Generation and Use of Recombinant Galectins.
01 Mar 2021-Vol. 1, Iss: 3
TL;DR: In this paper, the authors examined fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and -7 as examples.
Abstract: Galectins are soluble carbohydrate binding proteins that can bind β-galactose-containing glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host-pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan-binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and -7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin-1 and -9 as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Purification of galectins using lactosyl-sepharose affinity chromatography Basic Protocol 2: Purification of human galectin-7 using a nickel-NTA affinity chromatography column Alternate Protocol 1: Iodoacetamide alkylation of free sulfhydryls on galectin-1 Alternate Protocol 2: Purification of human galectin-9 using lactosyl-sepharose column chromatography.
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TL;DR: Galectin-3 (Gal-3) as mentioned in this paper was the first Galectin shown to engage microbial glycans, and exhibited high binding toward mammalian blood group A, B, and αGal antigens in a glycan microarray format.
Abstract: While adaptive immunity enables the recognition of a wide range of microbial antigens, immunological tolerance limits reactively toward self to reduce autoimmunity. Some bacteria decorate themselves with self-like antigens as a form of molecular mimicry to limit recognition by adaptive immunity. Recent studies suggest that galectin-4 (Gal-4) and galectin-8 (Gal-8) may provide a unique form of innate immunity against molecular mimicry by specifically targeting microbes that decorate themselves in self-like antigens. However, the binding specificity and antimicrobial activity of many human galectins remain incompletely explored. In this study, we defined the binding specificity of galectin-3 (Gal-3), the first galectin shown to engage microbial glycans. Gal-3 exhibited high binding toward mammalian blood group A, B, and αGal antigens in a glycan microarray format. In the absence of the N-terminal domain, the C-terminal domain of Gal-3 (Gal-3C) alone exhibited a similar overall binding pattern, but failed to display the same level of binding for glycans over a range of concentrations. Similar to the recognition of mammalian glycans, Gal-3 and Gal-3C also specifically engaged distinct microbial glycans isolated and printed in a microarray format, with Gal-3 exhibiting higher binding at lower concentrations toward microbial glycans than Gal-3C. Importantly, Gal-3 and Gal-3C interactions on the microbial microarray accurately predicted actual interactions toward intact microbes, with Gal-3 and Gal-3C displaying carbohydrate-dependent binding toward distinct strains of Providentia alcalifaciens and Klebsiella pneumoniae that express mammalian-like antigens, while failing to recognize similar strains that express unrelated antigens. While both Gal-3 and Gal-3C recognized specific strains of P. alcalifaciens and K. pneumoniae, only Gal-3 was able to exhibit antimicrobial activity even when evaluated at higher concentrations. These results demonstrate that while Gal-3 and Gal-3C specifically engage distinct mammalian and microbial glycans, Gal-3C alone does not possess antimicrobial activity.
10 citations
14 Jun 2021
TL;DR: In this paper, the functional relationships between galectin-7, p53 and matrix metalloproteinases (MMP-9) are discussed and a better understanding of these relationships will help to develop a working hypothesis and model that will provide the basis for further research in the hope of establishing a new paradigm for tackling the role of cancer.
Abstract: It has been almost 25 years since the discovery of galectin-7. This member of the galectin family has attracted interest from many working in the cancer field given its highly restricted expression profile in epithelial cells and the fact that cancers of epithelial origin (carcinoma) are among the most frequent and deadly cancer subtypes. Initially described as a p53-induced gene and associated with apoptosis, galectin-7 is now recognized as having a protumorigenic role in many cancer types. Several studies have indeed shown that galectin-7 is associated with aggressive behavior of cancer cells and induces expression of MMP-9, a member of the matrix metalloproteinases (MMP) family known to confer invasive behavior to cancer cells. It is therefore not surprising that many studies have examined its relationships with p53 and MMP-9. However, the relationships between galectin-7 and p53 and MMP-9 are not always clear. This is largely because p53 is often mutated in cancer cells and such mutations drastically change its functions and, consequently, its association with galectin-7. In this review, we discuss the functional relationships between galectin-7, p53 and MMP-9 and reconcile some apparently contradictory observations. A better understanding of these relationships will help to develop a working hypothesis and model that will provide the basis for further research in the hope of establishing a new paradigm for tackling the role of galectin-7 in cancer.
10 citations
TL;DR: Galectin-9 (Gal-9) has been shown to have strong binding preference for ABO(H) blood group antigens, with the C-terminal domain exhibiting higher binding to blood group B than the Nterminal Domain this paper .
8 citations
TL;DR: Galectin-7 (Gal-7) was shown to recognize a diverse range of microbes, each of which use molecular mimicry while failing to induce host cell injury as discussed by the authors .
5 citations
01 Jan 2022
TL;DR: This chapter is describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinereaGalectin 2 (CGL2), nickel-chromatography for histidine-tagged human galECTin-7, and glutathione-SepHarose chromation for Glutathione S-transferase-tagging (
4 citations
References
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TL;DR: A highly flexible method based on affinities which can be used in a more selective fashion by modern chromatographic techniques is described here.
Abstract: CONVENTIONAL nonspecific precipitation methods sometimes depend on affinities which can be used in a more selective fashion by modern chromatographic techniques. The affinity of proteins for heavy metal ions, for example, may provide a basis for their purification and analysis. A highly flexible method based on such affinities is described here.
2,063 citations
University of California, San Francisco1, University of Liège2, University of Oklahoma3, Columbia University4, Teikyo University5, Medical Research Council6, Scripps Research Institute7, University of Texas MD Anderson Cancer Center8, Harvard University9, Centre national de la recherche scientifique10, French Institute of Health and Medical Research11, Wayne State University12, University of Toronto13, Michigan State University14
1,098 citations
TL;DR: Galectin-1 induced apoptosis of activated human T cells and human T leukaemia cell lines and represents a new mechanism for regulating the immune response.
Abstract: Galectin-1, a member of the family of beta-galactoside binding proteins, has growth regulatory and immunomodulatory activities. We report here that galectin-1, expressed by stromal cells in human thymus and lymph nodes, is present at sites of cell death by apoptosis during normal T-cell development and maturation. Galectin-1 induced apoptosis of activated human T cells and human T leukaemia cell lines. Resting T cells also bound galectin-1, but did not undergo apoptosis. Human endothelial cells that expressed galectin-1 induced apoptosis of bound T cells. Galectin-1-induced apoptosis required expression of CD45, and was decreased when N-glycan elongation was blocked by treatment of the cells by swainsonine, whereas inhibition of O-glycan elongation potentiated the apoptotic effect of galectin-1. Induction of apoptosis by an endogenous mammalian lectin represents a new mechanism for regulating the immune response.
1,028 citations
TL;DR: It is found that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thiOREDoxin fusion proteins usually accumulate to high levels.
Abstract: We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.
973 citations
TL;DR: An increasing number of studies reveal the relevance of glycosylation to pathogen recognition, to the modulation of the innate immune system and to the control of immune cell homeostasis and inflammation.
Abstract: The importance of protein glycosylation in the migration of immune cells throughout the body has been extensively appreciated. However, our awareness of the impact of glycosylation on the regulation of innate and adaptive immune responses is relatively new. An increasing number of studies reveal the relevance of glycosylation to pathogen recognition, to the modulation of the innate immune system and to the control of immune cell homeostasis and inflammation. Similarly important is the effect of glycan-containing 'information' in the development of autoimmune diseases and cancer. In this review, we provide an overview of these new directions and their impact in the field of glycoimmunology.
672 citations