Enriching health-related research through glycobiological approaches

In the Laboratory of Mechanistic Glycobiology at Michigan Technological University, we have been working in four distinct but related areas. Our findings can significantly influence human health-related research. (1) We have documented a molecular strategy that can improve drug designing. (2) We have shown that the tumor-associated protein galectin-3 can create problems in cancer biomarker assays by hiding the biomarkers. (3) In another project, we reported that the role of galectin-3 in cancer could be more complicated than what is reported in the literature. (4) Our team also detected a novel natural product with anti-fungal and anti-cancer activities.https://digitalcommons.mtu.edu/techtalks/1012/thumbnail.jp

Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands †

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3′ of each of the two sugars, resulting in a 3′-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl β-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3′ position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series

Fine specificities of two lectins from Cymbosema roseum seeds: A lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fuc1,2Gal1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the (1,6) Man residue, the 3- and 4-hydroxyl group of the (1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewisx, Lewisy, Lewisa and Lewisb epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe. © 2011 The Author

The Reversible Two-State Unfolding of a Monocot Mannose-Binding Lectin from Garlic Bulbs Reveals the Dominant Role of the Dimeric Interface in Its Stabilization

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers $(A_2 \Longleftrightarrow\hspace{2mm}2U)$. Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding $(\Delta C_p)$ and also the temperature dependence of the thermodynamic parameters, namely, $\Delta G, \Delta H, and \hspace{2mm} \Delta S$. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters $\Delta H_g$, $\Delta S_g,$ and $\Delta C_p$ as $174.1 \hspace{2mm} kcal\hspace{1mm} mol^{-1}$, $0.512 \hspace{2mm}kcal \hspace{1mm}mol^{-1} K^{-1}$, and $3.41 \hspace{2mm}kcal \hspace{1mm} mol^{-1} K^{-1}$, respectively, at $T_g$ = 339.4 K. Also, the free energy of unfolding, $G_s,$ at its temperature of maximum stability $(T_s = 293 K)$ is $13.13 \hspace{2mm} kcal\hspace{1mm} mol^{-1}$. Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined $C_p (3.2 \pm 0.28 \hspace{2mm}kcal \hspace{1mm}mol^{-1} K^{-1})$ and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small $C_p$ for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein

The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2⇔2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (ΔCp) and also the temperature dependence of the thermodynamic parameters, namely, ΔG, ΔH, and ΔS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters ΔHg, ΔSg, and ΔCp as 174.1 kcal mol−1, 0.512 kcal mol−1 K−1, and 3.41 kcal mol−1 K−1, respectively, at Tg=339.4 K. Also, the free energy of unfolding, ΔGs, at its temperature of maximum stability (Ts=293 K) is 13.13 kcal mol−1. Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined ΔCp (3.2±0.28 kcal mol−1 K−1) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small ΔCp for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein

Revealing the Identity of Human Galectin-3 as a Glycosaminoglycan-Binding Protein

Human galectin-3 (Gal-3) is a β-galactoside-binding lectin. This multitasking protein preferentially interacts with N-acetyllactosamine moieties on glycoconjugates. Specific hydroxyl groups (4-OH, 6-OH of galactose, and 3-OH of glucose/N-acetylglucosamine) of lactose/LacNAc are essential for their binding to Gal-3. Through hemagglutination inhibition, microcalorimetry, and spectroscopy, we have shown that despite being a lectin, Gal-3 possesses the characteristics of a glycosaminoglycan (GAG)-binding protein (GAGBP). Gal-3 interacts with sulfated GAGs [heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC)] and chondroitin sulfate proteoglycans (CSPGs). Heparin, CSA, and CSC showed micromolar affinity for Gal-3, while the affinity of CSPGs for Gal-3 was much higher (nanomolar). Interestingly, CSA, CSC, and a bovine CSPG, not heparin and CSB, were multivalent ligands for Gal-3, and they formed reversible noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs to Gal-3 was completely inhibited when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was also reversed by β-lactose. These findings strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans. The GAG- and CSPG-binding properties of Gal-3 make the lectin a potential competitor/collaborator of other GAGBPs such as growth factors, cytokines, morphogens, and extracellular matrix proteins

Measuring Multivalent Binding Interactions by Isothermal Titration Calorimetry

© 2016 Elsevier Inc. All rights reserved. Multivalent glycoconjugate-protein interactions are central to many important biological processes. Isothermal titration calorimetry (ITC) can potentially reveal the molecular and thermodynamic basis of such interactions. However, calorimetric investigation of multivalency is challenging. Binding of multivalent glycoconjugates to proteins (lectins) often leads to a stoichiometry-dependent precipitation process due to noncovalent cross-linking between the reactants. Precipitation during ITC titration severely affects the quality of the baseline as well as the signals. Hence, the resulting thermodynamic data are not dependable. We have made some modifications to address this problem and successfully studied multivalent glycoconjugate binding to lectins. We have also modified the Hill plot equation to analyze high quality ITC raw data obtained from multivalent binding. As described in this chapter, ITC-driven thermodynamic parameters and Hill plot analysis of ITC raw data can provide valuable information about the molecular mechanism of multivalent lectin-glycoconjugate interactions. The methods described herein revealed (i) the importance of functional valence of multivalent glycoconjugates, (ii) that favorable entropic effects contribute to the enhanced affinities associated with multivalent binding, (iii) that with the progression of lectin binding, the microscopic affinities of the glycan epitopes of a multivalent glycoconjugate decrease (negative cooperativity), (iv) that lectin binding to multivalent glycoconjugates, especially to mucins, involves internal diffusion jumps, (bind and jump) and (v) that scaffolds of glycoconjugates influence their entropy of binding

Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin–glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin–mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions

Detection and purification of lectins and glycoproteins by a non-column chromatographic technique

Proteins including glycoproteins and lectins play important roles in many biological processes. Therefore, they are vigorously studied in academic, clinical and industrial research. Such research activities often need these proteins in their purest forms. Thus, protein purification constitutes an important step in many scientific projects. Conventional protein purification techniques include affinity chromatography, ion-exchange chromatography and size-exclusion chromatography, and electrophoresis. These techniques have their own limitations. Conventional approaches are generally tedious, multi-step, expensive and time consuming. They generally require elaborate infrastructure and larger starting crude materials. In addition, these techniques sometimes encounter non-specific binding. In order to overcome some of the limitations associated with these conventional methods, our lab developed a protein detection/purification method named “Capture and Release” (CaRe). In this method, a target capturing agent (TCA) captures a specific target (lectin or glycoprotein) in the crude solution and form insoluble complex. The complex is spun down while the other unwanted proteins are washed off. Captured target is released from the TCA by the addition of competitive monovalent ligand, separated by membrane filtration and visualized by gel electrophoresis. We were successful in purifying recombinant human Galectin-3 by CaRe. This method was able to purify glycoproteins as well. CaRe was validated by purifying known lectins and glycoproteins. Compare to conventional techniques, our method is relatively fast, simple, precise and less expensive and it can detect/purify lectins and glycoproteins even from a small volume (~1 ml) of starting material. Thus CaRe can serve as a valuable tool to discover unknown proteins and glycoproteins