAGs and CSPGs bind to the lactose/LacNAc binding site of Gal-3. Taken together, the current work reveals that the human lectin Gal-3 is capable of functioning as a lectin as well as a sulfated GAG-binding protein.

To the best of our knowledge, Gal-3 is shown to recognize 5 ACS Paragon Plus Environment

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desulfated chondroitin and dermatan but not sulfated GAGs such as unmodified chondroitin sulfates, dermatan sulfate and heparin

31

. An indirect inhibition assay showed that chemically

modified low-sulfated and ultra low-molecular heparin derivatives inhibited Gal-3 binding to asialofetuin. However, such inhibition was not observed with fully sulfated unfractionated and unmodified heparin 33. Another study reports that Gal-3 interacts with NG2 proteoglycan. However, the interaction occurred through N-linked oligosaccharides, not through the GAG chains of NG2 34. In contrast, the present study shows that human Gal-3 is capable of binding to unmodified and sulfated GAGs and proteoglycans. Isothermal titration calorimetry has been employed to study carbohydrate binding properties of Gal-3. However, none of these studies showed that Gal-3 could interact with sulfated GAGs and proteoglycans35

36

. Thus, our findings significantly extend the

number of biologically relevant binding partners of Gal-3. Experimental Procedures Materials-The plasmid expressing human galectin-3 (Gal-3) was obtained from DNA 2.0 (California, USA). BL21 (DE3) (New England Biolab) was transformed with this plasmid. Recombinant Gal-3 was purified by using a lactose-agarose column (Sigma Chemical Co.). The Cterminal carbohydrate recognition domain (CRD) of human Gal-3 was then produced by treating the intact full length Gal-3 with collagenase (type VII, Sigma). CRD was purified by lactose-agarose column. Subunit Gal-3 concentrations were determined spectrophotometrically at 280 nm using specific extinction coefficient of the protein (A1%280 or E1%1cm). A value of 6.1 (E1%1cm) was used for Gal-3, as determined from ITC experiments described previously

36

. Molecular mass of the Gal-3

subunit was 29 kDa. Reagents, GAGs and proteoglycans were obtained from Sigma Chemical Co, unless mentioned otherwise. The GAGs included the following: heparin from porcine intestinal mucosa (molecular weight (MW) 17 kDa to 19 kDa; average MW 18 kDa); HS from bovine kidney 6 ACS Paragon Plus Environment

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(MW 10 kDa to 14 kDa; average MW 12 kDa); chondroitin sulfate A from bovine trachea (MW 20 kDa to 40 kDa; average MW 30 kDa); chondroitin sulfate B from porcine intestinal mucosa MW 10 kDa to 40 kDa; average MW 25 kDa); chondroitin sulfate C from shark cartilage (MW 50 kDa to 60 kDa; average MW 55 kDa). The average MW of aggrecan (from bovine articular cartilage) was 2500 kDa including the core protein. Bovine proteoglycan (from bovine nasal septum) possessed similar MW. Molar concentrations of GAGs and proteoglycans were calculated based on the average molecular weights of the whole molecules. GAGs were dialyzed before use against 20 mM phosphate buffered saline (PBS) containing 150 mM NaCl and were rechromatographed as needed.

Hemagglutination Inhibition Assay-The assay was performed at room temperature using a 2-fold serial dilution technique and 2% (v/v) rabbit erythrocytes in PBS containing 150 mM NaCl. The minimum concentration of ligands required for complete inhibition of three hemagglutination doses was determined.

Isothermal Titration Microcalorimetry-ITC experiments were performed using a VP-ITC instrument from Microcal,Inc. (Northampton, MA). Injections of 4 µl of a GAG or proteoglycan solution were added from a computer-controlled microsyringe at an interval of 4 min into the solution of Gal-3 (cell volume 1.43 ml) with stirring at 310 rpm. Control experiments were performed by making identical injections of GAGs or proteoglycan solution into ITC cell containing buffer. Titrations were carried out at pH 7.4 using 20 mM PBS buffer. The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with ∆H (binding enthalpy in kilo calories per mole), Ka (association constant), and n (number of binding sites per monomer) as adjustable parameters. The instrument was calibrated using the calibration kit 7 ACS Paragon Plus Environment

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containing ribonuclease A (RNaseA) and cytidine 2’-monophosphate (2’-CMP) supplied by themanufacturer. Thermodynamic parameters were calculated from the Gibbs free energy equation ∆G=∆H - T∆S = RT lnKa, where ∆G, ∆H, and ∆S are the changes in free energy, enthalpy, and entropy of binding, respectively. T is the absolute temperature, and R = 1.98 cal mol-1 K-1. Multivalent ligand-receptor interaction often causes stoichiometry-dependent complex formation and precipitation. Precipitation during an ITC experiment adversely affects the quality of the data. We have shown that unambiguous ITC data of multivalent binding could be obtained by using low concentrations of lectins as well as their ligands and keeping their concentrations off the stoichimetric ratio 37. CSA, CSC and the bovine proteoglycan are multivalent ligands of Gal-3. We followed our previous approach 37 to arrest any possible complex formation during ITC titration. As a result, we obtained unambiguous ITC data (Figure 1, Figure 2 and Table 2).

Quantitative Precipitation Assays-The assays were performed in 20 mM PBS, pH 7.4, containing 0.15 M NaCl. Increasing amounts of GAGs or proteoglycan added to a series of tubes containing equal amount of Gal-3. The precipitation was allowed to occur for 5 to 20 h at room temperature. The optical density of the samples in each tube was measured at 420 nm. 200 mM lactose solution was added to the precipitates to check if the precipitation was glycan mediated. Experiments were repeated three times.

Kinetics of Precipitation-Measured volumes of GAGs or proteoglycan and Gal-3 solution at different ratios were mixed in a 1 ml quartz cuvette, and the time-dependent development of turbidity was measured at 420 nm. The buffer was 20 mM PBS (pH 7.4). All experiments were done at room temperature and repeated at least three times. Absorbance was monitored 8 ACS Paragon Plus Environment

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continuously until it remained constant for 30 min. After each experiment, a portion of the precipitate was treated with 200 mM lactose solution to check if the precipitation was glycan dependent.

Kinetics of dissolution of preformed CSA-Gal-3, CSC-Gal-3 and Gal-3-proteoglycan complexesGal-3 complexes with CSA, CSC and the ptoteoglycan were generated by mixing Gal-3 with the GAGs and proteoglycans at appropriate concentrations. Measured amount of lactose or GAGs solution was added to the preformed complexes in a 1 ml quartz cuvette. The time-dependent dissolution of the complex was measured at 420 nm. All experiments were repeated three times.

Quantitative elucidation of binding cooperativity by Hill plot analysis of ITC data. ITC binding data of CSA, CSC and the bovine proteoglycan were subjected to Hill plot analysis in order to understand if binding was associated with cooperitivity

38 39

. Hill plots were obtained by

plotting log[Y(i)/[1-Y(i)]] versus log[Xf(i)], where Y(i) is [Xb(i)](functional valency of ligand)/Mt(i) [where Xb(i) = concentration of bound ligand after the ith injection; Xf(i) = concentration of free ligand after the ith injection; Mt(i) = total lectin concentration after the ith injection]. These are modified versions of the Hill plots that accommodate multivalent binding by taking into account the functional valency of the ligand38 39. ITC driven functional valencies of CSA, CSC and the bovine proteoglycan for Gal-3 were used in these calculations. ITC raw data file after each experiment directly provides the following: (i) total concentration of ligand Xt(i) as well as lectin Mt(i) after the ith injection and (ii) the heat evolved on the ith injection


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Q(i). The concentration correction is automatically done by the Origin software. The concentration of bound ligand Xb(i) after the ith injection is Xb(i) = [Q(i)/(∆HVo)] + Xb(i - 1) (1) where Q(i) (µcal) is the heat evolved on ith injection, ∆H (cal mol-1) is the enthalpy change, Vo (mL) is the active cell volume, and Xb (mM) is the concentration of bound ligand. Mb (the concentration of bound lectin) is equal to Xb and in the present study of multivalent CSA, CSC and the bovine proteoglycan, the more general expression is Mb = (Xb)(functional valency of ligand). The concentration of free ligand (Xf) after the ith injection was was calculated from the following equation, Xf(i) = Xt(i) - Xb(i) (2) Microsoft Excel was used to generate the Hill plots. The values of total ligand as well as total lectin concentration and the amount of heat evolved after the ith injection were obtained directly from the ITC raw data file. Hill plots were further analyzed by using Microsoft Excel.

Results In order to study the GAG and proteoglycan binding properties of human Gal-3, we initially performed hemagglutination inhibition assays to screen proteoglycans, sulfated GAGs [heparin, HS, CSA, CSC and CSB (dermatan sulfate)] and some of their constituent mono- and disaccharides for their ability to interact with recombinant human Gal-3 (Table 1; Figure 1a). We then investigated the binding thermodynamics by isothermal titration calorimetry (ITC) (Figure 1 and Figure 2). Elucidation of non-covalent cross-linking of Gal-3 by GAGs and proteoglycans was done by quantitative precipitation and by precipitation kinetics studies (Figure 3 and Figure 4).


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Gal-3 binds to heparin at physiological pH and salt concentration- Hemagglutination inhibition data first indicate that Gal-3 binds to heparin, HS, CSs, aggrecan and a bovine proteoglycan (Figure 1a and Table 1). Our initial observation was further confirmed by ITC (Figure 1 and Figure 2). ITC data (Table 2) unequivocally demonstrate that Gal-3 binds to unmodified heparin (Figure 1b,c) with micromolar affinity in isotonic phosphate buffered saline (PBS) (pH 7.4 and 0.15M NaCl). Thermodynamic binding data of Gal-3 with heparin are shown in Table 2. Heparin interacts with Gal-3 with a Kd value of 8.1±0.5 µM (∆G = 7.0±0.03 kcal/mol), which is consistent with the hemagglutination inhibition data (Table 1). The ∆H and T∆S values are -2.3±0.1 kcal/mol and 4.7±0.2 kcal/mol, respectively (Table 2). The n value obtained from ITC (n= ~1.0) (Table 2) suggests that heparin is a monovalent ligand for Gal-3. This was further confirmed by the inability of heparin to precipitate Gal-3 in the precipitation studies described below.

Gal-3 binds to CSA and CSC at physiological pH and salt concentration- While hundreds of proteins are known to interact with heparin, only a few proteins are reported to have the ability to bind to CS with an affinity that is observed with heparin2. Our hemagglutination inhibition and calorimetric data show that Gal-3 binds to CSA and CSC (Figure 1a, Figure 1d and Figure 2a) with higher affinities than that of heparin (Table 1 and 2). The Kd of CSA and CSC, as determined by ITC are 2.7±0.06 µM (∆G = 7.6±0.02 kcal/mol) and 4.7±0.1 µM (∆G = 7.3±0.02), respectively (Table 2). Hemagglutination inhibition-derived minimum inhibitory concentrations of CSA and CSC (Table 1) agree with Kd values obtained with ITC. The ∆H and T∆S values of CSA are 21.7±0.8 kcal/mol and 14.1±0.8 kcal/mol, respectively (Table 2). The ∆H and T∆S values of CSC for Gal-3 are 28.0±1.3 kcal/mol and 20.5±1.3 kcal/mol, respectively (Table 2). Unlike heparin, CSA and CSC are multivalent ligands for Gal-3, as indicated by their fractional n values (0.28 and 0.27, 11 ACS Paragon Plus Environment

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respectively). Quantitative precipitation and precipitation kinetics data (shown below) demonstrate that CSA and CSC are indeed multivalent ligands as they precipitate Gal-3 through non-covalent cross-linking.

The affinity of Gal-3 for dermatan sulfate (CSB) is weaker than that of heparin, CSA and CSC- For some GAGBPs, the affinity of CSB is comparable to or higher than that of heparin 5. However, many GAGBPs show weaker binding affinity for CSB compared to heparin 5. We found through calorimetric (Figure 2b) and inhibition experiments that CSB was a weaker ligand of Gal-3, compared to heparin, CSA and CSC (Table 1 and 2). The minimum inhibitory concentration of CSB is 45 µM (Table 1), which is similar to ITC-derived Kd value (Table 2). CSB is a monovalent ligand (n value is 0.99±0.02) of Gal-3. As a result, CSB was unable to precipitate Gal-3 through non-covalent cross-linking. The ∆H and T∆S values of CSB are -2.7±0.2 kcal/mol and 3.3±0.2 kcal/mol, respectively. Both heparin and CSB are monovalent for Gal-3 and their binding thermodynamics are comparable. Binding entropies (T∆S) of both ligands are more positive compared to those of CSA and CSC (Table 2). It seems that the relatively lower affinity of CSB (compared to CSA and CSC) for Gal-3 is, at least in part, due to the presence of iduronic acid in CSB. Heparin is highly sulfated and it also contains iduronic acid along with glucuronic acid. However, the affinity of heparin for Gal-3 is four times more than that of CSB (Table 2).

Binding of Gal-3 to the constituent disaccharides of CS and selected sulfated monosaccharidesHemagglutination inhibition assays reveal that Gal-3 binds to two of the constituent disaccharides of CS (chondroitin disaccharide ∆di-0S, chondroitin disaccharide ∆di-4S) with low affinity (Table 12 ACS Paragon Plus Environment

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1). Glucuronic acid and some sulfated monosaccharides (N-acetyl-D-glucosamine 6-sulfate, Dgalactosamine-2-sulfate, N-acetyl D-galactosamine-4-sufate, N-acetyl D-galactosamine-6-sufate) were found to be weak ligands of Gal-3. Affinities of most of these sugars were too low to obtain reliable ITC data. The poor affinities of CS disaccharides indicate that they probably make very limited number of non-covalent bonds with the binding site and the length of the disaccharides is short enough to occupy the whole GAG-binding site on Gal-3.

Gal-3 binds to CS-containing proteoglycans with nanomolar affinity- Two CSPGs, namely, aggrecan and a proteoglycan from bovine nasal septum (bovine proteoglycan, BPG) bind to Gal-3 with nanomolar affinity as determined by hemagglutination inhibition studies (Table 1). The GAG chains of both proteoglycans are predominantly CS. The affinity of aggrecan and BPG are 16 nM and 20 nM, respectively (Table 1). ITC studies (Figure 2d) show that the Kd value of BPG for Gal3 is 14 nM (∆G = 10.7±0.03 kcal/mol) (Table 2), which is consistent with the hemagglutination inhibition data. The ∆H and T∆S values of BPG are 712.1±16.2 kcal/mol and 701.4±16.2 kcal/mol, respectively (Table 2). The n value (n = 0.01; valence = 1/n)

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indicated that up to ~100 Gal-3

monomer or ~20 Gal-3 pentamer can bind to a single molecule of BPG.

The GAG-binding site of Gal-3 is located in the carbohydrate recognition domain (CRD) of Gal-3Intact or full length Gal-3 is composed of a large N-terminal domain and a C-terminal carbohydrate recognition domain (CRD). The CRD contains the carbohydrate (lactose/LacNAc) binding site. The truncated version of Gal-3 contains the CRD region. In order to understand whether the CRD of Gal-3 contains the GAG-binding site, we studied the binding thermodynamics of heparin (Figure 1c) and CSC (Figure 2c) to the CRD of Gal-3. The Kd of heparin (9.4±0.5 µM; ∆G = 6.9±0.04 13 ACS Paragon Plus Environment

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kcal/mol) for the CRD is similar to that obtained with the intact Gal-3 (Table 2). This suggests that the binding site of heparin is located in the CRD of Gal-3. The ∆H (-7.3±0.2 kcal/mol) and T∆S (0.4±0.03 kcal/mol) indicate that the binding of heparin to the CRD is entropically less positive than its binding to intact Gal-3 (Table 2). As observed with the intact Gal-3, heparin is also a monovalent ligand of the CRD (n = 1.04) (Table 2). CSC possesses similar Kd (6.0±0.3 µM) and ∆G (7.1±0.05 kcal/mol) values for both intact Gal-3 and its CRD, indicating that the CRD contains the binding site of CSC. The ∆H and T∆S of CSC for CRD is -33.5±1.9 kcal/mol and 26.4±1.8 kcal/mol, respectively. Binding entropy of CSC is less positive for the CRD than what was obtained with full length Gal-3 (Table 2).

GAG binding site of Gal-3 overlaps with its lactose/LacNAc-binding site- We wanted to understand whether GAGs bind to Gal-3 through non-specific electrostatic interactions or these anionic polysaccharides specifically bind to the neutral sugar binding site on Gal-3. Gal-3 is a monomeric lectin but it assembles into multimers in the presence of multivalent ligands

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and thus strongly

agglutinates rabbit erythrocytes. This property allowed us to perform hemagglutination inhibition assays. Hemagglutination inhibition studies (Figure 1a) provided the first indication that GAGs occupy the lactose/LacNAc-binding site of Gal-3. In a hemagglutination inhibition experiment, if the carbohydrate-binding site of the lectin is occupied by a test ligand (GAG in this case), the red blood cells used in that assay, cannot bind to the lectin (Gal-3 in this case) and settle at the bottom of the microtiter plate forming “buttons”. We observed robust inhibition (Figure 1a) with CSPGs and GAGs indicating that the CSPGs and GAGs interact with the lactose/LacNAc-binding site of Gal-3. In hemagglutination assays, it is possible that GAGs could bind to the N-terminal of galectin3 and prevent pentamerization. As a result, agglutination could be disrupted. To address these 14 ACS Paragon Plus Environment

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issues, we performed ITC binding studies using the CRD of Gal-3 (Figure 1c and Figure 2c). The affinities of the GAGs for CRD were found to be similar to those obtained with intact Gal-3 (Table 2) suggesting that the CRD contains the GAG binding site. Pre-incubation of full length Gal-3 and Gal-3 CRD with β-lactose in ITC experiments completely abolished its GAG binding property (Figure 1e, Figure 1f and Figure 1g). These data suggest that GAGs bind to the lactose/LacNAcbinding site located on the CRD of Gal-3 and it is unlikely that GAGs interact with the N-terminal of Gal-3. Through multivalent binding, CSA, CSC and a CSPG are capable of forming cross-linked complexes with Gal-3 (described below). The ability of β-lactose to reverse such complex formation further strengthens the suggestion that CSA, CSC and BPG interact with the lactose/LacNAc-binding site of Gal-3.

Gal-3 forms reversible cross-linked complexes with CSA and CSC but not with heparin or CSBQuantitative precipitation and precipitation kinetics studies revealed that CSA and CSC were able to form insoluble cross-linked complexes with Gal-3 (Figure 3a and Figure 3b). In quantitative precipitation assays, the extent of cross-linking was robust and concentration (of CSA and CSC, respectively) dependent. At a Gal-3 concentration of 90 µM, the CSA-Gal-3 precipitation curve reached a plateau at a CSA concentration greater than ~30 µM (Figure 3a). With 100 µM Gal-3, the precipitation curve of CSC-Gal-3 plateaued when CSC concentration was >35 µM (Figure 3b). We then determined the kinetics of CSA-Gal-3 and CSC-Gal-3 cross-linking interactions by using fixed quantities of Gal-3 (90 µM), CSA and CSC. The spectroscopic scans at 420 nm showed substantial cross-linking of Gal-3 by a single dose of CSA (55 µM, final concentration) (Figure 4a) and CSC (29 µM, final concentration) (Figure 4b), respectively, within the first three minutes of interaction.


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The respective cross-linking interaction plateaued in 30 minutes (Figure 4a, Figure 4b). Heparin (up to 420 µM final concentration), HS (up to 50 µM final concentration) and CSB (up to 180 µM final concentration) did not produce any detectable cross-linked complex with Gal-3. Although the binding affinity of heparin (∆G = 7.0 kcal/mol), CSA (∆G = 7.6 kcal/mol), and CSC (∆G = 7.3 kcal/mol), are comparable, the former lacks the ability to form cross-linked complex with Gal-3. The difference in monosaccharide structures, sulfation level and chain length of heparin (compared to CSA and CSC) might prevent heparin-Gal-3 cross-linked complex formation. The weaker affinity of CSB and its structure could contribute to its inability to form cross-linked complex with Gal-3. ITC-driven n values (Table 2) show that while heparin and CSB are monovalent ligands (n = ~1.0) for Gal-3, CSA and CSC are multivalent (n = 0.28 and 0.27, respectively). While multivalent ligands often cross-link lectins, monovalent ligands lack the ability to do so. The ITC-driven valence of heparin, CSB, CSA and CSC is consistent with their respective cross-linking properties. It should be mentioned that the repeating disaccharide unit of CSA is GlcAβ1–3GalNAc4S, while CSC contains GlcAβ1–3GalNAc6S as its repeating unit. However, the affinities and cross-linking properties of CSA and CSC are comparable. Thus, the location of the sulfate groups (4S vs 6S) does not appear to change the properties of CSA and CSC in a significant way. Data obtained with CSA, CSB and CSC suggest that other chondroitin sulfates (CSD and CSE) could potentially interact with Gal-3 with different affinities and cross-linking properties.

CSA-Gal-3 and CSC-Gal-3 cross-linked complexes are dissolved by heparin and lactose- Both CSA-Gal-3 and CSC-Gal-3 cross-linked complexes showed sensitivity to lactose and heparin. In quantitative precipitation assays, preformed CSA-Gal-3 and CSC-Gal-3 complexes were dissolved by lactose in a dose-dependent manner (Figures 3a.II and Figure 3b.II). More than 50% of the 16 ACS Paragon Plus Environment

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Biochemistry

precipitate was dissolved at 2 mM β-lactose concentration (Figures 3a.II and Figure 3b.II). In kinetics experiments, a fixed concentration of heparin (900 µM, final concentration) rapidly dissolved 80% of preformed CSA-Gal-3 cross-linked complex (Figure 4C.II) and a fixed amount of β-lactose (2.5 mM, final concentration) instantly dissolved preformed CSA-Gal-3 complex (Figure 4D.II). Similar effects of heparin and β-lactose were also observed on CSC-Gal-3 cross-linked complex. These observations indicate that heparin, CSA and CSC bind to the same site on Gal-3 or their binding sites are significantly overlapped. As the CSA-Gal-3 and CSC-Gal-3 complexes are also dissolved by lactose, it can be assumed that heparin, CSA and CSC occupy the lactose-binding site on Gal-3. These results are consistent with the observation that pre-incubation of Gal-3 with lactose in ITC experiments completely abolished (Figures 1e, 1f and 1g) its ability to bind heparin and CSA.

Reversible cross-linking of Gal-3 by a CSPG- BPG, a CSPG, was able to precipitate Gal-3 through cross-linking interaction. Quantitative precipitation data show that the proteoglycan precipitates Gal-3 in a concentration dependent manner (Figure 3c.I). The precipitation of 90 µM Gal-3 saturated at a proteoglycan concentration of ~0.25 µM. Addition of lactose dissolved preformed BPG-Gal-3 complex in a dose dependent manner. 50% of the complex dissolved at 1.1 mM βlactose concentration (Figure 3c.II). In precipitation kinetics studies, more than 50% of crosslinking interaction between 90 µM Gal-3 and 264-280 nM BPG occurred in less than 1 minute (Figure 4e,f,g,h). Single doses of heparin (650 µM, final concentration) (Figure 4e.II), HS (113 µM, final concentration) (Figure 4f.II), CSC (215 µM, final concentration) (Figure 4g.II) and CSA (215 µM, final concentration) (Figure 4h.II) instantly dissolved substantial portions of preformed 17 ACS Paragon Plus Environment

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BPG-Gal-3 cross-linked complexes.

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The ability of lactose and the GAGs to dissolve the

precipitates further confirms that the proteoglycan and the GAGs bind to the lactose binding site of Gal-3 or their binding sites are significantly overlapped.

Binding of CSA, CSC and BPG to Gal-3 is associated with progressively increasing negative cooperativity.

In Hill plots, log [free ligand] is plotted versus log(fraction of bound

protein)/(fraction of free protein). These plots reveal positive or negative cooperativity (or absence of any cooperativity) in the binding of ligands to proteins. A Hill plot slope value of 1.0 indicates an absence of cooperativity in binding. When binding is associated with positive cooperativity, the slope value is greater than 1.0. When a ligand binds with negative cooperativity, the slope of the Hill plot is less than 1.0. Thus, the Hill plots can numerically determine the degree of cooperativity associated in ligand-receptor interactions. As Hill plots are logarithmic representations, they are capable of plotting of all theoretically obtainable data. Hill plots have been employed to reveal cooperativity associated with binding of multivalent ligands to lectins

38 39

When using Hill plot

analysis for multivalent binding, the term for the fraction of bound ligand, Xb/Mt, is corrected for the valency of the sugar to give (Xb)(functional valency of sugar)/Mt, which is a modification of the classical Hill plot

38 39

. Similar modification was done in the present study (see Materials and

Methods). Hill plots of the ITC data for CSA, CSC and BPG binding to Gal-3 are shown in Figure 5 (a, b, c). The plots are curvilinear and are disposed around the zero point on the ordinate, after correction for the functional valencies of the multivalent CSA, CSC and BPG. This provides confirmation of the ITC-derived functional valencies of CSA, CSC and BPG for Gal-3. In order to quantitate the progressive and dynamic changes in binding cooperativity, every successive three data points were 18 ACS Paragon Plus Environment

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separately fitted and the tangent slope value of each fitting was determined (Figure 5, d, e, f). Bar graphs showing the slope values are shown in Figure 5 (g, h, i). The initial tangent slope values of CSA, CSC and BPG are 0.91, 0.78 and 0.92, respectively, while the final tangent slope values are 0.26, 0.12 and 0.38, respectively. These data indicate that the binding of CSA, CSC and BPG to Gal-3 is associated with progressively increasing negative cooperativity.

Discussions GAGBPs and lectins represent two major but distinct groups of glycan binding proteins. Both groups possess unique characteristics with regard to binding site structures, ability to recognize charged glycans, multivalency and cross-linking properties. A member of one group rarely possesses the characteristics of the other. C-type lectins selectins are notable exceptions: they interact with GAGs in a divalent-cation-dependent manner2. However, the ability of the selectins to recognize sulfated GAGs is not unexpected given their known interaction with the sulfate groups of their ligands40. Gal-3 is a β-galactoside binding endogenous human lectin. It specifically interacts with LacNAc (N-acetyllactosamine) or polyLacNAc chains of N-linked oligosaccharides

32

. The

lectin also recognizes O-linked glycans. However it is not known as a member of the family of over 100 known GAGBPs. To our knowledge, this is the first communication that reveals the proteoglycan and sulfated GAG-binding properties of human Gal-3 through calorimetric and spectroscopic data. The present work also shows for the first time that CSA, CSC and a CSPG are multivalent ligands that are capable of cross-linking Gal-3. It seems that GAG binding property is not a common feature of all galectins, as we found that galectin-1 does not interact with GAGs.

Most reported GAGBPs interact with heparin, only a few of them bind to CS with affinities 19 ACS Paragon Plus Environment

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comparable to that of heparin. The present work shows that the affinities of CSA and CSC for Gal-3 are higher than that of heparin. Data obtained with Gal-3 CRD prove that the GAG binding site is located on the CRD. The ability of lactose to completely inhibit GAG binding (Figures 1e, 1f, 1g) and to dissolve CSA-Gal-3, CSC-Gal-3 and proteoglycan-Gal-3 cross-linked complexes (Figure 3 and Figure 4) specifically indicates that GAG-binding site and lactose/LacNAc binding site on Gal3 are the same or they are significantly overlapped. The affinity of CS disaccharides for Gal-3 is 4080-fold weaker than lactose/LacNAc (Table 1) indicating lesser molecular contacts between Gal-3 and CS disaccharides. Such low affinity is enhanced by the repetitive occurrence of disaccharides in a long GAG chain. Presence of multiple copies of GAG chains in a proteoglycan further increase its affinity for Gal-3. The higher affinity of unmodified GAG chains, especially heparin, CSA and CSC suggests that GAG chains not only bind to the primary carbohydrate binding site, they are well accommodated in the vicinity of the LacNAc binding site.

The lactose/LacNAc binding site of Gal-3 is an open ended cleft that can accommodate longer oligosaccharides. Arginine144 of Gal-3 is suitably positioned to interact with the extended part of a ligand. Moreover, a less bulky residue (Ala-146) in Gal-3 seems to allow Gal-3 to accommodate additional residues of an oligosaccharide 41. We anticipate that Arginine144 plays an important role in GAG binding.

Mouse and human Gal-3 are reported to interact with RNA. Structural support for this observation comes from the electrostatic potential calculations of Gal-3

41

. This calculation shows that the

binding site is lined with three linearly arranged arginine residues (Arg-186, Arg-162, and Arg144). These three residues have been suggested to be crucial for RNA binding 41. This observation 20 ACS Paragon Plus Environment

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Biochemistry

strongly suggests that the carbohydrate binding cleft of Gal-3 can accommodate negatively charged linear biopolymers such as RNA. We believe that the same amino acids (Arg-186, Arg-162, and Arg-144) in the lactose/LacNAc binding cleft of Gal-3 could play important roles in GAG binding. The inhibitory activity of soluble oligosaccharides on RNA splicing assays

41

and the loss of

binding activity of GAGs with Gal-3 pre-incubated with lactose/LacNAc (in the present study) support this view. Most of the HS binding proteins, a subgroup of GAGBPs, contain four to seven positively charged amino acids in their HS binding sites 5. Interestingly, the LacNAc binding site of Gal-3 has three Arg and one His residues 41. There is also a lysine residue, Lys176 at the vicinity of the sugar binding site of Gal-342. Our data show that lactose completely abolished GAG binding to Gal-3 and the cross-linked complexes of Gal-3 with CSA, CSC and the bovine proteoglycan were dissolved by lactose. Taken together, these observations strongly suggest that the LacNAc binding site of Gal-3 is also the site for GAG binding.

ITC and precipitation data unequivocally show that heparin and CSB are monovalent and lack the ability of cross-linking Gal-3, while CSA and CSC are multivalent ligands and they are capable of cross-linking Gal-3. Although a single CSB chain is monovalent, a proteoglycan with multiple CSB chain could be functionally multivalent for Gal-3. Gal-3 mediated cross-linking of glycoconjugates is important for many biological processes including signaling. Therefore, the differential valence and cross-linking properties of heparin, CSA, CSB and CSC indicate that these GAGs could potentially engage Gal-3 in different biological functions.

The bovine proteoglycan contains numerous CS chains and thus is a highly multivalent ligand of Gal-3. The nanomolar binding affinity of this CSPG and its cross-linking property result from its 21 ACS Paragon Plus Environment

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multivalent interaction with Gal-3. Due to their strategic location in ECM and on the surface of cells, it is highly probable that proteoglycans and Gal-3 could bind to each other and form crosslinked complex. The observed Gal-3 mediated cross-linking of proteoglycan through high affinity binding may have significance in the biological function of both partners of the complex. Proteoglycans often shed their constituent GAGs. The ability of heparin, HS, CSA and CSC chains to dissolve proteoglycan-Gal-3 complex, as shown in the present work, may potentially interfere with proteoglycan-Gal-3 complex stability in the ECM and at the surface of cells and thus can modulate Gal-3 mediated functions of proteoglycans.

Binding of multivalent ligands to lectins has been shown to be associated with progressively increasing negative cooperativity38 39, 43. CSA, CSC and BPG are multivalent ligands of Gal-3. Hill plot analysis shows that these three ligands bind to Gal-3 of with progressively increasing negative binding cooperativity. The reduction in functional valency of CSA, CSC and BPG, as they bind an increasing number of Gal-3 molecules, at least partially, explains this increasing negative binding cooperativity effect.The other factor that may contribute to this increasing negative binding cooperativity is the formation of noncovalent cross-linked complexes between Gal-3 and these three multivalent ligands (CSA, CSC and BPG). Formation of cross-linked network between Gal-3 and the multivalent ligands may interfere with their binding interactions with successive Gal-3 molecules. As a result, the negative cooperativity increases as observed in the Hill plots analysis. Experimental data suggest that lectins bind to and jump on and rebind to multivalent linear biopolymers such as mucins (called ‘bind and jump’ model) 43. Hill plot data in the present study indicate that Gal-3 may bind to CSA, CSC and BPG (which are linear biopolymers) through similar mechanism. 22 ACS Paragon Plus Environment

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Sulfated GAG-binding properties of Gal-3, as described in the present work, suggest that the known glycoprotein-Gal-3 and GAG-GAGBP interactions in biological systems may not be as straightforward as they seem. In the vicinity where all four entities are present, glycoprotein-Gal-3 interactions will be interrupted by competitive GAG, GAG-GAGBP interactions will be challenged by competitive Gal-3 and GAG-Gal-3 interactions will face competition from GAGBPs and Gal-3specific glycoproteins. Therefore, all the known glycoprotein-Gal-3 and GAG-GAGBP interactions might be far more complicated than they currently appear.

Unlike other groups of glycan binding proteins (for e.g. lectins), GAG-binding proteins do not possess a common structural element, rather they are structurally quite unrelated. This structural diversity suggests that GAG-binding proteins have evolved through convergent evolution 44. Gal-3 probably acquired its GAG-binding properties through convergent evolution. It is tempting to think Gal-3 as a ‘missing-link’ between GAGBPs and lectins as it possesses the binding properties of both groups.

Acknowledgement: The authors thank Prof. Stuart Kornfeld (Washington University School of Medicine) for reading the manuscript.


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galectins: evidence from frontal affinity chromatography, Biochem Biophys Res Commun 373, 206-212. (32) Cummings, R. D., and Liu, F. T. (2009) Galectins, In Essentials of Glycobiology (Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler, M. E., Eds.), Cold Spring Harbor Laboratory Press (33) Duckworth, C. A., Guimond, S. E., Sindrewicz, P., Hughes, A. J., French, N. S., Lian, L. Y., Yates, E. A., Pritchard, D. M., Rhodes, J. M., Turnbull, J. E., and Yu, L. G. (2015) Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis, Oncotarget 6, 2367123687. (34) Wen, Y., Makagiansar, I. T., Fukushi, J., Liu, F. T., Fukuda, M. N., and Stallcup, W. B. (2006) Molecular basis of interaction between NG2 proteoglycan and galectin-3, J Cell Biochem 98, 115-127. (35) Bachhawat-Sikder, K., Thomas, C. J., and Surolia, A. (2001) Thermodynamic analysis of the binding of galactose and poly-N-acetyllactosamine derivatives to human galectin-3, FEBS letters 500, 75-79. (36) Ahmad, N., Gabius, H. J., Sabesan, S., Oscarson, S., and Brewer, C. F. (2004) Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3, Glycobiology 14, 817-825. (37) Talaga, M. L., Fan, N., Fueri, A. L., Brown, R. K., Chabre, Y. M., Bandyopadhyay, P., Roy, R., and Dam, T. K. (2014) Significant other half of a glycoconjugate: contributions of scaffolds to lectin-glycoconjugate interactions, Biochemistry 53, 4445-4454. (38) Dam, T. K., Roy, R., Page, D., and Brewer, C. F. (2002) Negative cooperativity associated with binding of multivalent carbohydrates to lectins. Thermodynamic analysis of the "multivalency effect", Biochemistry 41, 1351-1358. (39) Patra, D., Mishra, P., Vijayan, M., and Surolia, A. (2016) Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains, Biochemistry 55, 49-61. (40) Cummings, R. D., and McEver, R. P. (2009) C-type Lectins, In Essentials of Glycobiology (Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler, M. E., Eds.), Cold Spring Harbor Laboratory Press (41) Seetharaman, J., Kanigsberg, A., Slaaby, R., Leffler, H., Barondes, S. H., and Rini, J. M. (1998) X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution, J Biol Chem 273, 13047-13052. (42) Salomonsson, E., Carlsson, M. C., Osla, V., Hendus-Altenburger, R., Kahl-Knutson, B., Oberg, C. T., Sundin, A., Nilsson, R., Nordberg-Karlsson, E., Nilsson, U. J., Karlsson, A., Rini, J. M., and Leffler, H. (2010) Mutational tuning of galectin-3 specificity and biological function, J Biol Chem 285, 35079-35091. (43) Dam, T. K., and Brewer, C. F. (2008) Effects of clustered epitopes in multivalent ligandreceptor interactions, Biochemistry 47, 8470-8476. (44) Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler, M. E. (2009) Essentials of Glycobiology Second ed., Cold Spring Harbor Laboratory Press, New York 26 ACS Paragon Plus Environment

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Table 1 Inhibition of Gal-3 mediated agglutination of rabbit erythrocytes by mono- and disaccharides at pH 7.2 and 0.15 M NaCl. Ligand

Minimum inhibitory concentration * mM

N-Acetyl-D-glucosamine 6-sulfate

65.0±6.0

D- Glucuronic Acid

48.0±4.5

N-Acetyl-D-galactosamine 4-sulfate

47.0±3.3

N-Acetyl-D-galactosamine 6-sulfate

46.0±4.1

D-Galactosamine-2-sulfate

63.0±4.2

Chondroitin disaccharide ∆di-0S

20.0±1.9

Chondroitin disaccharide ∆di-4S

40.0±2.7

N-acetyllactosamine

0.5±0.02 µM

Heparin

14.2±1.0

Heparan sulfate

62.0±4.8

Chondroitin sulfate A

3.9±0.2

Chondroitin sulfate B

45.0±2.7

Chondroitin sulfate C

7.0±0.5

Bovine proteoglycan

0.02±0.0009

Aggrecan

0.016±0.0005

* Each data represents a mean value of a particular parameter obtained from three different experiments. The Table shows the mean values and standard errors. Molar concentrations of the 27 ACS Paragon Plus Environment

Biochemistry

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GAGs and proteoglycans were calculated based on the average molecular weight of the whole molecules. The above ligands did not bind to galectin-1

Table 2 Thermodynamic binding parameters of full length galectin-3 and its CRD obtained with sulfated glycosaminoglycans and a proteoglycan at 27oC* Ligand

Ka

Kd

(M-1x10-4)

(µM)

-∆G

-∆H

(kcal/mol)

-T∆

(kcal/mol) (kcal/mol)

n (sites/monomer)

Gal-3 (full length) Heparin

12.4±0.8

8.1±0.5

7.0±0.03

2.3±0.1

-4.7±0.2

1.01±0.02

CSA

37.8±0.9

2.7±0.06

7.6±0.02

21.7±0.8

14.1±0.8

0.28±0.01

CSB

2.5±0.2

41.0±3.3

6.0±0.05

2.7±0.2

-3.3±0.2

0.99±0.02

CSC

21.3±0.4

4.7±0.1

7.3±0.02

28.0±1.3

20.5±1.3

0.27±0.02

712.1±16.2 701.4±16.2

0.01±0.0005

BPG

7142.0±311 0.014±0.0009

10.7±0.03

Carbohydrate recognition domain (CRD) of Gal-3 Heparin

10.6±0.5

9.4±0.5

6.9±0.04

7.3±0.2

0.4±0.03

1.04±0.03

CSC

16.6±0.8

6.0±0.3

7.1±0.05

33.5±1.9

26.4±1.8

0.41±0.03

* Each data represents a mean value of a particular parameter obtained from three different experiments. Ka, -∆H and “n” values are the mean values of respective parameters from three experiments. -∆G and –T∆S were calculated separately for each experiment and then the mean 28 ACS Paragon Plus Environment

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Biochemistry

values of -∆Gs (from 3 experiments) and T∆Ss (from three experiments) were determined. The Table shows those mean values and standard errors. CSA, chondroitin sulfate A; CSB, Chondroitin sulfate B; CSC, Chondroitin sulfate C; BPG, bovine proteoglycan. Molar concentrations of the GAGs and proteoglycans were calculated based on the average molecular weight of the whole molecules.


Biochemistry

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Figure Legends FGURE 1. Binding of heparin and CSA to Gal-3 and Gal-3 CRD. (a) Representative hemagglutination profiles of Gal-3 with heparin (I), bovine proteoglycan (II) and CSC (III). The buffer used was PBS, pH 7.4 with 0.15 M NaCl. Isothermal titration calorimetric (ITC) profiles of (b) intact human Gal-3 (51 µM) and (c) Gal-3 CRD (58 µM) with unmodified heparin (2.6 mM) at 27 °C and pH 7.4. The top panels of b and c show data obtained for automatic injections (4 µL each) of heparin. (d) ITC profile of intact human Gal-3 (35 µM) with unmodified CSA (0.52 mM) at 27 °C and pH 7.4. Data obtained for automatic injections (4 µL each) of CSA shown at the top panel of d. The integrated curve showing experimental points and the best fit are shown in the bottom panels of b, c and d. Heparin (2.6 mM) showed no binding when injected into (e) intact Gal-3 (51 µM) and (f) Gal-3 CRD (58 µM), both pre-incubated with 200 mM β-lactose. (g) 0.52 mM of CSA showed no binding when injected into intact Gal-3 (35 µM) pre-incubated with 200 mM β-lactose.

FIGURE 2. Binding of CSB, CSC and bovine proteoglycan to Gal-3. (a) ITC profiles at 27 °C and pH 7.4 of (a) intact human Gal-3 (30 µM) with unmodified CSC (0.82 mM), (b) intact human Gal-3 (36 µM) with unmodified CSB (1.8 mM), (c) human Gal-3 CRD (70 µM) with unmodified CSC (0.58 mM), and (d) human Gal-3 (25 µM) with bovine proteoglycan (5.0 µM). The top panels of a, b, c and d shows data obtained for automatic injections (4 µL each) of CSC, CSB, CSC and bovine proteoglycan, respectively. The integrated curve showing experimental points and the best fit are shown in the bottom panels of a, b, c and d. 30 ACS Paragon Plus Environment

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Biochemistry

FIGURE 3. Cross-linking Gal-3 by CSA, CSC and bovine proteoglycan. Quantitative precipitation profile of Gal-3 (90 µM) with (a. I) CSA, (b. I) CSC, and (c. I) bovine proteoglycan. Dose dependent dissolution of pre-formed (a. II) CSA-Gal-3, (b. II) CSC-Gal-3 and (c. II) Gal-3bovine proteoglycan cross-linked complexes with β-lactose.

FIGURE 4. Kinetics of Cross-linking of Gal-3 by CSA, CSC and bovine proteoglycan. Kinetics of cross-linked complex formation of Gal-3 (90 µM) with (a) a single dose of CSA (55 µM, final concentration) and (b) a single dose of CSC (29 µM, final concentration). (c) I. Kinetics of cross-linked complex formation of Gal-3 (60 µM) with a single dose of CSA (35 µM, final concentration); II. When the precipitation reached a plateau, a single dose of heparin (900 µM, final concentration) was added to the precipitate and a substantial portion of the complex dissolved instantly. (d) I. Cross-linked complex formation of Gal-3 (80 µM) with a single dose of CSA (35 µM). II. A single dose of β-lactose (2.5 mM, final concentration) rapidly dissolved the precipitate. Cross-linking kinetics of Gal-3 (90 µM) with a single dose of bovine proteoglycan [264 nM, final concentration (e I); 275 nM, final concentration (f I); 280 nM, final concentration (g I) and 275 nM, final concentration (h I)]. Kinetics of dissolution of the Gal-3-proteoglycan cross-linked complex with a single dose of heparin (650 µM, final concentration) (e II), HS (113 µM, final concentration) (f II), CSC (215 µM, final concentration) (g II) and CSA (215 µM, final concentration) (h II).

FIGURE 5. Hill plots of the ITC data for CSA (a), CSC (b) and bovine proteoglycan (c) binding to Gal-3. In the plot, Y = (Xb)(functional valency of ligand)/Mt, where Xb is the 31 ACS Paragon Plus Environment

Biochemistry

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concentration of bound ligand, Mt is the concentration of total lectin, and Xf is the concentration of free ligands. Progressive three-points were fitted to determine the changing tangent slope values of the Hill plot for CSA (d), CSC (e) and bovine proteoglycan (f). Bar graphs showing the three-point tangent slope values (determined in d, e and f) of the Hill plots of CSA (g), CSC (h) and bovine proteoglycan (i).

Figure 1


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Biochemistry

Figure 2


Biochemistry

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Figure 3


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Biochemistry

Figure 4


Biochemistry

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Figure 5


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Biochemistry

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