In recent years, there has been a growing recognition that free glycans are used as signals for the initiation of a wide variety of biological processes. Such signaling events are found in development, in defense responses of plants and animals, and in interactions of organisms with one another. This chapter covers the state of current information on this emerging field of study.

NATURE AND SCOPE OF GLYCAN SIGNALING SYSTEMS

Glycan signaling systems are diverse. Simple sugars (such as glucose, fructose, and sucrose) employ various sensing systems often linked with the metabolism of the sugar and form complex webs of signaling events linked to hormones. Glycan signaling systems can also involve various glycoconjugates. For example, the addition of O-GlcNAc to cytoplasmic and nuclear proteins results in changes in the cytoskeleton, gene transcription, and enzyme activation (see Chapter 18). Glycosphingolipids can form lipid rafts, which act as a platform for sequestering signaling receptors, or can associate with receptor tyrosine kinases and modulate their activity (see Chapter 10). Membrane proteoglycans containing sulfated glycosaminoglycans (such as syndecans and phosphacan) may act as signaling molecules by interactions with kinases or phosphatidylinositol-4,5-bisphosphate (see Chapters 16 and 35). Most plasma membrane signaling receptors, including receptor tyrosine kinases and G-protein-coupled receptors, contain N-glycans and O-glycans that can modulate their stability and activity (see Chapters 8 and 9). These types of signaling events are covered in other chapters in this book and are not discussed further here.

In recent years, it has become clear that specific free glycans in picomolar to micromolar concentrations can act as signals in the initiation of a number of biological processes. These signals were first discovered as part of the defense response in plants and are now found to play roles in both plant and animal development, innate immunity, and the initiation of the nitrogen-fixing Rhizobium–legume symbiosis. Present knowledge regarding each of these areas is described in the following sections. In most of these processes, the glycans that serve as signals have been identified, but we are still in the beginning stages of identifying and characterizing their receptors and the mechanisms of signal transduction.

GLYCAN SIGNALS TRIGGER THE INITIATION OF THE PLANT DEFENSE RESPONSE

The plant defense response is a multifaceted one that includes the recognition of a pathogen, changes in ion flux across the plasma membrane, formation of reactive oxygen species, the activation of a number of genes that lead to changes in the plant cell wall, the production of glycanases that can degrade the pathogen cell wall, and the production of phytoalexins, which are antimicrobial components that kill the invading pathogen (Figure 37.1). This response leads to the formation of necrotic spots at the site of infection and it limits the spread of the pathogen. Early studies of this defense response showed that a variety of oligosaccharides derived from plant or pathogen cell wall glycans have the ability to elicit this response. This was the first indication that glycans may play signaling roles in nature, and these signals were initially called “oligosaccharins.”

FIGURE 37.1

Plant defense response. This response is initiated by a glycan elicitor produced either by the pathogen or following the interaction of the pathogen with the cell wall. The elicitor interacts with a membrane receptor to initiate a variety of cell responses (more...)

Several different oligosaccharides from plant and fungal cell walls have been found to be extremely effective elicitors at low nanomolar concentrations (~10 nM) (Figure 37.2). One of these is a heptaglucoside. This glycan was isolated from a mixture estimated to contain about 300 inactive structural isomers of this compound. Comparisons of the biological activity of this heptaglucoside with the activities of other isolated or chemically synthesized isomers and derivatives showed that both the size and position of the branches were essential for activity (Figure 37.3). Elongating the oligosaccharide at the reducing end had no significant effect on activity. Most activity was also retained by removal of a single glucose from the reducing end. However, the hexaglucoside was found to be the minimal structure that has appreciable activity.

FIGURE 37.2

Oligosaccharide elicitors of the plant defense response. (For the monosaccharide symbol code, see Figure 1.5, which is also reproduced on the inside front cover.) Symbol Key:

FIGURE 37.3

Relative biological activities of synthetic oligosaccharide isomers and derivatives of heptaglucoside. (Adapted, with permission, from Darvill A., Augur C., Bergmann C., et al. 1992. Glycobiology 2: 181–198, © Oxford University Press.) (more...)

Other glycan elicitors that have been found are oligogalacturonides, chitosan, and chitin oligosaccharides (Figure 37.2). These glycans are linear homopolymers of various lengths, and studies have focused primarily on the degree of polymerization (dp) necessary for biological activity. Such studies suggest that a dp of 10–14 is necessary to elicit a defense response by oligogalacturonides. In contrast, dps of greater than 7 and greater than 4 are necessary for the oligochitosans and oligochitins, respectively.

The low quantities of glycan signal molecules that are necessary to elicit a defense response and the different types of oligosaccharides that can act as elicitors suggest that specific receptors are available on the plasma membrane that recognize these glycans. Indeed, the plant defense system may have similarities to the innate immune system in animals, in which specific pattern recognition occurs (see below and Chapter 39).

Studies with plant cell cultures and isolated plasma membranes have demonstrated the existence of specific cell-surface or membrane-binding sites that are saturable and have binding specificities similar to those required for biological behavior. Recently, several proteins have been identified that are candidates for such receptors. One such protein is CEBiP, a chitin oligosaccharide elicitor binding protein that binds chitin elicitors and was first identified by affinity labeling as a 75-kD plasma membrane protein from cultured rice cells. This protein was recently purified and its gene was cloned. RNA interference (RNAi) studies showed that reducing expression of the gene resulted in suppression of the defense response. Although it is a membrane protein, there is no appreciable portion on the cytoplasmic side of the membrane. This protein may thus be part of an elicitor–receptor complex, but the other components in the system have not been identified.

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

The interaction between Rhizobium and leguminous plants is one of the most agriculturally and economically important symbiotic relationships that occur in nature, because it leads to the fixation of atmospheric nitrogen. A major step in the initiation of this process is the recognition by the plant of a lipooligosaccharide signal, called the Nod factor, which is produced by the bacteria. This signal stimulates a number of changes in the roots of the plant, including the formation of a new organ (the nodule) and changes in the root hairs that enable the bacteria to enter the plant and migrate to the nodule where nitrogen fixation occurs. The importance of the Nod factors in this symbiosis is demonstrated by the finding that picomolar to nanomolar levels of purified Nod factor applied to the roots of an appropriate legume species can initiate the plant responses that lead to nodule formation.

Both the initiation of nodule formation and Rhizobium entry are host strain–specific; this specificity is determined by the type of Nod factor produced by a particular Rhizobium strain and the ability of a leguminous species to recognize that signal. All Nod factors consist of a short chitin oligosaccharide backbone, but they differ among Rhizobium strains in the types of modification of this backbone. A generic structure of a Nod factor is shown in Figure 37.4 with sites of potential modification. The number of N-acetylglucosamine residues in this backbone has been found to vary from three to five. Modifications that have been identified to date include methylation, acylation (usually with a C₁₆ or C₁₈ fatty acid), acetylation, carbamylation, sulfation, glycosylation, and the addition of glycerol.

FIGURE 37.4

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

Both genetic and biochemical approaches have been useful in identifying potential Nod factor receptors in plant roots. Genetic analyses have identified a few genes from mutants of different legume species that encode proteins that may play a role in this signaling system. It is of interest that these proteins are transmembrane proteins with a serine/threonine receptor kinase motif on the cytoplasmic side of the membrane and lysine motif (LysM) domains that may recognize glycans on the exterior of the membrane. It has been postulated that these LysM domains may bind to Nod factors, although no carbohydrate-binding studies have been conducted. On the other hand, a biochemical study identified a novel lectin nucleotide phosphohydrolase (LNP) from the roots of a legume that binds Nod factors from Rhizobium symbionts of the plant species from which it was obtained. LNP is a peripheral membrane protein and it is possible that it may function in a type of receptor complex with one or more of the above proteins.

OLIGOSACCHARIDE SIGNALS IN EARLY PLANT AND ANIMAL DEVELOPMENT

Some of the glycan signals described above were found to have effects on plant growth and organogenesis. For example, oligogalacturonides (see Figure 37.2) with a dp of 12–14 are active in inducing flower formation, and oligogalacturonides with a dp of 11–14 can inhibit root formation. These glycans exert their effect on organogenesis at a concentration of approximately 400 nM and are mainly studied using tissue explants. Pectin fragments (see Chapter 22) have also been shown to affect plant growth and development by enhancing expansion of the cell, and a nonasaccharide-rich fragment of xyloglucan at a concentration of approximately 10⁻⁸ M inhibited auxin-induced elongation of pea stem segments (Figure 37.5). Some studies have shown that Nod factors can activate developmental pathways in nonlegumes, which has led to the suggestion that plants may utilize endogenous Nod-factor-like signals to regulate growth and organogenesis.

FIGURE 37.5

A nonasaccharide from xyloglucan that exhibits signaling properties. Symbol Key:

Chitin oligosaccharides may also play a role in animal embryogenesis. A gene called DG42 has chitin synthase activity and it is expressed briefly in Xenopus endoderm cells during the mid-late gastrulation stage. As discussed in Chapter 25, this gene is homologous to the Rhizobium NodC gene, which encodes a chitin synthase. Homologs of this gene were found in zebrafish and mice, and transgenic expression of the gene resulted in chitin oligomers that were digestible by chitinase. However, this gene is also homologous to a gene responsible for the synthesis of hyaluronan, and studies have shown that this gene may be able to assemble both chitin and hyaluronan. Although the biochemical function of DG42 is not certain, injection of chitinases or expression of NodZ (which encodes a fucosyltransferase that can modify chitin) also has profound effects on development. Thus, chitin oligosaccharides are examples of free glycans that act as intracellular signaling molecules.

GLYCOSAMINOGLYCANS AND CELL SIGNALING

Glycosaminoglycans (GAGs) can be considered signaling glycans because they interact with receptor tyrosine kinases and/or their ligands and facilitate changes in cell behavior (see Chapters 15, 16, and 35). For example, hyaluronan oligosaccharides can bind to specific membrane proteins, such as CD44. In some cells, binding results in 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 37.6). Like the systems described above, signaling by hyaluronan oligosaccharides depends on the degree of polymerization of the glycans, with low-molecular-weight chains more active than high-molecular-weight chains.

FIGURE 37.6

Schematic diagram of signaling pathways activated by binding of hyaluronan to CD44. In tumor cells, activation results in proliferation and invasion. In embryonic stem cells, it can result in epithelial to mesenchymal transition. Symbol Key:

In contrast to hyaluronan-dependent signal transduction, signaling via sulfated GAGs (heparan sulfate [HS] and chondroitin/dermatan sulfate) occurs by a more indirect mechanism. Few membrane receptors have been described where binding actually 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. No significant conformational change in the ligand occurs upon 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 could 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.

BACTERIAL GLYCANS AND ACTIVATION OF INNATE IMMUNITY

The innate immune system developed early in evolution as the first line of defense of eukaryotes against infection by microorganisms. A key prerequisite of this system is the ability to distinguish self from infectious nonself. In higher eukaryotes, this has been accomplished by the evolution of a range of receptors that recognize conserved molecular patterns on pathogens that are not found in the host. The term pathogen-associated molecular patterns (PAMPs) has been coined to refer to such motifs. The cognate receptors on the host cells are referred to as pattern-recognition receptors (PRRs). Many of these PAMPs are glycans found on the surfaces of bacteria that are not produced by the host. Examples are the lipopolysaccharides (LPS) of Gram-negative bacteria and the peptidoglycans of Gram-positive bacteria (see Chapter 20). A variety of PRRs are present in mammals and they recognize various PAMPs and induce host-defense pathways (see Chapter 39).

One of the best-studied models of innate immunity is the LPS of Gram-negative bacteria, which plays a role in causing septic shock (see Chapter 20). Lipid A (endotoxin), which is the glucosamine-based phospholipid anchor of LPS, is responsible for activating the innate immune system. Lipid A has a highly conserved structure among Gram-negative bacteria, which makes it an excellent PAMP. This glycan is detected at picomolar levels by a PRR, called TLR-4, which is one of the Toll-like receptors (see Figure 39.2). It is first opsonized and complexed with another host cell-surface protein, CD14. The Toll-like receptors are a major class of PRRs found on the surfaces of cells and, upon recognizing PAMPs, they activate various signaling pathways that induce inflammation and antimicrobial effector responses. Some of these receptors are expressed on antigen-presenting cells and help in the activation of the adaptive immune response. A number of other glycan PAMPs have been identified, such as peptidoglycan (which is recognized by TLR-2) and mannans (which are recognized by a soluble PRR that is a mannan-binding lectin).

FURTHER READING

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2. Denarie J, Debelle F, Prome JC. Rhizobium lipochitooligosaccharide nodulation factors: Signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem. 1996;65:503–535. [PubMed: 8811188]
3. Ebel J. Oligoglucoside elicitor-mediated activation of plant defense. Bioessays. 1998;20:569–576. [PubMed: 9723006]
4. Bakkers J, Kijne JW, Spaink HP. Function of chitin oligosaccharides in plant and animal development. EXS. 1999;87:71–83. [PubMed: 10906952]
5. Cullimore JV, Ranjeva R, Bono J.-J. Perception of lipochitooligosaccharidic Nod factors in legumes. Trends Plant Sci. 2001;6:24–30. [PubMed: 11164374]
6. Rolland F, Winderickx J, Thevelein JM. Glucose-sensing mechanisms in eukaryotic cells. Trends Biochem Sci. 2001;26:310–317. [PubMed: 11343924]
7. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003;21:335–376. [PubMed: 12524386]
8. Nurnberger T, Brunner F. Innate immunity in plants and animals: Emerging parallels between the recognition of general elicitors and pathogen-associated molecular patterns. Curr Opin Plant Biol. 2005;5:318–324. [PubMed: 12179965]
9. Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol. 2007;23:435–461. [PubMed: 17506690]
10. Raetz CRH, Reynolds CM, Tent MS, Bishop RE. Lipid A modification systems in Gram-negative bacteria. Annu Rev Biochem. 2007;76:295–329. [PMC free article: PMC2569861] [PubMed: 17362200]