Aberrant GPA expression and regulatory function of red blood cells in sickle cell disease.

ABSTRACT: Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans.

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Galectin-1 mediates interactions between polymorphonuclear leukocytes and vascular endothelial cells, and promotes their extravasation during lipopolysaccharide-induced acute lung injury.

The lung airway epithelial surface is heavily covered with sialic acids as the terminal carbohydrate on most cell surface glycoconjugates and can be removed by microbial neuraminidases or endogenous sialidases. By desialylating the lung epithelial surface, neuraminidase acts as an important virulence factor in many mucosal pathogens, such as influenza and S. pneumoniae. Desialylation exposes the subterminal galactosyl moieties - the binding glycotopes for galectins, a family of carbohydrate-recognition proteins playing important roles in various aspects of immune responses. Galectin-1 and galectin-3 have been extensively studied in their roles related to host immune responses, but some questions about their role(s) in leukocyte recruitment during lung bacterial infection remain unanswered. In this study, we found that both galectin-1 and galectin-3 bind to polymorphonuclear leukocytes (PMNs) and enhance the interaction of endothelial intercellular adhesion molecule-1 (ICAM-1) with PMNs, which is further increased by PMN desialylation. In addition, we observed that in vitro galectin-1 mediates the binding of PMNs, particularly desialylated PMNs, onto the endothelial cells. Finally, in a murine model for LPS-mediated acute lung injury, we observed that galectin-1 modulates PMN infiltration to the lung without altering the expression of chemoattractant cytokines. We conclude that galectins, particularly galectin-1, may function as adhesion molecules that mediate PMN-endothelial cell interactions, and modulate PMN infiltration during acute lung injury.

Synthesis, binding affinity, and inhibitory capacity of cyclodextrin-based multivalent glycan ligands for human galectin-3.

Human galectin 3 (Gal-3) has been implicated to play important roles in different biological recognition processes such as tumor growth and cancer metastasis. High-affinity Gal-3 ligands are desirable for functional studies and as inhibitors for potential therapeutic development. We report here a facile synthesis of ß-cyclodextrin (CD)-based Tn and TF antigen-containing multivalent ligands via a click reaction. Binding studies indicated that the synthetic multivalent glycan ligands demonstrated a clear clustering effect in binding to human Gal-3, with up to 153-fold enhanced relative affinity in comparison with the monomeric glycan ligand. The GalNAc (Tn antigen) containing heptavalent ligand showed the highest affinity for human Gal-3 among the synthetic ligands tested, with an EC50 of 1.4 µM in binding to human Gal-3. A cell-based assay revealed that the synthetic CD-based multivalent ligands could efficiently inhibit Gal-3 binding to human airway epithelial cells, with an inhibitory capacity consistent with their binding affinity measured by SPR. The synthetic cyclodextrin-based ligands described in this study should be valuable for functional studies of human Gal-3 and potentially for therapeutic applications.

Manipulating Galectin Expression in Zebrafish (Danio rerio).

Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are ß-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.

In Structural Glycobiology, Deuterium provides the Details.

In this issue of Structure, Gadjos et al. (2021b) determine the structure of a bacterial lectin in complex with L-fucose by neutron diffraction of both perdeuterated protein and carbohydrate ligand. The structure provides insight into lectin-ligand interactions, opening avenues for drug design targeting bacterial lectins for intervention in infectious disease.

F-type lectin from serum of the Antarctic teleost fish Trematomus bernacchii (Boulenger, 1902): Purification, structural characterization, and bacterial agglutinating activity.

The increasing availability of sequenced genomes has enabled a deeper understanding of the complexity of fish lectin repertoires involved in early development and immune recognition. The teleost fucose-type lectin (FTL) family includes proteins that preferentially bind fucose and display tandemly arrayed carbohydrate-recognition domains (CRDs) or are found in mosaic combinations with other domains. They function as opsonins, promoting phagocytosis and the clearance of microbial pathogens. The Antarctic fish Trematomus bernacchii is a Perciforme living at extremely low temperatures (-1.68 °C) which is considered a model for studying adaptability to the variability of environmental waters. Here, we isolated a Ca++-independent fucose-binding protein from the serum of T. bernacchii by affinity chromatography with apparent molecular weights of 32 and 30 kDa under reducing and non-reducing conditions, respectively. We have characterized its carbohydrate binding properties, thermal stability and potential ability to recognize bacterial pathogens. In western blot analysis, the protein showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, its molecular and structural aspects, showing that it contains two CRD-FTLs confirmed that T. bernacchii FTL (TbFTL) is a bona fide member of the FTL family, with binding activity at low temperatures and the ability to agglutinate bacteria, thereby suggesting it participates in host-pathogen interactions in low temperature environments.

Galectin-mediated immune recognition: Opsonic roles with contrasting outcomes in selected shrimp and bivalve mollusk species.

Galectins are a structurally conserved family of ß-galactoside-binding lectins characterized by a unique sequence motif in the carbohydrate recognition domain, and of wide taxonomic distribution, from fungi to mammals. Their biological functions, initially described as key to embryogenesis and early development via recognition of endogenous ("self") carbohydrate moieties, are currently understood as also encompassing tissue repair, cancer metastasis, angiogenesis, adipogenesis, and regulation of immune homeostasis. More recently, however, numerous studies have contributed to establish a new paradigm by revealing that galectins can also bind to exogenous ("non-self") glycans on the surface of potentially pathogenic virus, bacteria, and eukaryotic parasites, and function both as pathogen recognition receptors (PRRs) and effector factors in innate immunity. Our studies on a galectin from the kuruma shrimp Marsupenaeus japonicus (MjGal), revealed that it functions as a typical PRR. Expression of MjGal is upregulated by infectious challenge, and can recognize both Gram (+) and Gram (-) bacteria. MjGal also recognizes carbohydrates on the shrimp hemocyte surface, and can cross-link microbial pathogens to the hemocytes, promoting their phagocytosis and clearance from circulation. Therefore, MjGal contributes to the shrimp's immune defense against infectious challenge both as a PRR and effector factor. Our studies on galectins from the bivalve mollusks, however, have shown that although they can function in immune defense as MjGal, protistan parasites take advantage of the recognition roles of the host galectins, for successful attachment and host infection. We identified in the eastern oyster Crassostrea virginica two galectins (CvGal1 and CvGal2) that not only recognize a large variety of bacterial species, but also the protozoan parasite Perkinsus marinus. Like the shrimp MjGal, both oyster galectins function as opsonins, and promote parasite adhesion and phagocytosis. However, P. marinus survives intrahemocytic oxidative killing and proliferates, eventually causing systemic infection and death of the oyster host. In the softshell clam Mya arenaria we identified a galectin (MaGal1) that displays carbohydrate specificity and recognition properties for sympatric Perkinsus species (P. marinus and P. chesapeaki), that are different from CvGal1 and CvGal2. Our results suggest that although galectins from bivalves can function as PRRs, Perkinsus parasites have co-evolved with their hosts to subvert the galectins' immune functions for host infection by acquisition of carbohydrate-based mimicry.

The functional relevance of shrimp C-type lectins in host-pathogen interactions.

C-type lectins (CTLs) are key recognition proteins in shrimp immunity. A few years ago we reviewed sequence information, ligand specificity, expression profiles and specific functions of the shrimp CTLs. Since then, multiple integrated studies that implemented biochemical approaches using both the native and recombinant proteins, functional genetic approaches using RNA interference, and mechanistic studies by analyzing protein-protein interactions were carried out. Results from these rigorous studies revealed the functions and mechanisms of action of selected members of the shrimp CTL family. This review focuses on this new knowledge, that includes unique structural aspects, functions, and mechanisms in host-pathogen interactions, the functional relevance of regions other than the C-type lectin domain, and the regulation of transcription of shrimp CTLs. Thus, this review aims to provide a detailed update of recent studies that have contributed to our better understanding of the shrimp immune events that involve CTL functions.

Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 µM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.

F-Type Lectins: Structure, Function, and Evolution.

F-type lectins (FTLs) are characterized by a fucose recognition domain (F-type lectin domain; FTLD) that displays a novel jellyroll fold ("F-type" fold) and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains. Further, differences in carbohydrate specificity among tandemly arrayed FTLDs present in any FTL polypeptide subunit, together with the expression of multiple FTL isoforms in a single individual supports a striking diversity in ligand recognition. Functions of FTLs in self/nonself recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others, revealing an extensive structural/functional diversification. The taxonomic distribution of FTLDs is surprisingly discontinuous, suggesting that this lectin family has been subject to secondary loss, lateral transfer, and functional co-option along evolutionary lineages.

Purification and Biochemical Characterization of Selected F-Type Lectins.

The purification of fucose-binding lectins from the liver of striped bass (Morone saxatilis), a teleost fish, and the identification of a novel lectin sequence motif led to the identification of a new family of lectins, the F-type lectins (FTLs) (see overview of the FTL family in Chapter 23 ). Isolation and purification of these proteins from liver extracts of striped bass was accomplished by affinity chromatography and size exclusion, and their identification as FTLs, by direct Edman sequencing, and protein, transcript, and gene sequence analysis. The development of specific antibodies against the M. saxatilis FTL provided an additional tool for the identification of FTLs. These methods have been successfully used for the purification of the FTL family members from tissues and body fluids of various animal species. Production and characterization of FTLs has been facilitated by the expression of the recombinant proteins. In this chapter, the biochemical characterization of FTLs is focused on the analysis of their carbohydrate specificity.

Biochemical Characterization of Oyster and Clam Galectins: Selective Recognition of Carbohydrate Ligands on Host Hemocytes and Perkinsus Parasites.

Both vertebrates and invertebrates display active innate immune mechanisms for defense against microbial infection, including diversified repertoires of soluble and cell-associated lectins that can effect recognition and binding to potential pathogens, and trigger downstream effector pathways that clear them from the host internal milieu. Galectins are widely distributed and highly conserved lectins that have key regulatory effects on both innate and adaptive immune responses. In addition, galectins can bind to exogenous ("non-self") carbohydrates on the surface of bacteria, enveloped viruses, parasites, and fungi, and function as recognition receptors and effector factors in innate immunity. Like most invertebrates, eastern oysters (Crassostrea virginica) and softshell clams (Mya arenaria) can effectively respond to most immune challenges through soluble and hemocyte-associated lectins. The protozoan parasite Perkinsus marinus, however, can infect eastern oysters and cause "Dermo" disease, which is highly detrimental to both natural and farmed oyster populations. The sympatric Perkinsus chesapeaki, initially isolated from infected M. arenaria clams, can also be present in oysters, and there is little evidence of pathogenicity in either clams or oysters. In this review, we discuss selected observations from our studies on the mechanisms of Perkinsus recognition that are mediated by galectin-carbohydrate interactions. We identified in the oyster two galectins that we designated CvGal1 and CvGal2, which strongly recognize P. marinus trophozoites. In the clam we also identified galectin sequences, and focused on one (that we named MaGal1) that also recognizes Perkinsus species. Here we describe the biochemical characterization of CvGal1, CvGal2, and MaGal1 with focus on the detailed study of the carbohydrate specificity, and the glycosylated moieties on the surfaces of the oyster hemocytes and the two Perkinsus species (P. marinus and P. chesapeaki). Our goal is to gain further understanding of the biochemical basis for the interactions that lead to recognition and opsonization of the Perkinsus trophozoites by the bivalve hemocytes. These basic studies on the biology of host-parasite interactions may contribute to the development of novel intervention strategies for parasitic diseases of biomedical interest.

Galectins in Host-Pathogen Interactions: Structural, Functional and Evolutionary Aspects.

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 new paradigm 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.

Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase.

BACKGROUND: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? RESULTS: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. CONCLUSIONS: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Pregnancy Galectinology: Insights Into a Complex Network of Glycan Binding Proteins.

Galectins are a phylogenetically conserved family of soluble ß-galactoside binding proteins, consisting of 15 different types, each with a specific function. Galectins contribute to placentation by regulating trophoblast development, migration, and invasion during early pregnancy. In addition, galectins are critical players regulating maternal immune tolerance to the embedded embryo. Recently, the role of galectins in angiogenesis during decidualization and in placenta formation has gained attention. Altered expression of galectins is associated with abnormal pregnancies and infertility. This review focuses on the role of galectins in pregnancy-associated processes and discusses the relevance of galectin-glycan interactions as potential therapeutic targets in pregnancy disorders.

Structure of the zebrafish galectin-1-L2 and model of its interaction with the infectious hematopoietic necrosis virus (IHNV) envelope glycoprotein.

Galectins, highly conserved ß-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.