NATURE AND SCOPE OF GLYCAN SIGNALING SYSTEMS

Glycan signaling systems are diverse. Sugars (glucose, fructose, and sucrose) may be used by sensing systems, which are typically linked with the metabolism of the sugar, and form complex webs of signaling events linked to hormones. Glycan-signaling systems also involve various glycoconjugates. The addition of O-linked N-acetylglucosamine (GlcNAc) to cytoplasmic and nuclear proteins results in changes in the cytoskeleton, gene transcription, and enzyme activation (Chapter 19). Glycosphingolipids may form lipid rafts, which act as a platform for sequestering signaling receptors or can associate with receptor tyrosine kinases and modulate their activity (Chapter 11). Membrane proteoglycans containing sulfated glycosaminoglycans—including syndecans, glypicans, and phosphacan—may act as signaling molecules by interacting with kinases or phosphatidylinositol-4,5-bisphosphate (Chapters 17 and 38). Most plasma membrane signaling receptors, including receptor tyrosine kinases and G-protein-coupled receptors, contain N-glycans and O-glycans that modulate their stability and activity (Chapters 9 and 10). Binding of galectins to these glycans (Chapter 36) or removal of sialic acids by cell-surface sialidases (Chapter 15) is also thought to modulate signaling. These and other signaling processes are described elsewhere in this book and are not discussed further here.

There is increasing evidence that low concentrations of specific free glycans are signals that initiate numerous biological processes. The first evidence for such signals was obtained during studies of defense responses in plants. Subsequently, glycan perception and signaling have been shown to be important in plant and animal development, in innate immunity, and in the initiation of the nitrogen-fixing Rhizobium–legume symbiosis. The glycans that function as signals have been identified in many of these processes and have been given the generic term “oligosaccharins,” regardless of their species of origin or the biological process(es) in which they participate. In contrast, only a few of the receptors and mechanisms of signal transduction have been identified and characterized.

GLYCAN SIGNALS TRIGGER THE INITIATION OF THE PLANT DEFENSE RESPONSE

Plants have several surveillance and defensive systems to control infection by pathogens. One of these systems shows similarities with the mammalian innate immune system and consists of plasma membrane-localized pattern-recognition receptors (PRRs) that trigger defense responses upon perception of specific molecules by their extracellular ectodomains (ECDs). These ECDs can bind nonself molecules derived from pathogens (pathogen-associated molecular patterns, PAMPs) or self-compounds that are released from plant cells or synthesized on infection by pathogens (named generically damage-associated molecular patterns or DAMPs; Figure 40.1). Among the molecules perceived by PRRs are glycans (oligosaccharides) released from microbial and plant cell wall polysaccharides by hydrolytic enzymes originating from the plant or the pathogen. On binding of a ligand by its specific PRR, a PRR coreceptor is often recruited to form a protein complex, which leads to the activation of the receptor's cytoplasmic kinase domain and the initiation of defense responses. Defense response signaling can involve changes in ion flux across the plasma membrane (including a cytoplasmic Ca⁺⁺ burst), the formation of reactive oxygen species (ROS) by NADPH oxidases (RBOHs), the phosphorylation and activation of mitogen-activated and calcium-dependent protein kinases (MPKs and CDPKs, respectively) and the up-regulation of defense-associated genes (Figure 40.1). These defense responses can lead to changes in the plant cell wall, the production of glycanases that fragment the pathogen's cell wall, and the synthesis of metabolites (phytoalexins) and antimicrobial proteins/peptides that inhibit pathogen growth. These defense responses often lead to localized cell death in the plant tissue, which is visible as necrotic spots at the site of infection and limits the pathogen's spread. Of the vast number of ligands that ECD-PRRs can perceive, those composed of carbohydrate moieties are poorly studied. Only a limited number of PRR/glycan pairs have been identified and their triggered defense responses characterized. There is evidence for some similarities between the plant defense responses and animal innate immune system, most notably where specific pattern recognition occurs (Chapter 42).

FIGURE 40.1.

Plant defense responses upon glycan perception by pattern-recognition receptors (PRRs). These responses are initiated by a glycan generated by glycanases that fragment either the pathogen cell walls or the plant cell walls. The glycan interacts with specific (more...)

Early studies showed that oligosaccharides derived from cell wall glycans of the plant or the pathogen elicit numerous plant defense responses at nanomolar/micromolar concentrations. Oligogalacturonides composed of 1-4-linked α-GalA residues are one example of oligosaccharins released from plant cell wall polysaccharides, in this specific case from homogalacturonans by endopolygalacturonases (EPGs) secreted by the pathogen. The activities of such pathogen-derived EPGs are frequently inhibited or modulated by polygalacturonase-inhibiting proteins (PGIP) produced by plants. Similarly, oligoglucosides composed of 1-6- and 1-3-linked β-Glc residues released from the mycelial walls of the soybean pathogen Phytophthora sojae by plant endo-glucanases were an early example of oligosaccharins generated from a pathogen's cell wall.

Other pathogen-derived oligosaccharins are the PAMPs derived from linear homo-oligomers from fungal/oomycete cell walls that include chitin [1,4-β-D-(GlcNAc)_n], and its deacetylated form (chitosan), and β-1,3-glucan oligosaccharides (Figure 40.2). More recently, additional plant cell wall–derived oligosaccharins have been shown to trigger defense responses, including cellulose-derived oligomers (β-1,4-glucans), mixed-linkage glucans (MLGs: β-1,4/β-1,3 glucans), and oligosaccharides derived from xyloglucans, mannans, xylans, or callose, which trigger signaling cascades in Arabidopsis and other plant species, including crops (Figure 40.2). It has recently been shown that at least some “self” immune-active plant glycans are released in a regulated manner as part of normal plant developmental pathways and are not necessarily tied to cell-wall damage pathways (see below). It is also noteworthy that some immune-active oligosaccharides can be released from polymers present in the walls of both the pathogen and the plant. For example, β-1,3-glucan oligosaccharides are released from callose, which is produced by plants and is also a component of many fungal and oomycete cell walls. Similarly, β-1,4-glucan oligosaccharides are released from cellulose that is present in the cell walls of plants and some oomycetes.

FIGURE 40.2.

Oligosaccharins that are active in plants. Examples of oligosaccharides derived from fungal, oomycete, and plant cell walls are shown. The degree of polymerization (DP) of the minimally active oligosaccharide structures triggering some responses in plants (more...)

The composition (monosaccharide units), degree of polymerization (DP), and branching of the oligosaccharins determine their biological activity in triggering plant defense. For example, oligogalacturonides that are biologically active typically require a DP of 10–14 for activity, whereas DPs of >7 and >4 are necessary for the oligochitosans and oligochitins, respectively (Figure 40.2). A single active hepta-glucoside from oomycete walls was isolated from a mixture of approximately 300 inactive structural isomers and found to trigger defense responses in soybean. Both the DP and location of the β-1-3 branches in this oligosaccharide are important for its biological activity (Figure 40.2). Elongating the oligosaccharide at the reducing end had no discernible effect on bioactivity and activity was also largely retained by removal of a single glucose from the reducing end. However, the hexa-glucoside was the minimal structure of this oligosaccharide that had appreciable activity in inducing defense responses in soybean.

The low quantities and different types of glycan signal molecules that elicit defense responses suggest that these glycans are recognized by specific plasma membrane-localized receptors. A 75-kDa plasma membrane protein with high-binding affinity for chitin elicitors was identified in rice cell plasma membranes and proposed to be involved in oligosaccharide perception and signal transduction. This rice chitin oligosaccharide elicitor-binding protein (CEBiP) contains a lysozyme motif (LysM) on its ECD and no appreciable portion of the protein on the cytoplasmic side of the membrane. Similarly, a plasma membrane-localized receptor-like kinase (CERK1) with a LysM-ECD is required for chitin-induced signaling in Arabidopsis. In both rice and Arabidopsis, the perception of chito-oligosaccharins involves an additional LysM-containing receptor, CERK1 and LYK5, respectively (Figure 40.2). A similar situation has been shown to exist in legumes (Medicago), which requires LYK9 (a AtCERK1 homolog) and LYR4 (a AtLYK5 homolog) for chito-oligosaccharide (DP=8) activation of defense signaling. Members of the LysM family of Arabidopsis are also involved, probably as coreceptors, in the perception of β-1,3-glucan and β-1,4/β-1,3-glucan oligosaccharides. Recognition of pectin-derived oligogalacturonides in Arabidopsis involves wall-associated receptor-like kinases (WAKs; WAK1) that have an ECD with similarities to the epidermal growth factor (EGF) domain of mammals. Reducing or impairing expression of genes encoding these receptors in plants by RNA interference (RNAi) or mutation results in suppression of defense responses.

Nod FACTORS ARE SIGNALS FOR THE INITIATION OF THE NITROGEN-FIXING RHIZOBIUM–LEGUME SYMBIOSIS

The interaction between Rhizobium and the roots of legumes is an agriculturally and economically important symbiotic relationship because it enables the plant to fix atmospheric nitrogen. An early step in this process is the plant's recognition of lipooligosaccharide signals (Nod factors), which are produced by the bacteria (Figure 40.3). Nod factors have a chitin oligosaccharide backbone containing from three to five GlcNAc residues. However, the types of modifications of this backbone, which include methylation, acylation (typically with a C₁₆ or C₁₈ fatty acid), acetylation, carbamylation, sulfation, glycosylation, and the addition of glycerol, differ among Rhizobium strains.

FIGURE 40.3.

Generic structure of a Nod factor. Sites on the molecule where species-specific modifications can occur are designated by R1–R7. R1 = H or methyl; R2 = C16:2, C16:3, C18:1, C18:3, C18:4, C20:3, or C20:4; R3 = H, carbamate; R4 = H, carbamate; R5 (more...)

Nod factors are effective at subnanomolar concentrations, are host-specific, and stimulate numerous changes in the plant's root hairs and roots that allow the bacteria to enter the root cortex and induce the formation of nodules where nitrogen fixation occurs. The initiation of nodule formation and Rhizobium entry into the root are host strain–specific; this specificity is determined by the structure of the Nod factor produced by a particular Rhizobium strain and the ability of a leguminous species to recognize that signal.

Genetic and biochemical approaches have been used to identify potential plant root Nod factor receptors and proteins involved in the signaling events. The putative receptors are transmembrane proteins with a serine/threonine receptor kinase motif on the cytoplasmic side of the membrane and LysM domains that may recognize Nod factors on the exterior of the membrane. Two receptors (NFR5 and NFR1) have been reported to bind Nod factor directly at high-affinity binding sites, although only limited carbohydrate-binding studies were conducted. A lectin nucleotide phosphohydrolase (LNP) has been identified in legume roots and reported to bind Nod factors from Rhizobium symbionts of the plant species from which it was obtained. LNP is a peripheral membrane protein that may function in a receptor complex with one or more LysM-type proteins or act downstream of the Nod factor receptors.

Rhizobium exopolysaccharides (EPS) also have important roles in the development of nitrogen-fixing root nodules. A root receptor–like kinase (EPR3) has been identified and shown to have a role in the recognition of the bacterial EPS. Thus, receptor-mediated recognition of Nod factors and EPS signals may be involved in plant-bacterial compatibility and bacterial access to legume roots.

OLIGOSACCHARIDE SIGNALS IN PLANT AND ANIMAL DEVELOPMENT

Several glycans have been shown to affect plant growth and plant organogenesis. Nanomolar concentrations of oligogalacturonides with a DP between 12 and 14 (Figure 40.2) induce flower formation but inhibit root formation. Oligogalacturonides also enhance cell expansion and thereby affect plant growth and development. Many of these effects may result from the ability of oligogalacturonides to alter the plant's responses to the hormone auxin. Oligogalacturonide receptor proteins (WAKs) have been reported to bind to cell wall pectin and thereby affect plant cell expansion. Auxin-induced elongation of pea stem segments is inhibited by nanomolar concentrations of a nonasaccharide-rich fragment of xyloglucan (Figure 40.2). Plants may also use endogenous Nod-factor-like signals to regulate their growth and development. Recent work on plants with altered lignin structure/composition suggests that plants have the ability to monitor changes in the structures of their walls and to trigger responses distinct from those involved in plant defense. In this case, the released active oligosaccharides appear to be fragments of rhamnogalacturonans, which are distinct from the oligogalacturonides involved in plant defense. These results suggest that plants are capable of using the substantial informational content of their wall glycans to release signals for diverse cellular pathways and responses.

Chitin oligosaccharides may have a role in animal embryogenesis. The Xenopus gene DG42 encodes a protein with chitin synthase activity and is transiently expressed in endoderm cells during the mid-late gastrulation stage (Chapter 27). Homologs of DG42 have also been identified in zebrafish and mice. The DG42 protein has sequence homology with the Rhizobium NodC chitin synthase. Transgenic expression of DG42 results in the formation of glycans that are fragmented by chitinase. DG42 is also homologous to a gene encoding a hyaluronan synthase, and studies suggest that the DG42 protein synthesizes chitin and hyaluronan, with the former perhaps as an initiation primer (Chapter 16). Injection of chitinases or expression of NodZ (which encodes a fucosyltransferase that can modify chitin) in animal cells has profound effects on development. Thus, chitin oligosaccharides are examples of free glycans that appear to act as intracellular signaling molecules in animals.

Human milk contains numerous compounds that affect newborn health including lactose, lipids, and the third most abundant component, human milk oligosaccharides (HMOs). HMOs are a set of more than 150 unique oligosaccharides synthesized from lactose in the mammary gland that are essentially not digested and function, among others, as prebiotics that selectively promote growth of mutualist intestinal microbes. HMOs contain lactose at their reducing end, may be fucosylated at O-2 of Gal (2′-fucosyllactose), at O-3 (3′-fucosyllactose), or may be sialylated at O-6 or O-3 of Gal. They may also be elongated by β1-3- or β1-6-linked lacto-N-biose or N-acetyllactosamine. HMOs can be linear or branched with α1-2, α1-3, or α1-4 fucosylation and/or α2-3 or α2-6 sialylation and may contain from three to more than 30 sugar units. The amount and composition of HMOs is genetically determined, varying among women and mirroring blood group characteristics. Increasing evidence suggests that HMOs protect breastfed infants from microbial infection through cell signaling and cell–cell recognition events resulting in enrichment of protective gut microbiota and inhibition of pathogenic microbe growth, adhesion, and invasion into the intestinal mucosa. For example, 2′-fucosyllactose, representing ∼30% of HMOs in human milk, has been shown to inhibit binding and infection of distinct enteropathogens (Chapter 42) by competing for binding of microbes to mucosal surface human receptors terminating in α1-2-linked fucose, thereby inhibiting the first step of pathogenesis. Although more research is required to understand the mechanisms of HMO action, current data indicate that HMOs stimulate immunomodulatory activity at the neonatal intestinal surface and modulate cytokine production.

The presence of abnormal glycans or the accumulation of glycans in the wrong place may negatively impact signaling pathways in animal cells. Three prime repair exonuclease 1 (TREX1) is an ER-associated negative regulator of innate immunity. Mutations that affect TREX1 function are associated with numerous autoimmune and autoinflammatory diseases (Chapter 45). The ER-localized carboxyl terminus of TREX1 has been proposed to interact with, and stabilize, the catalytic activity of the ER oligosaccharyltransferase (OST) complex and thereby suppress immune activation. The OST complex becomes dysfunctional in the presence of carboxy-terminal truncated TREX1. This leads to the release of free glycans from dolichol-linked oligosaccharides, which has been hypothesized to lead to the activation of genes with immune system-related functions and the production of autoantibodies. Thus, TREX1 may safeguard the cell against free glycan buildup in the ER and thereby prevent glycan and glycosylation defects that can lead to immune disorders.

N-linked glycans have a role in the correct folding of glycoproteins in the ER. Misfolded glycoproteins are targeted for degradation by an ER-associated degradation (ERAD) process in which they are retrotranslocated into the cytosol (Chapter 39). The glycans are then released from the glycoprotein by the N-glycanase NGLY1. The protein is degraded by the proteasome, whereas the released glycans are likely partially de-mannosylated in the cytosol and then transported to the lysosomes by an as yet unidentified oligosaccharide transporter. It is not known if these free glycans have any signaling functions in the cytosolic/nuclear compartment. In the lysosome, glycosidases hydrolyze the glycans into monomeric sugars that can then be reused by the cell. Mutations that disrupt NGLY1 function may cause severe health problems in humans. Studies with Ngly1 mutant mice cells suggest that in the absence of NGLY1, ERAD becomes dysfunctional because a cytosolic endo-β-N-acetylglucosaminidase generates proteins that contain only a single Asn-linked GlcNAc instead of completely deglycosylated proteins. The accumulation of these GlcNAc-proteins may result in the formation of aggregates that are harmful to the cell or they may interfere with intracellular signaling processes.

GLYCOSAMINOGLYCANS AND CELL SIGNALING

Glycosaminoglycans (GAGs) are signaling glycans because they interact with receptor tyrosine kinases and/or their ligands and facilitate changes in cell behavior (Chapters 16, 17, and 38). Hyaluronan oligosaccharides bind to specific membrane proteins, including CD44. In some cells, this binding leads to clustering of CD44, which activates kinases such as c-Src and focal adhesion kinase (FAK). Phosphorylation alters the interaction of the cytoplasmic tail of CD44 with regulatory and adaptor molecules that modulate cytoskeletal assembly/disassembly and cell survival and proliferation (Figure 16.6). Signaling by hyaluronan oligosaccharides depends on the DP of the glycans. Low molecular weight glycans are more active in triggering danger responses via binding to Toll-like receptors (TLRs).

In contrast to hyaluronan-dependent signal transduction, signaling via sulfated GAGs such as heparan sulfate (HS) and chondroitin/dermatan sulfate occurs by an indirect mechanism. Indeed, few membrane receptors have been described in which sulfated GAGs binding causes a specific downstream response, such as phosphorylation of the receptor or activation of a kinase. Instead, sulfated GAGs bind to many ligand/receptor pairs, thereby lowering the effective concentration of ligand required to engage the receptor or increasing the duration of the response. An example of this is the ability of exogenous heparin or endogenous HS proteoglycans to activate fibroblast growth factor (FGF) receptors by FGF (Chapter 38). No substantial conformational change in the ligand occurs on binding to sulfated GAG, consistent with the idea that the glycan primarily aids in the juxtaposition of components of the signal transduction pathway. Free HS oligosaccharides can be released by the action of secreted heparanase. These glycans may facilitate signaling through the mechanism described above or by the release of growth factors from stored depots in the extracellular matrix. Sulfated GAGs also facilitate the formation of morphogen gradients in tissues during early development. Because the gradient determines cell specification during development, the glycan indirectly affects signaling responses in receptive cells. These examples do not exclude the possibility that sulfated glycosaminoglycans may act as ligands and induce signaling directly (e.g., by ligating receptors).

GLYCANS AS MODULATORS OF INNATE IMMUNITY

In addition to mucins (Chapter 10), the innate immune system developed early in eukaryote evolution is a first line of defense against infection by microorganisms. A key feature of this system is its ability to distinguish self from infectious nonself. In more advanced eukaryotes, this is accomplished by receptors that recognize conserved molecular patterns specific to the pathogens. Many of these PAMPs are glycans located on the surfaces of the microorganism. The glycans include the lipopolysaccharides (LPS) of Gram-negative bacteria, the peptidoglycans and techoic acids of Gram-positive bacteria (Chapters 21 and 22) and the mannans and glucans of fungi, perceived by PRRs. Just as in plants (as described above), numerous PRRs are present in mammals that recognize diverse PAMPs and induce host-defense pathways, including TLRs and mannan-binding lectin (Chapter 42). Binding of PAMPs to TLRs activates various signaling pathways that induce inflammation and antimicrobial effector responses. Some TLRs are present on antigen-presenting cells and help to activate the adaptive immune response. TLRs also respond to tissue injury via binding of released hyaluronan fragments as DAMPs (Figure 16.6).

One of the best-studied models of mammal innate immunity involves the LPS of Gram-negative bacteria, which has a role in causing septic shock (see Chapter 42). Lipid A (endotoxin) is the glucosamine-based phospholipid anchor of LPS responsible for activating the innate immune system. Lipid A is an excellent PAMP as its structure is highly conserved among Gram-negative bacteria. Picomolar concentrations of lipid A are detected by TLR-4. The LPS is first opsonized and complexed with another host cell-surface protein, CD14. The binding of LPS leads to recruitment of the adaptor proteins MyD88 and IRAK. This complex initiates a signaling cascade of phosphorylation events that ultimately lead to the transcription of proinflammatory genes.

In contrast to PAMPs and DAMPs, inhibitory Siglec receptors (Chapter 35) on mammal innate immune cells recognize endogenous sialylated glycoconjugates as self-associated molecular patterns (SAMPs) and dampen unwanted immune reactions against the host. Details of the sialoglycan specificity involved require further investigation, but pathogens take advantage of the system via molecular mimicry (Chapters 7 and 42).

Plants also exhibit a type of innate immunity that on activation could confer resistance to pathogen attack to the entire plant. Preparations of β-1,3-glucans, MLGs and xyloglucan oligosaccharides (Figure 40.2) triggering defensive responses in plants are able to confer protection against different pathogens when applied exogenously to crops. Xyloglucan oligosaccharides effectively protected grapevine and Arabidopsis against the fungal/oomycete pathogens, whereas β-1,3-glucans improved, among others, tobacco, and grapevine protection against bacterial, fungal, and oomycete pathogens. Given the high abundance of β-1,3-glucans in brown seaweed, laminarin-based products have been successfully developed for use in agriculture as activators of plant natural defense against pathogens. Similarly, pretreatments with cellobiose reduced growth of some pathogens on Arabidopsis seedlings, although high doses were required to observe such effects. In all of these cases, PRRs involved in the perception of these oligosaccharide structures that are active in generating these resistance responses in plants have not yet been characterized in detail.

The examples given in this chapter indicate the diversity of free glycan structures that can function as signaling molecules. It is likely that further examples of glycan signals in both plant and animal cells, as well as in their interactions with microbes, will become apparent in the future.

ACKNOWLEDGMENTS

The authors appreciate the helpful comments, suggestions, and contributions from Xi Chen and Gabriel A. Rabinovich and by Laura Bacete.

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