LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 1. THE FOUNDATIONS OF BIOCHEMISTRY: STRUCTURE AND CATALYSIS - 2011

PART I. STRUCTURE AND CATALYSIS

7. CARBOHYDRATES AND GLYCOBIOLOGY

7.4. Carbohydrates as Information Molecules: The Sugar Code

Glycobiology is The Study of the Structure and function of Glycoconjugates, and today it stands as one of the most rapidly advancing and fascinating fields in biochemistry and Cell biology. It is now well established that Cells use specific Oligosaccharides to encode crucial information regarding intracellular protein trafficking, cell-cell interactions, tissue development, and extracellular signaling. We will Touch upon just a few Examples illustrating the structural diversity and functional range of glycoconjugates. In Chapter 20, we will discuss the details of Polysaccharide Biosynthesis, including peptidoglycans, and in Chapter 27, we will examine the mechanisms of oligosaccharide chain assembly in Glycoproteins.

Advances in analytical Methods for determining The structure of oligosaccharides and Polysaccharides have revealed an astonishing complexity and diversity of oligosaccharide chains in glycoproteins and Glycolipids. Consider the typical glycoprotein oligosaccharides shown in Fig. 7-29. The most complex among them contains 14 monosaccharide residues of four different types, linked by 1 —> 2, 1 —> 3, 1 —> 4, 1 —> 6, 2 —> 3, and 2 —> 6 bonds, with some having an α- and others a β-configuration. Branched structures, which do not occur in Nucleic Acids and Proteins, are quite common in oligosaccharides. Assuming that 20 different monosaccharide units could be used to build an oligosaccharide (which is entirely plausible), the theoretical number of distinct hexameric oligosaccharides is 1.44 • 10¹⁵. In comparison, the number of hexapeptides composed of 20 Amino Acids is 6.4 • 10⁷ (20⁶), and the number of hexanucleotides composed of four nucleotide units is 4,096 (4⁶). If we further account for the possibility of sugar residue sulfation, this figure increases by another two orders of magnitude. Oligosaccharides are exceptionally rich in structural information; the Amount of Information they carry is not only comparable to, but far exceeds, that contained in a nucleic acid molecule of average size. Each of the oligosaccharides depicted in Fig. 7-29 features a unique three-dimensional structure—a "sugar code"—that is deciphered by proteins capable of interacting with them.

Lectins are proteins that "read" the sugar code and participate in many biological processes

Lectins, found in all organisms, are proteins that bind with exceptionally high affinity and Specificity to particular CARBOHYDRATES (Table 7-3). Lectins play a role in numerous recognition, signaling, and Cell Adhesion processes, as well as in the Intracellular Transport of Newly synthesized proteins. In the laboratory, purified lectins are used to detect and fractionate glycoproteins with differing oligosaccharide chains. Here, we will focus on just a few examples illustrating The Importance of lectins in animal cells.

Class="center">Table 7-3. Some Lectins and Their Oligosaccharide Ligands

Certain Peptide Hormones circulating in the bloodstream contain sugar residues that determine their half-life. Luteinizing hormone and thyrotropin (polypeptide hormones from the anterior pituitary) contain N-linked oligosaccharides terminating in the disaccharide GalNAc4S(β1—> 4)GlcNAc, which is recognized by a hepatocyte lectin (receptor). (GalNAc4S is N-acetylgalactosamine sulfated at the C-4 hydroxyl group.) The interaction between the receptor and the hormone regulates the uptake and degradation of luteinizing hormone and thyrotropin, thereby controlling their concentration in the Blood. Thus, the circulating levels of these hormones rise As a result of pituitary secretion and fall due to continuous degradation in hepatocytes.

Sialic acid residues (Neu5Ac) located at the termini of the oligosaccharide chains of many plasma glycoproteins (Fig. 7-29) protect these proteins from degradation in the Liver. For instance, the copper-binding serum glycoprotein ceruloplasmin has several oligosaccharide chains terminating in Neu5Ac. The removal of sialic acid residues by the enzyme sialidase (also known as neuraminidase) is used by The Cell to tag "Aging" proteins destined for destruction and replacement. The Plasma Membrane of hepatocytes contains lectin molecules known as asialoglycoprotein receptors (i.e., receptors for desialylated glycoproteins). These receptors specifically bind oligosaccharide chains in which galactose residues are no longer protected by a terminal Neu5Ac residue. The interaction between the receptor and ceruloplasmin leads to the endocytosis and destruction of the latter.

A strikingly similar mechanism appears to operate in mammals for clearing aged erythrocytes from the Circulation. Young erythrocytes bear several membrane glycoproteins with oligosaccharide chains terminating in Neu5Ac. If sialic acid residues are enzymatically removed from a blood sample in vitro using sialidase and this blood is then returned to the circulation, the treated erythrocytes disappear within a few hours. Erythrocytes with intact oligosaccharide chains (those collected and reinfused without sialidase Treatment) continue to circulate for several more days.

Cell-surface lectins play a vital role in the Pathogenesis of various human diseases, involving both host lectins and those of infecting agents. Plasma membrane lectins that mediate Cell Recognition and adhesion across a wide spectrum of cellular processes are called selectins. One such process is the migration of immune system cells (neutrophils) from the blood through capillary walls to inflamed or infected tissue sites (Fig. 7-31). At an inflammatory site, P-selectin On the surface of capillary endothelial cells interacts with a specific oligosaccharide on a glycoprotein of the circulating neutrophil. This interaction slows the neutrophil down and causes it to "roll" along the capillary endothelium. Next, an interaction occurs between integrin molecules (p. 641) on the neutrophil plasma membrane and adhesion proteins on the endothelial surface. This interaction brings the neutrophil to a complete halt, allowing it to squeeze through the Capillary Wall into the infected tissue to mount an immune attack. Two Other types of selectins participate in this process (known as lymphocyte homing): E-selectins on endothelial cells and L-selectins on leukocytes bind specific oligosaccharides on neutrophils and endothelial cells, respectively.

Fig. 7-31. Lectin-ligand interactions during lymphocyte migration to sites of inflammation or injury. The motion of a neutrophil in a capillary is slowed down by transient interactions between P-selectin molecules on the Plasma Membranes of capillary endothelial cells and P-selectin ligands on the neutrophil surface. As the neutrophil continues to interact with the P-selectins on the endothelial surface, its rolling along the capillary wall decelerates. At the site of inflammation, stronger interactions between Integrins on the neutrophil surface and their endothelial ligands completely arrest the neutrophil's movement. Triggered by signals from the inflammatory site, extravasation occurs—the neutrophil migrates through the capillary wall to the site of injury.

Human selectins are implicated in the inflammatory processes of rheumatoid Arthritis, asthma, psoriasis, multiple sclerosis, and transplant rejection. This underscores the urgent need to develop drugs that inhibit selectin-mediated cell adhesion. Many carcinoma cells express an antigen normally found only on embryonic cells (sialyl-Lewis x, or sialyl-Lex), which, when present in the circulation, facilitates Cancer cell dissemination and metastasis. Carbohydrate derivatives that mimic sialyl-Lex within sialoglycoproteins, as well as agents that alter oligosaccharide biosynthetic pathways, could prove to be effective therapeutics for treating chronic Inflammatory Diseases or combating cancer metastasis.

Certain animal viruses, including the influenza virus, attach to host cells via cell-surface oligosaccharides. The influenza virus lectin, known as the HA protein (hemagglutinin), plays a critical role in viral entry and infection. Once the virus has entered the host cell and replicated, newly synthesized viral progeny bud from the cell, acquiring an envelope derived from pieces of the host plasma membrane. Viral sialidase (neuraminidase) cleaves terminal sialic acid residues from host cell surface oligosaccharides, releasing the Viral Particles from the cell and preventing them from clumping together. A new round of infection can then begin. The antiviral drugs oseltamivir (Tamiflu) and zanamivir (Relenza) are used to treat influenza. These sugar derivatives inhibit the viral sialidase by competing for binding to host cell oligosaccharides, thereby preventing viral release from infected cells and causing viral particles to aggregate, which blocks the subsequent stage of infection.

The initial stage of the infection cycle for herpes simplex viruses HSV-1 and HSV-2 (the causative agents of oral and genital herpes, respectively) involves the specific binding of their lectins to heparan sulfate on the host cell surface, with the precise arrangement of sulfate groups within this polymer being essential for successful infection.

Pathogenic microbes frequently use lectins to attach to host cells or deliver their toxins. As discovered in the 1980s by Barry J. Marshall and J. Robin Warren, the bacterium Helicobacter pylori—the CAUSATIVE AGENT OF most Stomach ulcers—adheres to the gastric lining through interactions between bacterial coat lectins and specific oligosaccharides present in gastric epithelial membrane glycoproteins (Fig. 7-32). One of the recognition sites for H. pylori is the Le^b oligosaccharide, a structural fragment of the blood group O determinant. This finding helps explain why stomach ulcers occur several times more frequently in individuals with blood group O than in those with Blood Groups A or B. Chemically synthesized Le^b analogues could prove useful in treating these conditions; by competing with gastric glycoproteins for binding to bacterial lectins, they can prevent bacterial adhesion and, consequently, infection.

Fig. 7-32. Formation of a gastric ulcer. Helicobacter pylori cells attached to The Stomach wall. The ulcer results from the interaction between bacterial lectins and the Le^b oligosaccharide (blood group O antigen) on The surface of the gastric epithelium.

Some of the most severe human parasitic infections, prevalent mainly in developing countries, are caused by eukaryotic microorganisms that feature unusual oligosaccharides on their surface, which in some cases serve a protective function for the parasite cells. Among these organisms are trypanosomes, responsible for African sleeping sickness and Chagas disease, Plasmodium falciparum, which causes malaria, and Entamoeba histolytica, the causative agent of amoebic dysentery. Driven by the search for drugs that can halt the synthesis of these unusual oligosaccharide chains and inhibit parasite Replication, numerous studies have focused on the biosynthetic pathways of these oligosaccharides.

Cholera toxin, produced by Vibrio cholerae, enters intestinal cells responsible for Water absorption and induces severe diarrhea. The toxin binds to target cells via the oligosaccharide of ganglioside GM1, a membrane phospholipid on the surface of the intestinal epithelium (the structure of GM1 is shown in Addendum 10-2,

Fig. 1). Similarly, the toxin produced by the pertussis pathogen, Bordetella pertussis, enters target cells only after interacting with one or possibly several oligosaccharides terminating in a sialic acid residue. Understanding the exact STRUCTURE OF THE oligosaccharide-binding sites in these toxins (lectins) can aid in The Development of engineered toxin variants for vaccination. Engineered toxins lacking the carbohydrate-binding domain are safe for humans because they cannot bind to cells or enter them, yet they can still elicit an Immune Response that protects the individual against subsequent exposure to the native toxin. It is also conceivable to design drugs that mimic cell-surface oligosaccharides, bind to bacterial lectins, and prevent their productive attachment to the cell surface.

Lectins also exert their activity intracellularly. Within the Golgi apparatus, a mannose-6-phosphate-containing oligosaccharide tags newly synthesized proteins destined for transport to Lysosomes (Fig. 27-39). A common structural feature of these glycoproteins—a distinct signal patch—ensures their recognition by an enzyme that phosphorylates the mannose residue at the end of the oligosaccharide chain. The resulting mannose-6-phosphate residue is recognized by the cation-dependent mannose-6-phosphate receptor, a membrane-bound lectin possessing a mannose-phosphate-binding site on the luminal surface of the Golgi apparatus. When the region of the Golgi containing this receptor buds off to form a transport vesicle, proteins bearing mannose-phosphate residues are drawn into the forming vesicle via the interaction between mannose-6-phosphate and its receptor. The vesicles subsequently fuse with lysosomes and release their contents. Many, if not all, lysosomal hydrolytic Enzymes are captured and transported via precisely this mechanism. Some mannose-6-phosphate receptors can capture enzymes containing mannose-6-phosphate residues and target them to lysosomes. This process forms The basis of "enzyme replacement therapy," which aims to correct human disorders associated with defective lysosomal function. ■ Furthermore, lectins participate in other protein-sorting processes. Any newly synthesized protein in The Endoplasmic reticulum is already linked to a specific oligosaccharide that can bind via one of two ER lectins (which also act as chaperones): membrane-bound calnexin and soluble calreticulin. These lectins couple the nascent protein to an enzyme that catalyzes rapid disulfide exchange during protein folding, ultimately leading to the native conformation. At this point, ER enzymes process the oligosaccharide residues so that they are recognized by another lectin, ERGIC53, which routes the properly folded protein (glycoprotein) to the Golgi apparatus for further maturation. If a protein is misfolded, the oligosaccharides are processed differently and recognized by the lectin EDEM, which targets the aberrantly folded protein to the Cytosol for degradation. Thus, protein glycosylation in the ER serves as a critical quality-control signal that allows the cell to eliminate improperly folded proteins (this process is discussed in detail in Chapter 27).

Lectins bind carbohydrates with high affinity and exquisite specificity.

In all the lectin functions described above, as well as in many other processes involving interactions between lectins and oligosaccharides, the unique structure of the oligosaccharide is crucial for ensuring high binding specificity. The information encoded in oligosaccharides is expressed as a distinct code comprising a virtually endless array of short "words" recognized by specific proteins. The oligosaccharide-binding sites in lectin molecules are complementary to strictly defined carbohydrate sequences, making lectin-oligosaccharide interactions exceptionally specific. Although the affinity of an oligosaccharide for an individual carbohydrate-binding domain of a lectin is often modest (with K_d values in the micromolar range), the effective affinity is amplified manifold by lectin multivalency, since each lectin molecule possesses multiple carbohydrate-binding domains. In oligosaccharide clusters (which typically form on the membrane surface), for instance, each oligosaccharide can bind to one of the lectin's carbohydrate-binding domains, reinforcing the interaction. If a multitude of receptors exists on the cell surface, the avidity of the interaction can be very high, driving highly Cooperative processes such as cell adhesion and rolling (see Fig. 7-31).

X-ray crystallographic analysis of several lectin-carbohydrate complexes has provided a detailed view of these interactions (Fig. 7-33). A family of 11 lectins that bind oligosaccharide chains terminating in a sialic acid residue performs several vital biological functions in The Human Body. All these lectins bind sialic acids within $\beta$-sandwich domains similar to those found in IMMUNOGLOBULINS (this motif is illustrated in the CD8 protein in Fig. 4-21). Collectively known as siglecs (sialic acid-recognizing Ig-superfamily lectins), the members of this family are numbered 1 through 11 and are sometimes referred to as sialoadhesins. The interaction between siglecs and sialic acid ($N$-acetylneuraminic acid, Neu5Ac) involves all substituents on the Neu5Ac ring: the acetamido group at C-5 participates in hydrogen bonding and Van der Waals interactions with the protein, the carboxyl group forms a salt bridge with a conserved Arg residue, and the glycerol hydroxyl groups form Hydrogen Bonds with the protein. Siglecs regulate processes in the immune and nervous systems and also participate in blood cell development. For example, siglec-7 binds to a specific ganglioside (GD3) containing two sialic acid residues and inhibits The activity of natural killer (NK) cells, thereby protecting cells that would otherwise be destroyed by The Immune System. Elevated levels of GD3 in certain cancers, such as malignant melanoma and neuroblastoma, may enable cancer cells to evade immune system defenses.

Figure 7-33. Details of a lectin-carbohydrate interaction, (a) Bovine mannose-6-phosphate receptor complexed with mannose-6-phosphate (PDB ID 1M6P) shown with surface electrostatic potential: red regions are predominantly negatively charged, and blue regions are predominantly positively charged. The mannose-6-phosphate molecule is depicted in stick representation; the manganese ion is highlighted in purple, (b) Close-up view of the binding site. Mannose-6-phosphate forms a Hydrogen bond with the Arg¹¹¹ residue and a coordination bond with the Mn²⁺ ion (shown smaller than its van der Waals radius for clarity). The His¹⁰⁵ residue forms a hydrogen bond with one of the oxygen atoms of the phosphate group. Protonation of this residue at the lower pH of the lysosome likely triggers the release of mannose-6-phosphate from the receptor.

The structure of another lectin, the mannose-6-phosphate receptor, has also been determined by X-ray crystallography. This work revealed the fine details of the interaction between this lectin and mannose-6-phosphate, explaining binding specificity and the essential requirement for a divalent cation in the interaction (Fig. 7-33a). The Arg¹¹¹ residue in the receptor molecule forms a hydrogen bond with the hydroxyl group at C-2 of the mannose residue, which is coordinated by the Mn²⁺ ion. The His¹⁰⁵ residue forms a hydrogen bond with one of the phosphate oxygen atoms (Fig. 7-33b). When a protein bound to mannose-6-phosphate enters a lysosome—where the pH is lower than in the Golgi apparatus—the receptor's affinity for mannose-6-phosphate decreases, likely due to the protonation of the His¹⁰⁵ residue.

In addition to these specific interactions, more general interactions also contribute to the binding of many carbohydrates to their respective lectins. For instance, many sugar residues possess more polar and less polar faces (Fig. 7-34). The more polar regions form hydrogen bonds with the lectin, whereas the less polar regions engage in hydrophobic interactions with nonpolar amino acid residues of the protein. The combination of all these interactions results in high affinity (often K_d $\le 10^{-8}$ M) and high binding specificity. This represents a fundamental information-transfer mechanism that is pivotal in numerous intracellular processes and cell-cell interactions. Figure 7-35 illustrates several biological processes in which sugars act as information carriers.

Figure 7-34. Hydrophobic interactions of sugar residues. Monomeric sugar units, such as galactose, have a more polar face (at the top of the chair conformation, where the ring oxygen and hydroxyl groups are located) that participates in hydrogen bonding with the lectin, and a less polar face that can engage in hydrophobic interactions with nonpolar amino acid residues, such as the indole ring of Tryptophan.

Figure 7-35. Involvement of sugars in cell-surface recognition and adhesion processes, (a) Oligosaccharides with unique structures (depicted as sequences of six-membered rings) incorporated into various glycoproteins or glycolipids on the outer surface of the plasma membrane interact with high affinity and specificity with extracellular lectins, (b) Animal viruses, such as influenza virus, bind to cell-surface glycoproteins during the initial stage of the infection cycle, (c) Bacterial toxins (e.g., cholera or pertussis toxins) bind to cell-surface glycolipids to gain entry into the cell, (d) Certain bacteria (such as Helicobacter pylori) attach to host cells prior to colonization or infection, (e) Selectins (lectins) in the plasma membrane of certain cells mediate cell-cell interactions, such as the binding of T lymphocytes to capillary endothelial cells at sites of inflammation, (f) The mannose-6-phosphate receptor (a mannose-binding lectin) in the trans-Golgi network binds to the oligosaccharide of a lysosomal enzyme and directs its transport to the lysosome.

Summary of Section 7.4 Carbohydrates as Information Molecules: The Sugar Code

■ Monosaccharides can be assembled into an almost endless variety of oligosaccharide combinations that differ in stereochemistry, glycosidic linkage positions, types and orientations of substituents, and the number and types of branches. Oligosaccharides carry vastly more biological information than nucleic acids or proteins.

■ Lectins—proteins with specific carbohydrate-binding sites—are typically located on the outer surface of the cell, where they initiate cell-cell interactions. Vertebrate lectins recognize specific oligosaccharide "tags" on peptide hormones, circulating Plasma Proteins, and Blood Cells, thereby regulating their clearance.

■ The attachment of pathogenic viruses, bacteria, and certain eukaryotic parasites to host cells occurs through the binding of pathogen-associated lectins to oligosaccharides on the host cell surface.

■ Intracellular lectins participate in the delivery of intracellular proteins to specific Organelles or to their secretion sites.

■ X-ray crystallographic studies have provided detailed insights into lectin-carbohydrate complexes, establishing the molecular complementarity that accounts for the strength and specificity of their interactions.