7.4: The Sugar Code and Lectin Decoding

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

The Glycan Code and Glycan-Binding Protein Families

• Explain the "glycan code" concept — that the structural diversity of glycans (arising from monosaccharide identity, anomeric configuration, linkage position, branching, chain length, and chemical modifications including sulfation and sialylation) encodes biological information that is "written" by glycosyltransferases and glycan-modifying enzymes without a genetic template and "read" by glycan-binding proteins (GBPs) — and distinguish the nine classes of GBPs (including viral, bacterial, and eukaryotic lectins), defining a lectin as any protein that specifically recognizes and binds a glycan motif without chemically modifying it.
• Distinguish the structural features and Ca²⁺ dependence of the three major animal GBP families: C-type lectins (largest family, Ca²⁺-dependent carbohydrate recognition domain with EPN and WND motifs that determine mannose/GlcNAc/Fuc vs. Gal/GalNAc specificity, respectively), galectins (Ca²⁺-independent β-sandwich jellyroll CRD that binds Galβ1,3GlcNAc or Galβ1,4GlcNAc disaccharides, expressed in virtually all cells), and siglecs (sialic acid-binding immunoglobulin-like lectins with N-terminal V-set Ig domain expressed on immune cells, recognizing sialylated oligosaccharides through a conserved Arg residue, with cytoplasmic ITIM domains that transduce inhibitory signals).

P-Selectins, Integrins, and Cell Adhesion

• Describe the molecular mechanism of leukocyte rolling and capture during inflammation — explaining how P-selectin (a C-type lectin stored in intracellular granules of platelets and endothelial cells and rapidly translocated to the cell surface on activation) binds the SiaLewX (sialyl-Lewis X) glycan on P-selectin glycoprotein ligand 1 (PSGL-1) on circulating leukocytes to mediate low-affinity transient rolling, and how tyrosine sulfation at Tyr 607 of PSGL-1 dramatically increases binding affinity by adding electrostatic interactions with the positively charged P-selectin surface — explaining why the selectin domain structure (CLECT + EGF + multiple CCP domains + transmembrane domain) is suited for cell adhesion at physiological flow rates.
• Explain the sequential steps of leukocyte extravasation — from selectin-mediated rolling (low-affinity carbohydrate-protein interaction) to integrin activation (triggered by selectin engagement) to firm adhesion (integrin binding to ICAM/VCAM through protein-protein interactions involving RGD and LDV motifs on extracellular matrix proteins) to transendothelial migration — and connect this cascade to L-selectin-mediated blastocyst implantation in the uterus as a biologically analogous non-immune example of glycan-mediated cell capture.

Galectins, Siglecs, and Glycan-Mediated Immune Regulation

• Explain the immunological roles of siglecs and galectins — describing how Siglec-8 on basophils, mast cells, and eosinophils is engaged by multivalent sialylated mucin glycoproteins bearing 6′-sulfo sLex epitopes during inflammation, triggering ITIM-mediated inhibitory signaling that restrains or terminates immune cell function — and connect the sialic acid type (Neu5Ac in humans vs. Neu5Gc in chimps) and differential siglec expression on T cells to the striking difference in susceptibility to inflammatory diseases and HIV infection between humans and great apes, noting also that hypersialylated CD43 on acute myeloid leukemia cells creates a "glyco-immune barrier" that shields cancer cells from macrophage phagocytosis.
• Describe the mannose receptor (CD206) as an example of a multidomain C-type lectin that reads diverse glycan codes — explaining how its CLECT domains (particularly CLECT 4) bind mannose, GlcNAc, and fucose in a Ca²⁺-dependent manner (recognizing equatorial OHs at C3/C4), while its fibronectin type 2 (FN2) cysteine-rich domain binds sulfated glycans like 3-SO₄-Lewis X in a Ca²⁺-independent, non-mannose-containing manner — and connect this dual specificity to the mannose receptor's dual roles as (1) a scavenger receptor that clears asialoglycoproteins and aged circulating glycoproteins from blood, and (2) a pattern recognition receptor on macrophages/dendritic cells that mediates phagocytosis of mannose-displaying fungal and bacterial pathogens.

Introduction

By now, you should be convinced that the structures of glycans are extraordinarily complex and, in many ways, much more complicated than proteins and nucleic acids. Their structural diversity is staggering, given the number of different sugar monomers, stereocenters, linkages, lengths, conformers, dynamic flexibility, and chemical modifications. Yet evolution has allowed this astronomical diversity, which must serve more than just simple functions such as protecting proteins from degradation. Much of the diversity derives from a lack of an equivalent genetic code for glycan synthesis.

Since all biological events start with a binding interaction, let's ponder the binding of glycans to partner "ligands" such as proteins, lipids, and nucleic acids. A binding site on a glycan could be a single monosaccharide or a much larger and more complex interface. Figure \(\PageIndex{1}\) shows an interactive iCn3D model of one of the few glycoproteins with pdb coordinates, the unliganded simian immunodeficiency virus (SIV) gp120 core glycoprotein (3fus).

Figure \(\PageIndex{1}\): Simian immunodeficiency virus (SIV) gp120 core glycoprotein (3fus). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?DzZYhLp8JL1vPYYw6

The protein surface is shown in ivory, and the glycans are shown as color-coded sticks, with the correct symbolic spheres or cubes around them.

Now let's convert in our imagination an image file showing one face of the protein to a black and white QR code as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): An imaginary QR code for the surface of a glycoprotein.

Computers can recognize information encoded in QR codes and decode it into another form, such as a restaurant menu. Likewise, organisms have evolved "readers" to decode the glycan code written by enzymes (glycan synthases, hydrolases, and modifying enzymes). The glycan code is written onto the 3D surfaces of polysaccharides, glycoproteins, glycolipids, and proteoglycans. It should be no surprise that the biological readers of the glycan code are mostly proteins, which locate and bind to the correct "QR" code displayed on the glycan surface.

Luckily, the QR code metaphor for the glycan code is a bit exaggerated since the readers of the glycan code, glycan-binding proteins, seem to recognize just small sections of a glycan. They can be compared to antibodies, which bind to foreign molecules, such as proteins. The binding site on a foreign protein recognized by an antibody is called an epitope. Epitopes can be continuous (linear) stretches of the foreign protein sequence or discontinuous (conformational), made of some continuous stretches of amino acid and some further away in the sequence but close in the 3D folded protein. The average continuous epitope is often 5-6 amino acids long. Yet that might be an underestimation, since an analysis of all contact residues (within a conservative 4 Å distance) for target proteins and their bound antibodies in the Protein Data Bank found that each is around 18-19 amino acids (Stave and Lindpaintner). Glycan-binding proteins presumably also bind a mixture of continuous and discontinuous glycan sequences. Linear one would be much easier to determine and study.

Now, let's explore the family of these glycan-binding proteins (GBP), the readers of the glycan code.

Glycan-Binding Proteins (GBPs)

There appear to be nine types of glycan-binding proteins (GBPs). These include nonenveloped capsid virus GBPs,enveloped-virus GBPs (ex., influenza and coronaviruses), eukaryotic microbial GBPs (ex, yeast), and bacterial toxin GBPs (ex, botulinum toxin). Bacterial adhesins (parts of organelles like flagella), lectins (soluble proteins), and lectin domain-containing proteins are also examples of glycan-binding proteins (GBP). We will discuss in more detail three other types: C-type lectins, galectins, and siglecs.

In the broadest sense, if a lectin is a protein that binds a specific carbohydrate motif (i.e., a glycan code) without modifying the motif, then any glycan-binding protein could be called a lectin. This excludes enzymes that synthesize, degrade, or modify glycans, as well as antibodies that recognize foreign or self-glycan sequences. Table \(\PageIndex{1}\) below shows some lectins and their target glycan ligand from plants, animals, viruses, and bacteria.

Lectin Family/Lectin	Abbreviation	Ligand(s)
Plants
Concanavalin A	ConA	Man α1- OCH3
Griffonia simplicifolia lectin 4	GS4	Lewis b (Leb) tetrasaccharide
Wheat germ agglutinin	WGA	Ner5Ac(α 2,3)Gal(β 1,4)GlcGlcNAc(β1,4)GlcNAc
Ricin		Gal(β 1,4)Glc
Animals
Galectin-1		Gal(β 1,4)Glc
Mannose-binding protein	MBP-A	High Mannose Octasaccahride
Viral
Influenza Virus hemagglutinin	HA	Neu5Ac(α 2,6)Gal(β1,4)Glc
Polyoma virus protein 1	VP1	Neu5Ac(α 2,3)Gal(β1,4)Glc
Bacterial
Enterotoxin	LT	Gal
Cholera toxin	CT	GM1 pentasaccharide

Table \(\PageIndex{1}\): Lectin families and their ligands.

In animals, lectins facilitate cell-cell interactions by forming multiple but weak interactions (also called multivalent interactions) between the protein and many sugars on the ligand to which it binds.

Now, let's consider the other three classes of glycan-binding proteins (or lectins), C-type lectins, galectins, and siglecs, in more detail. Focus on the very different structures of the carbohydrate-binding domains.

C-Type Lectins

C-type lectins comprise the largest number of glycan-binding proteins. These proteins have a glycan or carbohydrate-recognition domain that depends on Ca²⁺. They bind self-glycans and those on pathogens, thereby targeting viruses to specific cells. Many are on the surface of immune cells. They have an N-terminal glycan-binding domain called a C-lectin (CLECT) or carbohydrate recognition domain (CRD). However, some proteins with the domain do not appear to bind either Ca²⁺ or glycans. They serve as adhesion molecules and are also involved in cell signaling. Some residues in the lectin-binding domain appear critical for binding lectins. These include an EPN motif, which interacts with Man, GlcNAc, Fuc, and Glc, and a WND motif that binds Gal and GalNAc.

Let's now look at one example of a C-type lectin: the selectins.

P-Selectins

These are involved in the interaction of immune cells in the blood with endothelial cells that line the blood vessel wall. Consider the challenges an immune cell faces as it moves from the blood into a tissue where an infection might occur! Blood flow in vessels is at a rate inversely proportional to the total cross-sectional area of the blood vessel. That rate is about 5-20 cm/sec in arteries, 1.5-7 cm/sec in veins, and about 1 mm/sec (1000 μm)/sec) in capillaries. Assuming a lymphocyte's average diameter of 10 μm, the cell would move about 100 cell lengths per second. An equivalent speed for a human with an arm span of 6 feet (approximately fitting into a circle of diameter = 6 feet as drawn by Leonardo da Vinci) would be around 600 feet/second. The cell must go from its typical circulating speeds to a stop before moving through the blood vessel wall into tissues. Nature has solved this by providing a way to slow down the moving cell until its final capture. The cells roll along the endothelial cells, making transient low-affinity interactions that slow it down enough for high-affinity interactions to effectively stop it (unless it dissociates first).

Also, you wouldn't want immune cells to stop and move into tissue without an infection signaled by mediator molecules. Another problem solved! P-selectins are stored in the intracellular granules of platelets (the source of the name P-selectin) and endothelial cells, so moving immune cells are not spuriously captured without some signal. In the presence of the appropriate chemical signal, endothelial cells and platelets become activated, and P-selectin is rapidly transported to the cell surface, where "capture" occurs before the cell can move into the underlying tissue. P-selectin mediates the first transient interactions and subsequent rolling of immune cells on activated platelets and endothelial cells.

Figure \(\PageIndex{3}\) is a video animating the rolling and "capture" of a lymphocyte by endothelial cells. (See the video for the reference.) Note that cancer cells can also move through the endothelial cells of blood vessels in forming metastases.

Figure \(\PageIndex{3}\): Video animation of a lymphocyte rolling and being captured by endothelial cells lining blood vessel walls

P-selectins, hence, are receptors for molecules on immune cells. They bind Ca²⁺ ions, which helps create an active conformation. Their binding ligands are glycan codes and the nearby sections of the protein to which they are attached. The glycan ligand on the surface of a circulating immune cell is the sialyl-Lewis X (SiaLew^X) glycan or a derivative of it. One of the immune membrane proteins with SiaLew^X is the P-selectin glycoprotein ligand 1 (PSGL-1, the gene name), also called SELPLG. It mediates rapid rolling of leukocytes over vascular surfaces during the initial steps in inflammation through interaction with SELPLG"

P-selectin is a mediator of cell adhesion (to other cells). As such, it could also be classified as an adhesion protein. The three main types of selectins:

1. L-selectins: found on leukocytes ("white" blood cells that are circulating immune cells).
2. P-selectins: found on activated platelets (which can aggregate to form a type of blood clot) and activated endothelial cells. Activation occurs during the inflammatory response, which can lead to the rapid movement of pre-formed selectins from the cytoplasm to the membrane. In addition, their expression can be induced.
3. E-selectins: found on activated endothelial cells only after the cells have been induced to form them by certain immune hormones called cytokines, released by immune cells during an inflammatory response.

Figure \(\PageIndex{4}\) shows the domain structure of human P-selectin.

Figure \(\PageIndex{4}\): Domain structure of human P-selectin (from https://smart.embl.de/)

It contains an N-terminal C-Lectin (CLECT) domain, also called the carbohydrate-recognition domain (CRD) or C-type lectin domain (CTLD). In addition, it has an epidermal growth factor (EGF) domain, nine complement control protein (CCP) domains, and a blue transmembrane domain.

Figure \(\PageIndex{5}\) shows the structure of the SLew^x glycan along with its symbol nomenclature for glycans (SNFG) representation.

Figure \(\PageIndex{5}\): Structure of the SLew^x glycan

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the crystal structure of P-selectin lectin/EGF domains complexed with SLeX (1g1r) to which P-selectin binds with weak affinity. Fucose interacts with the Ca²⁺ ion. The glycan interacts with the CLECT domain.

Figure \(\PageIndex{6}\): P-selectin lectin/EGF domains complexed with SLeX (1g1r). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..s1GYbAfXfnuSz9

SLew^x is not present in isolation but rather attached to a membrane protein on an immune cell, which serves as a ligand for the P-selectin on activated endothelial cells or platelets. (The SLew^x can also be part of a glycolipid.) Now let's contrast the interactions of the P-selectin LE domain with "naked" SLewx with those between P-selectin LE and a higher-affinity natural binding ligand, human P-selectin glycoprotein ligand 1 (PSGL-1), an integral membrane protein of immune cells. PSGL-1 is expressed on neutrophils, monocytes, and most lymphocytes. The P-selectin:PSGL-1 complex has a much lower K_D (higher affinity) than the binding of the unmodified SLe^X (1g1s). PSGL-1 is a disulfide-linked homodimer. When sulfated on a specific Tyr (48), the protein displays high affinity for P-selectin. In contrast, when sulfated on a different Tyr (51), it displays a high affinity for L-selectin instead.

The SLex^X type glycan O-linked to the peptide is a bit more complicated than the simple SLex^X ligand, as it is connected to a protein through an O-linked bond at a threonine. The SNFG is shown in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): SNFG if SLex^X type glycan O-linked to a peptide

The crystal structure of a trisulfated, SLew^x-modified peptide from the N-terminal region of PSGL-1 (1G1S) bound to P-selectin lectin and EGF domains (P-LE) has been solved. Figure \(\PageIndex{8}\) shows an interactive iCn3D model that shows some of the interactions between the PSGL-1 peptide (green backbone) and P-LE (magenta backbone).

Figure \(\PageIndex{8}\): P-selectin Lectin/EGF domains and bound PSGL-1 peptide (1G1S). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..E2mVULZc8WqVR9

In the crystal structure, the peptide from the P-selectin ligand (which again is a membrane protein) contains three sulfated tyrosine residues (605, 607, and 610), which correspond to amino acids 5, 7, and 10 in the peptide. No electron density for the side chain of Tyr 605 was seen. Tyr 607 binds through multiple interactions to the P-selectin LE domain and is most likely responsible for the high-affinity interaction of P-selectin with the P-selectin glycoprotein ligand (again represented by the green chain). In contrast, Tyr 610 interacts with the peptide glycan SLex^X via an intermediary water molecule.

Figure \(\PageIndex{9}\) shows the electrostatic surface potential map of one of the dimers of the P-select LE domains. Blue represents positive potential, and red represents negative. The backbone of the P-selectin ligand peptide is green, with all negatively charged side chains (Tyr, Asp, and Glu) shown in stick representation, with CPK colors. Note that these amino acids are all bound in blue (positive) regions of P-selection. The glycan portion attached to the peptide (stick, CPK color) is positioned mostly over negative potential, allowing hydrogen bonding between the sugar OHs and the protein.

Figure \(\PageIndex{9}\): Electrostatic surface potential map of one monomer of the P-selectin LE domains with bound P-selectin ligand peptide.

You could surmise that the blue region of positive potential could also bind other strongly negatively charged ligands (such as heparin and other glycosaminoglycans), which could inhibit the function of this protein, as it would prevent binding of the PSGL-1.

Figure \(\PageIndex{10}\) shows an interactive iCn3D model, which shows the surface electrostatic potential of the P-selectin Lectin/EGF domains and bound PSGL-1 peptide

Figure \(\PageIndex{10}\): Electrostatic potential of the P-selectin Lectin/EGF domains and bound PSGL-1 peptide (1G1S). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..X2CSQQzgbFn2VA

The blue represents positive potential, and the red represents negative. The backbone of the P-selectin ligand peptide is green, with all negatively charged side chains (Tyr, Asp, and Glu) shown in stick representation with CPK colors.

There are also nonpolar interactions that are not shown in the figure and model above. The aromatic ring of Tyr 607 (7) interacts with the nonpolar parts of a Ser (-CH₂) and Lys (-CH₂)₄ side chains, and the ring of Tyr 610 (10) interacts with two leucine side chains.

The selectins are also part of a class of molecules called adhesion molecules. As described for the selectins, adhesion molecules contain

• an extracellular CHO binding domain (the lectin domain), which mediates binding to adjacent cells or the extracellular matrix;
• a transmembrane domain;
• and a cytoplasmic domain, which often interacts with the cytoskeleton within the cell.

This initial binding mediated by selectin-CHO interactions activates the expression of another adhesion molecule on the leukocyte, integrin, a heterodimer with an a and b chain. These cause strong leukocyte-endothelial cell interactions, leading to leukocyte movement through the vessel wall. Other classes of adhesion molecules (in addition to selectins and integrins) are cadherins (calcium-dependent adhesion molecules) and the immunoglobulin-like superfamily (ICAM1, ICAM2, VCAM). VCAM (Vascular Cell Adhesion Molecule) binds to integrins expressed on activated lymphocytes, allowing lymphocytes to pass from the vessel lumen into the tissues. Integrins appear to bind proteins in the extracellular matrix through RGD (Arg-Gly-Asp) and also through LDV (Leu-Asp-Val) motifs on the proteins, including fibronectin (RGD), thrombospondin (RGD & LDV), fibrinogen (RGD & LDV), von Willebrand Factor (RGD), and vitronectin (RGD). They also bind other matrix proteins with an "alpha domain," including collagen and laminin. Integrin/Adhesion molecule interactions involve protein/protein interactions.

A fertilized egg (in the blastocyst stage, which is ready for implantation in the uterine cell wall) expresses L-selectin, which allows a low-affinity (rolling-type) interaction of the fertilized egg with the uterine epithelial cells. These cells expressed the CHO ligands on their surface, which bind to the L-selectin on the blastocyst. The CHO ligands are only transiently expressed on the surface of the epithelial cells of the uterus, presumably only when the uterus is primed for implantation. After the initial interaction of the blastocyst and epithelial cells, further expression of integrins on the blastocyst surface might result. Problems in any of these molecular steps could result in infertility. Figure \(\PageIndex{11}\) shows endothelial cell/leukocyte interactions mediated through selectins, integrins, and ICAMs.

Figure \(\PageIndex{11}\): Endothelial cell/leukocyte Interactions mediated through selectins, integrins, and ICAMs

Post-translational modifications of proteins (such as glycosylation) can confer new binding and biological functions. Site-directed mutagenesis can replace surface amino acids with cysteine or methionine with nonnatural amino acid analogs that contain azide or alkyne groups. These modified groups could then direct the location of chemical-modifying reagents (such as sugars) to these sites. A protein completely unrelated to PSGL-1 has been selectively modified to contain covalently attached glycans and sulfated tyrosine side chains—the unrelated protein bound to P-selectin.

Mannose Receptor.

What do you do with a protein that no longer has the correct structure(s) to perform its designed function(s)? Proteins, as with any molecule, undergo chemical changes during their biological lifetime. They must be recognized as aberrant and then removed from "service," ultimately being degraded into component amino acids for reuse. There are no repair enzymes for proteins as there are for DNA. One modification that changes glycoproteins and signals the need for their removal is the removal of the terminal sialic acid residue, forming asialoglycoproteins, whose glycans end in galactose, as you can envision from Figure \(\PageIndex{12}\), which shows a typical structure of an N-linked glycoprotein.

Figure \(\PageIndex{12}\): Typical structure of a N-linked glycoprotein

The asialoglycoprotein receptor, a member of the C-type lectin family, is a transmembrane protein that binds terminal galactose and N-acetylgalactenables macrophages (immune cellsialoglycoproteins, leading to their endocytosis into the cell. It is expressed on the surface of hepatocytes (liver cells). Receptors of this type are also called scavenger receptors, as they remove proteins from circulation.

The mannose receptor (also called CD206), also expressed in liver endothelial cells, is another C-type lectin involved in the binding and removal of glycoproteins from the circulation. It binds both sulfated and non-sulfated glycans. It is also a receptor that allows macrophages (immune cells) and dendritic cells to bind and phagocytose bacterial and fungal pathogens. Unfortunately, tumor cells also interact with macrophages using the mannose receptor (CD206/MRC1) expressed on tumor-associated macrophages (TAMs).  This promotes metastasis, blood vessel growth, and, in general, immunosuppressive activities. The protein binds and scavenges sulfated glycoprotein hormones, mannose-bearing glycoproteins released during inflammation, lysosomal enzymes released from cells on injury, and collagen fragments.

Figure \(\PageIndex{13}\) shows the domain structure of the human mannose receptor.

Figure \(\PageIndex{13}\): Domain structure of the human mannose receptor.

Given the large number of CLECT domains, you might surmise that this protein could bind many different target glycans from both self and pathogens. What is different about the domain structure compared to P-selectin is the presence of an N-terminal Ricin and a Fibronectin type 2 (FN2) domain. The FN2 domain has two cystines from the four conserved cysteines involved in disulfide bonds. What's so interesting about the mannose receptor is that it binds glycans both in the CLECT domains and in the FN2 domain.

Glycan binding at the CLECT domain: The CLECT domain binds targets containing mannose, fucose, and N-acetylglucosamine with a preference for Man(α1,2)Man or fucose. Figure \(\PageIndex{14}\) shows an interactive iCn3D model. of the CLECT 4 domain of the mannose receptor complexed with Man(α1,2)Man (7jue). Interactions of fucose ligands, such as the Lewis a trisaccharide, strengthen binding.

Figure \(\PageIndex{14}\): CLECT 4 domain of the mannose receptor complexed with Man(α1,2)Man (7jue). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..jhn5eMLMrG6mX7

The receptor can bind a variety of glycans. Both mannose and N-acetylglucosamine interact with bound Ca²⁺ through equatorial OHs on carbon 3 and 4 of the ring, while fucose uses OHs on carbon 2 and 3, or 3 and 4.

The interactions with fungal pathogens are medically important. Fungi, like yeast, have an outer structure composed of a membrane bilayer and a mixture of glycans, which deploys an incredibly complex "glycan code" to host cells infected by them, as illustrated in Figure \(\PageIndex{15}\).

Figure \(\PageIndex{15}\): Structure of fungal cell wall. Kang, X., Kirui, A., Muszyński, A. et al. Molecular architecture of fungal cell walls revealed by solid-state NMR. Nat Commun 9, 2747 (2018). https://doi.org/10.1038/s41467-018-05199-0. Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0/

Mannans, polymers of just mannose, differ widely in structure. Their main backbone can be DMan(α-1,6)DMan or DMan(β-1,4)DMan with many branches.

Glcyan binding at the FN2 (Cysteine-Rich) Domain (1FWU): The mannose receptor can also bind non-mannose sulfated glycans, such as 3-SO4-LEWIS(X), for which the SNFG representation is shown in Figure \(\PageIndex{16}\).

Figure \(\PageIndex{16}\): 3-SO4-LEWIS(X) non-mannose sulfated glycans for mannose receptor

The mannose receptor binds this glycan, which does not even contain mannose, through the FN2 domain (which contains four disulfide bonds) and not the CLECT calcium-dependent carbohydrate-binding domain. Hence, the protein can bind both sulfated and nonsulfated glycans.

Figure \(\PageIndex{17}\) shows an interactive iCn3D model of the complex of the FN2 domain of the mannose receptor with the non-mannose containing 3-SO4-LEWIS(X) glycan (1fwu).

Figure \(\PageIndex{17}\): FN2 domain of the mannose receptor with the non-mannose containing 3-SO4-LEWIS(X) glycan (1fwu). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..kXn2Rz8qMzebo9

Look at the number of CLECT domains in the domain structure diagram for the mannose receptor above. Along with interactions of sulfated glycans at the FN2 domain, these would enable the binding of widely diverse glycan structures. Reported ligands for the mannose receptor include those with high mannose content released during inflammation (lysosomal hydrolases, collagen peptides, and tissue plasminogen activator), and sulfated ones (including the pituitary hormones lutropin and thyrotropin.

Galectins

This family of glycan-binding proteins contains a common carbohydrate recognition domain (CRD) of about 130 amino acids, which binds Galβ1,3GlcNAc or Galβ1,4GlcNAc disaccharides (hence the name galectins) as well as other glycan motifs. They are expressed in almost all cells and multicellular organisms. There are 15 different types grouped in how the CRD is functionally expressed (as dimers, tandem repeats, or chimeras), as illustrated in Figure \(\PageIndex{18}\). The figure also shows their role in cancer biology.

Figure \(\PageIndex{18}\): Galnectins structure and function Shimada, C.; Xu, R.; Al-Alem, L.; Stasenko, M.; Spriggs, D.R.; Rueda, B.R. Galectins and Ovarian Cancer. Cancers 2020, 12, 1421. https://doi.org/10.3390/cancers12061421. Creative Commons Attribution License

The carbohydrate-binding domain of the galactins has a jellyroll-like protein architecture with two anti-parallel β-sheets forming a β-sandwich.

Galectin I

This protein is secreted and found in the extracellular matrix and the cytoplasm. It induces apoptosis in T-cells. It binds beta-galactosides and other glycans. The main ligand of galectin-1 has a Galβ1-4GlcNAc (or LacNAc) structure. Figure \(\PageIndex{19}\) shows an interactive iCn3D model of Human Galectin-1 in Complex with Type 1 N-acetyllactosamine (Gal(β1,3)GlcNAc), which binds less tightly than Galβ1,4GlcNAc (Type 2)(4XBL)

Figure \(\PageIndex{19}\): Human Galectin-1 in Complex with Type 1 N-acetyllactosamine (Gal(β1,3)GlcNAc) (4XBL). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..MTyV7QvPKqqibA

A comparison of the crystal structures shows different phi/psi angles for bound Type I (135°) versus the more tightly bound Type 2 (-108°), highlighting the nuance in binding conformations in the interactions between glycans and glycan-binding proteins.

Siglecs

The proteins are sialic acid-binding immunoglobulin (Ig)-like lectins found on immune cells like basophils, macrophages, mast cells, and eosinophils. One type (Siglec-4) is found in myelinated structures of the central and peripheral nervous systems. They all have an N-terminal extracellular immunoglobulin domain (abbreviated as IG or V-Set) and a differing number of IG-like domains, also called C2-set Ig domains. The glycan binding epitope recognized by Siglecs is sialylated oligosaccharides on a protein section containing a conserved arginine. Figure \(\PageIndex{20}\) compares the domain structures of the human Siglec family.

Figure \(\PageIndex{20}\): Domain structures of the human Siglec family Siddiqui, S.S.; Matar, R.; Merheb, M.; Hodeify, R.; Vazhappilly, C.G.; Marton, J.; Shamsuddin, S.A.; Al Zouabi, H. Siglecs in Brain Function and Neurological Disorders. Cells 2019, 8, 1125. https://doi.org/10.3390/cells8101125. Open access article distributed under the Creative Commons Attribution License

Here is one example of a Siglec.

Siglec-8

This protein is expressed on the surface of immune cells like basophils, mast cells, and eosinophils. When activated by infection and prolonged inflammation, they release the contents of intracellular granules, which have potent physiological effects that can lead to allergic and asthmatic responses. In infection and other inflammatory states, immune cytokines are released, which, through signaling, trigger the release of sialoglycans that act as ligands, binding to Siglec-8 on the surface of immune cells. One type of sialoglycan released is mucins, which are very large glycoproteins with many 6′S sLe^x glycans attached. These "multivalent" glycan epitopes can bind to Siglec-8, leading to signaling in the cells and ultimate inhibition of cell function (including by death or apoptosis). The mucins in mucus (cross-linked mucins), which cover epithelial cells and airways, also act as a first line of defense, as they can bind viruses via multiple-contact (multivalent) binding sites, effectively trapping them. The glycan structure recognized by Siglec-8 is sialic acid and sulfate (NeuAcα2-3[6S]Galβ1-4G[Fucα1-3]GlcNAc-). Given their role in inhibiting and inducing apoptosis in immune cells, the family of siglecs is likely involved in checkpoint signaling, which is important in cancer and inflammatory conditions.

Figure \(\PageIndex{21}\) shows the domain structure of Siglec-8

Figure \(\PageIndex{20}\): Domain structure of Siglec-8

Note that there is no CLECT domain, but rather immunoglobulin (IG) or IG-like domains, which seems logical given their role in binding glycan "epitopes". The IG domain is also called the immunoglobulin V-set domain (V-Set). The blue rectangle represents the transmembrane domain (single helix). The cytoplasm contains a tyrosine-inhibitory motif (ITIM) involved in transducing the signal on binding 6′S sLe^x glycans to the IG domains.

As discussed above, humans lack the hydrolase gene necessary for the hydroxylation of Neu5Ac to Neu5Gc, which is present in chimps and other primates. Chimp's immune systems seem to confer protection from acquiring simian versions of AIDS, cirrhosis, and other diseases which humans acquire when they are infected with the human versions of HIV, hepatitis B or C, or other viruses. These diseases and others associated with overactive T cells (rheumatoid arthritis, asthma, Type 1 diabetes) are uncommon in chimps. It turns out that there is a link between the type of sialic acid and the expression of siglecs.  This influences differences in our disease propensity. Varki et al. have shown that chimps and gorillas exhibit much higher levels of siglecs on T cells, which are critical regulatory and effector cells of the immune system. When siglecs on T cells are activated, T-cell responses are down-regulated. Although HIV ultimately kills T helper cells, the virus initially activates them upon infection, leading to their proliferation and the production of more cells for the virus to infect.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of human Siglec-8 lectin domain in complex with 6'sulfo sialyl Lewisx (2N7B)

Figure \(\PageIndex{19}\): Human Siglec-8 lectin domain in complex with 6'sulfo sialyl Lewisx (2N7B). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i..Gaa93cu86jMPA9

 

Acute myeloid leukemia cells have a protective "shield" of high-sialylated CD43 proteins on their cell surface.  This shield helps protect cancer cells from immune surveillance and macrophage phagocytosis.  Removing the barrier enables phagocytosis and destruction of these cells.  Jooho Chung et al.  Sialylated CD43 forms a glyco-immune barrier that restrains antileukemic immunity. Science 392,  (2026).DOI:10.1126/science.ady5196.  [As a reminder, myeloid (like red blood cells, platelets, and some white blood cells, such as granulocytes, including neutrophils, eosinophils, and basophils), and monocytes are not lymphocytes (like T and B cells). The term leukocytes is used to include all white blood cells, including lymphocytes.]  

Normally, another surface protein, CD47, acts as a "do not eat me" signal to inhibit phagocytosis.  Human acute myeloid leukemia cells don't use CD47 to escape phagocytosis; rather, they use CD43.  The protein is also called Leukosialin, the major sialoprotein on leukocytes that regulates their interaction with T cells. 

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter develops the concept of the "glycan code" — the idea that the extraordinary structural complexity of glycans constitutes an information-rich molecular language that is read by a diverse family of glycan-binding proteins (GBPs) — and examines in detail the molecular basis of glycan recognition by three major families of animal lectins: C-type lectins, galectins, and siglecs.

The glycan code arises from the combinatorial diversity of monosaccharide identities, anomeric configurations (α or β), glycosidic linkage positions (1→2, 1→3, 1→4, 1→6), branching patterns, chain lengths, and chemical modifications (sulfation, sialylation, fucosylation, acetylation) that make glycan surfaces uniquely "information-rich" compared to the simpler linear information encoding of nucleic acids or proteins. Unlike the genetic code, glycan structure is not templated by DNA but is determined by the spatial and temporal expression of glycosyltransferases, hydrolases, and modifying enzymes — making the glycan code highly cell-type specific and dynamically regulated. Using the QR code metaphor, organisms have evolved molecular "readers" (GBPs) that decode specific local glycan patterns on the surfaces of polysaccharides, glycoproteins, glycolipids, and proteoglycans. Nine distinct classes of GBPs have been identified, ranging from plant and bacterial lectins to viral surface proteins (influenza HA, coronavirus spike) to animal C-type lectins, galectins, and siglecs. In the broadest definition, any protein that specifically binds a glycan motif without modifying it can be called a lectin. Glycan recognition typically involves a small continuous or discontinuous epitope (analogous to the 5–19 amino acid epitopes recognized by antibodies), and biological specificity is often enhanced by multivalent binding — multiple weak interactions between a GBP and many glycan units on a ligand surface summing to high-avidity binding.

C-type lectins constitute the largest class of animal GBPs and are defined by a carbohydrate recognition domain (CRD, also called CLECT or CTLD) that requires Ca²⁺ for glycan binding. Ca²⁺ coordinates the equatorial hydroxyl groups of sugar residues, with the EPN amino acid motif (Glu-Pro-Asn) conferring specificity for mannose, GlcNAc, fucose, and glucose, while the WND motif (Trp-Asn-Asp) confers specificity for galactose and GalNAc. P-selectin, a prototypical C-type lectin expressed on the surfaces of activated platelets and endothelial cells, illustrates the central role of glycan-protein interactions in inflammation. Normally stored in intracellular secretory granules, P-selectin is rapidly translocated to the cell surface on inflammatory stimulation, where it binds the sialyl-Lewis X (SiaLewX) glycan — a tetrasaccharide of Neu5Ac(α2,3)Gal(β1,4)[Fuc(α1,3)]GlcNAc — on circulating leukocytes, mediating low-affinity transient interactions that slow ("roll") the leukocyte along the vessel wall. The natural high-affinity ligand is P-selectin glycoprotein ligand 1 (PSGL-1), whose interaction with P-selectin is dramatically enhanced by sulfation of Tyr 607 (but not Tyr 605 or 610 alone); the sulfo-tyrosine interacts with the positively charged surface of the P-selectin lectin-EGF domain, while the SiaLewX O-linked glycan interacts with the more negatively charged region. Leukocyte rolling transitions to firm adhesion when selectin signaling activates integrin expression on the leukocyte surface; integrins then mediate high-affinity protein-protein interactions with ICAM-1/2 (via the integrin αL/β2 heterodimer) and VCAM (via α4/β1) on endothelial cells, completing the multi-step "capture" process. The mannose receptor (CD206), another C-type lectin on macrophages and liver endothelial cells, reads both nonsulfated glycans (through multiple CLECT domains, particularly CLECT 4, binding Man(α1→2)Man, GlcNAc, and fucose in a Ca²⁺-dependent fashion through equatorial C3/C4 OH contacts) and sulfated glycans that do not even contain mannose (through the fibronectin type 2/cysteine-rich domain in a Ca²⁺-independent manner). This dual binding capacity underlies the mannose receptor's dual roles: scavenging circulating asialoglycoproteins and aged glycoproteins (including tissue plasminogen activator, collagen fragments, and sulfated pituitary hormones), and serving as a pattern recognition receptor for the mannose-rich cell walls of fungi and bacteria.

Galectins are a family of 15 GBPs united by a conserved ~130-amino acid β-sandwich carbohydrate recognition domain (a "jellyroll" of two antiparallel β-sheets) that binds β-galactosides — preferentially Galβ1,4GlcNAc (Type 2 LacNAc) or Galβ1,3GlcNAc (Type 1, which binds with slightly different φ/ψ angles) — in a Ca²⁺-independent manner. Unlike C-type lectins, galectins can also bind a broad range of glycan motifs beyond simple galactosides. They are expressed intracellularly in essentially all cells and multicellularly in all animals, and are secreted into the extracellular matrix where they participate in cell adhesion, apoptosis induction (galectin-1 induces apoptosis in T cells), and cancer biology; galectin expression is elevated in many tumors, where they promote immune evasion, angiogenesis, and metastasis.

Siglecs are sialic acid-binding immunoglobulin-like lectins, expressed predominantly on immune cells (Siglec-8 on basophils, mast cells, and eosinophils; Siglec-4 in myelinated neural tissue), with an N-terminal V-set immunoglobulin domain that binds sialylated glycan epitopes through a conserved arginine residue, and varying numbers of C2-set Ig-like domains. Their cytoplasmic ITIM (immunoreceptor tyrosine-based inhibitory motif) domains transduce inhibitory signals on glycan binding. Siglec-8 binds the highly specific glycan 6′-sulfo sialyl Lewis X (NeuAcα2-3[6S]Galβ1-4[Fucα1-3]GlcNAc), a sulfated sialylated glycan on mucins secreted during inflammation; multivalent engagement of Siglec-8 by these mucin glycoproteins triggers ITIM-mediated signaling that inhibits or kills the immune cell, serving as a checkpoint that limits excessive inflammatory responses. The biological significance of siglecs extends to the striking differences in disease susceptibility between humans and chimpanzees: chimps and gorillas express far higher levels of siglecs on T cells, whose engagement down-regulates T-cell responses; the difference in sialic acid type (Neu5Gc in chimps, Neu5Ac in humans, due to loss of the CMAH gene in the human lineage) alters siglec recognition and contributes to the differential susceptibility of humans (but not chimps) to HIV, inflammatory diseases like rheumatoid arthritis and asthma, and type 1 diabetes. Most recently, high sialylation of CD43 on acute myeloid leukemia cells was identified as a "glyco-immune barrier" that shields these cancer cells from macrophage phagocytosis — a mechanism distinct from the CD47 "don't eat me" signal used by other cancers, opening new therapeutic avenues through targeting sialylated CD43 to restore immune surveillance.