Galectins are a family of ß-galactoside-binding lectins characterized by
a unique sequence motif in the carbohydrate recognition domain, and evolutionary
and structural conservation from fungi to invertebrates and vertebrates, including
mammals. Their biological roles, initially understood as limited to recognition of
endogenous (“self”) carbohydrate ligands in embryogenesis and early development,
dramatically expanded in later years by the discovery of their roles in tissue repair,
cancer, adipogenesis, and regulation of immune homeostasis. In recent years, however,
evidence has also accumulated to support the notion that galectins can bind
(“non-self”) glycans on the surface of potentially pathogenic microbes, and function
as recognition and effector factors in innate immunity. Thus, this evidence has established
a newparadigm by which galectins can function not only as pattern recognition
receptors but also as effector factors, by binding to the microbial surface and inhibiting
adhesion and/or entry into the host cell, directly killing the potential pathogen
by disrupting its surface structures, or by promoting phagocytosis, encapsulation,
autophagy, and pathogen clearance from circulation. Strikingly, some viruses, bacteria, and protistan parasites take advantage of the aforementioned recognition roles of the vector/host galectins, for successful attachment and invasion. These recent findings suggest that galectin-mediated innate immune recognition and effector mechanisms, which throughout evolution have remained effective for preventing or fighting viral, bacterial, and parasitic infection, have been “subverted” by certain pathogens by unique evolutionary adaptations of their surface glycome to gain host entry, and the acquisition of effective mechanisms to evade the host’s immune responses.
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Structure of the zebrafish galectin-1-L2 and model of its interaction with the infectious hematopoietic necrosis virus (IHNV) envelope glycoprotein
Abstract Galectins, highly conserved β-galactoside-binding lectins, have diverse regulatory roles in development and immune homeostasis and can mediate protective functions during microbial infection. In recent years, the role of galectins in viral infection has generated considerable interest. Studies on highly pathogenic viruses have provided invaluable insight into the participation of galectins in various stages of viral infection, including attachment and entry. Detailed mechanistic and structural aspects of these processes remain undetermined. To address some of these gaps in knowledge, we used Zebrafish as a model system to examine the role of galectins in infection by infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that is responsible for significant losses in both farmed and wild salmonid fish. Like other rhabdoviruses, IHNV is characterized by an envelope consisting of trimers of a glycoprotein that display multiple N-linked oligosaccharides and play an integral role in viral infection by mediating the virus attachment and fusion. Zebrafish’s proto-typical galectin Drgal1-L2 and the chimeric-type galectin Drgal3-L1 interact directly with the glycosylated envelope of IHNV, and significantly reduce viral attachment. In this study, we report the structure of the complex of Drgal1-L2 with N-acetyl-d-lactosamine at 2.0 Å resolution. To gain structural insight into the inhibitory effect of these galectins on IHNV attachment to the zebrafish epithelial cells, we modeled Drgal3-L1 based on human galectin-3, as well as, the ectodomain of the IHNV glycoprotein. These models suggest mechanisms for which the binding of these galectins to the IHNV glycoprotein hinders with different potencies the viral attachment required for infection.
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- Award ID(s):
- 1656720
- PAR ID:
- 10169577
- Date Published:
- Journal Name:
- Glycobiology
- Volume:
- 29
- Issue:
- 5
- ISSN:
- 1460-2423
- Page Range / eLocation ID:
- 419 to 430
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.
This article was corrected on 20 July 2022. See the end of the full text for details.
Basic Protocol 1 : Purification of galectins using lactosyl‐sepharose affinity chromatographyBasic Protocol 2 : Purification of human galectin‐7 using a nickel‐NTA affinity chromatography columnAlternate Protocol 1 : Iodoacetamide alkylation of free sulfhydryls on galectin‐1Alternate Protocol 2 : Purification of human galectin‐9 using lactosyl‐sepharose column chromatography -
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Abstract Within this work, a new class of sequence‐defined heteromultivalent glycomacromolecules bearing lactose residues and nonglycosidic motifs for probing glycoconjugate recognition in carbohydrate recognition domain (CRD) of galectin‐3 is presented. Galectins, a family of β‐galactoside‐binding proteins, are known to play crucial roles in different signaling pathways involved in tumor biology. Thus, research has focused on the design and synthesis of galectin‐targeting ligands for use as diagnostic markers or potential therapeutics. Heteromultivalent precision glycomacromolecules have the potential to serve as ligands for galectins. In this work, multivalency and the introduction of nonglycosidic motifs bearing either neutral, amine, or sulfonated/sulfated groups are used to better understand binding in the galectin‐3 CRD. Enzyme‐linked immunosorbent assays and surface plasmon resonance studies are performed, revealing a positive impact of the sulfonated/sulfated nonglycosidic motifs on galectin‐3 binding but not on galectin‐1 binding. Selected compounds are then tested with galectin‐3 positive MCF 7 breast cancer cells using an in vitro would scratch assay. Preliminary results demonstrate a differential biological effect on MCF 7 cells with high galectin‐3 expression in comparison to an HEK 293 control with low galectin‐3 expression, indicating the potential for sulfonated/sulfated heteromultivalent glycomacromolecules to serve as preferential ligands for galectin‐3 targeting.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/glycob/cwz015
