Rapid Detection and Purification of Galectin-3 by the Capture and Release (CaRe) Method.

Specific interactions between lectins and glycoproteins determine the outcomes of numerous biological processes. To elucidate the roles of lectins and glycoproteins in those processes, it is essential to detect these proteins in biological samples and purify them to homogeneity. Conventional protein detection and purification techniques are multi-step, time-intensive, and expensive. They often require rigorous trial and error experimentations and fairly larger volumes of crude extracts. To minimize some of these challenges, we recently formulated a new method named Capture and Release (CaRe). This method is rapid, facile, precise, and inexpensive, and it works even when the sample volume is smaller. We developed this method to detect and purify recombinant human Galectin-3 and subsequently validated this method by purifying several other lectins. Besides lectins, CaRe is capable of detecting/purifying glycoproteins. In this method, targets (lectins and glycoproteins) are captured by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. CaRe can potentially be used as a tool to discover new lectins and glycoconjugates and elucidate their functions.

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

Human galectin-3 (Gal-3) is a ß-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 ß-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was also reversed by ß-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.

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.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

A capture and release method based on noncovalent ligand cross-linking and facile filtration for purification of lectins and glycoproteins.

Glycan-binding proteins such as lectins are ubiquitous proteins that mediate many biological functions. To study their various biological activities and structure-function relationships, researchers must use lectins in their purest form. Conventional purification techniques, especially affinity column chromatography, have been instrumental in isolating numerous lectins and glycoproteins. These approaches, however, are time-consuming, consist of multiple steps, and often require extensive trial-and-error experimentation. Therefore, techniques that are relatively rapid and facile are needed. Here we describe such a technique, called capture and release (CaRe). The strength of this approach is rooted in its simplicity and accuracy. CaRe purifies lectins by utilizing their ability to form spontaneous noncovalently cross-linked complexes with specific multivalent ligands. The lectins are captured in the solution phase by multivalent capturing agents, released by competitive monovalent ligands, and then separated by filtration. CaRe does not require antibodies, solid affinity matrices, specialized detectors, a customized apparatus, controlled environments, or functionalization or covalent modification of reagents. CaRe is a time-saving procedure that can purify lectins even from a few milliliters of crude protein extracts. We validated CaRe by purifying recombinant human galectin-3 and five other known lectins and also tested CaRe's ability to purify glycoproteins. Besides purifying lectins and glycoproteins, CaRe has the potential to purify other glycoconjugates, including proteoglycans. This technique could also be used for nonlectin proteins that bind multivalent ligands. Given the ubiquity of glycosylation in nature, we anticipate that CaRe has broad utility.