STRUCTURE AND PROPERTIES OF BIOMOLECULES - A. E. Zemlyakov - 2017

12. CARBOHYDRATE-CONTAINING COMPLEXES

The structural diversity of Monosaccharides and the possibility of forming several types of glycosidic bonds during carbohydrate chain assembly allow them to encode significantly more information compared to Peptides or Nucleic Acids. For example, a single elementary unit (an amino acid or nucleotide) can form only one dipeptide and one dinucleotide, whereas two identical monosaccharides can form 42 Disaccharides.

Class="center">

It has been calculated that 20 standard Amino Acids can form ~6,4 10⁷ hexapeptides, a set of 4 standard NUCLEOTIDES yields ~4,1 10³ hexanucleotides, whereas 20 monosaccharides can produce ~1,4 10¹⁵ hexasaccharides. Partly for this reason, carbohydrate chains are responsible in nature for "fingerprint" Functions—the Cells of each individual possess a unique set of carbohydrate structures, and each strain of Gram-negative Bacteria has its own specific lipopolysaccharide.

Most often, CARBOHYDRATES perform their functions not as free oligo- and Polysaccharides, but as complexes with other Biomolecules.

❖ Classification.

♦ Carbohydrate-Structure/178.html">Protein Complexes. Based on the protein-to-carbohydrate ratio, carbohydrate-protein complexes are subdivided into Glycoproteins (predominantly protein) and Proteoglycans (predominantly carbohydrate). In reality, there is no strict boundary between these two classes; for instance, mucins, which contain several hundred carbohydrate chains, are traditionally classified as glycoproteins.

The carbohydrate-protein complex group also includes peptidoglycans, in which polysaccharide chains are combined with short peptide chains, and glycopeptides, representing combinations of mono- and Oligosaccharides with peptides.

♦ Carbohydrate-lipid complexes. These include Glycolipids and lipopolysaccharides.

❖ Glycoproteins. In these structures, one or more oligosaccharide chains are typically attached to a protein molecule; for example, interleukin-2 contains a single carbohydrate chain, whereas immunoglobulin M contains 10 chains. Glycoproteins feature A wide variety of functions: Enzymes, Hormones, Cell Membrane Receptors, immune system Proteins, and Blood Plasma components.

The attachment of carbohydrate chains to Polypeptides occurs via a glycosidic bond. To date, several ways of linking monosaccharides to Amino acids have been identified. The two most common types of attachment are N- and O-glycosidic bonds. Less common are S-glycosidic linkages via Cysteine residues and C-glycosidic bonds between D-mannose and Tryptophan residues.

✵ N-Glycosidic bond — the most frequent linkage involves an N-acetylglucosamine residue attached via a β-glycosidic bond to the nitrogen atom of an asparagine side chain. This type of bond is found in IMMUNOGLOBULINS, y-interferon, and major Histocompatibility Antigens. Other variants of N-glycosidic bonds are less common, for example, D-GalNAc-β1—> Asn, D-Glc- β1 —> Asn, L-Rha-α1 —> Asn, or D-Glc-β1 —> Arg.

Typically, another similar monosaccharide and a D-mannose trisaccharide fragment are attached to N-acetylglucosamine, forming a standard

oligosaccharide core from which further oligosaccharide chains can branch. Depending on the monosaccharide composition, complex-type chains (mannose-rich) and hybrid-type chains are distinguished.

The first type includes, in particular, the oligosaccharide chains of immunoglobulin G.

✵ O-Glycosidic bond — a typical linkage involves an N-acetylgalactosamine residue attached via an α-glycosidic bond to the oxygen atom of Serine or Threonine. This type of bond is characteristic of blood group substances, among others. The hydroxyl group of these amino acids can also be glycosylated with D-xylose (heparin, chondroitin sulfate), β-N-acetylglucosamine (cytoplasmic and Nuclear Proteins), β-D-glucose, α-D-galactose, α-D-mannose, or α-L-fucose.

Even less common are conjugates of β-N-acetylglucosamine, α-D-galactose, or β-D-arabinose with hydroxyproline; β-D-glucose, α- or β-D-galactose with Tyrosine; and β-D-galactose with 5-hydroxylysine (Collagen).

The N-acetylgalactosamine residue is, in turn, glycosylated with D-galactose, forming a standard disaccharide core. Further elaboration of the carbohydrate chains occurs by attaching N-acetyllactosamine disaccharide units and terminal residues of L-fucose or N-acetylneuraminic acid to the core.

❖ Antifreeze glycoproteins. The blood of polar fish contains several types of proteins with molecular weights of 3–16 kDa, as well as glycoproteins with an MW of 2.6–33 kDa, at concentrations up to 25 mg/mL, which enable these animals to survive at temperatures below 0 °C. It is believed that these compounds prevent The formation of ice crystals.

The antifreeze glycoprotein is a block polymer of Ala-Ala-Thr, glycosylated with the β-Gal-(1—>3)-α-GlcNAc disaccharide.

Later, such compounds were discovered in certain insects, plants, Fungi, and even bacteria.

❖ Blood group substances — immunogenic structures (carbohydrates, proteins, glycoproteins, etc.) located On the surface of erythrocytes that determine blood type. The main blood group system is the ABO (H) system.

As early as the beginning of the twentieth century, the Austrian scientist K. Landsteiner (Nobel Prize in Physiology or Medicine, 1930) hypothesized that Two Types of structures—determinants A and B, which are responsible for immunological processes—may be present or absent on The surface of red Blood Cells.

The absence of these determinants, the presence of only one determinant, or the simultaneous presence of both determinants A and B explains the existence of 4 Blood Groups (correspondingly, blood groups I, II, III, and IV, or 0 (H), A, B, and AB).

Table 8. The ABO blood group system

Human blood plasma was found to contain two types of protein molecules capable of binding to these determinants, forming a stable complex and causing erythrocyte agglutination. These compounds were named α- and β-agglutinins. Later, it turned out that they are antibodies directed against these determinants: anti-A and anti-B. This explains the incompatibility of blood transfusions between individuals with different blood groups. Even blood group I, formerly known as the "universal donor" group, may contain a significant amount of antibodies, which prevents its transfusion to individuals of other blood groups.

Later studies showed that the 0 (H) carbohydrate determinant also exists. It is the disaccharide O-L-fucopyranosyl-(β1—>2)-D-galactopyranose, and the entire difference between determinants A and B is that in the former case, α-D-N-acetylgalactosamine is additionally attached to the 0 determinant via the hydroxyl group at the C3 position of the galactose residue, whereas in the latter case, α-D-galactose is attached. Even such minor structural differences result in significant variations in immunological reactions.

On the lipid membrane of erythrocytes, determinant chains can be anchored either as glycolipids or via membrane glycoproteins. In the first case, glucocerebroside acts as the lipid "anchor." Between the determinant and the "anchor," there are several repeating units [—>4)-β-GlcNAc-(1—>3)-β-Gal-(1—>].

The integral glycoprotein has a Molecular Weight of ~43 kDa. A connecting trisaccharide unit with a terminal oligosaccharide group-specific determinant is attached to it via an O-glycosidic bond.

The Biosynthesis of ABH antigens is carried out by specific Glycosyltransferases, the synthesis of which is encoded by specific genes in the DNA.

Determinant groups are found not only on the surface of red blood cells. Similar carbohydrate chains are present in the oligosaccharide fragments of mucins—glycoproteins secreted by various body Tissues (saliva, mucus) that line the cavities of the respiratory and digestive systems.

Along with the blood group substances of the ABO (H) system, more than 45 other blood group systems have been identified (see Table 9), in which various Biopolymers act as antigenic determinants. Their combination results in the formation of a unique antigenic profile on The Cell surface of every individual.

Table 9. Some blood group antigen systems

✵ Rhesus factor — the second most important blood group antigen system. It is formed by a group of proteins on the erythrocyte surface. The presence of the D antigen (cofactor) is of the greatest immunological importance. The absence of this antigen is often denoted by the symbol d (cofactor). Rhesus negativity is observed in ~15% of Caucasians, ~7% of Negroids, and only ~0.5% of Mongoloids. Differences in Rhesus factors must be taken into account during blood transfusions.

In women with the dd homozygous genotype, if the father is Rhesus-positive with a heterozygous Dd genotype, There is a 50% probability that the child will inherit the Rh factor (and 100% if the father has the homozygous DD genotype). This can cause a Rhesus incompatibility reaction (The production of anti-Rh⁺ antibodies in the mother's blood), leading to various pathologies, including hemolytic disease of the newborn.

The name of the factor stems from its discovery in 1942 (by Karl Landsteiner in collaboration with the American immunologist Alexander Wiener) using serum obtained by immunizing rabbits with erythrocytes from rhesus macaques.

❖ Mucins. Animal epithelial cells produce mucin glycoproteins. These glycoproteins are the primary components of tissue mucous membranes. Mucins provide these membranes with moisture and elasticity, perform protective tissue functions—such as shielding against the proteolytic action of gastric and intestinal juices—participate in mineralization processes, and are capable of binding pathogens. The molecular weight of mucin complexes reaches up to 20 MDa.

The structure of mucins is highly diverse and heterogeneous. For example, the MUC1 mucin is an O-glycosylated protein embedded in the lipid membrane, with a high content of Serine and threonine.

The MUC2 mucin molecule has a more complex structure. The backbone of the molecule consists of a protein containing repetitive blocks rich in threonine (serine) and Proline. Oligo- and polysaccharide chains (comprising up to 20 monosaccharides, including sialic acids) are attached via O-glycosidic bonds. Cysteine-rich regions are also distinguished within the mucin structure. The N- and C-terminal domains contain a relatively small number of N-glycosylated oligosaccharides.

Located at the C-terminus, the "cysteine knot" enables the macromolecule to di- and oligomerize through the formation of Disulfide Bonds. Disulfide bridges, along with hydrophobic and ionic interactions, further drive the assembly of the three-dimensional gel structure of mucins.

❖ Cartilage proteoglycan is a complex supramolecular assembly of protein and carbohydrate components. Up to 100 chains of the aggrecan biopolymer are attached to the hyaluronic acid polysaccharide backbone via link proteins.

Hyaluronic acid (from Greek hyalos – Glass) is a polymer composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. The number of disaccharide residues in the chain can reach 25,000, with a molecular weight of up to 10⁷ Da. Due to its polyanionic nature, the polysaccharide forms a Hydration shell up to 500 nm in diameter.

Aggrecan consists of a core protein molecule with a molecular weight of approximately 250 kDa, which carries keratan sulfate and chondroitin sulfate polysaccharide chains.

Chondroitin sulfate chains (from Greek chondros – cartilage) consist of repeating units of glucuronic acid and N-acetylgalactosamine (n = 10–100), irregularly sulfated at the C4 and/or C6 hydroxyl groups of the galactosamine residues. Approximately 100 chains of this polysaccharide are attached via O-glycosidic bonds to serine residues in the aggrecan core protein.

Aggrecan also contains up to 30 keratan sulfate chains (from Greek keras – horn)—a block polymer of galactose and 6-O-sulfate-N-acetylglucosamine (n = 8–40), partially sulfated at the C6 hydroxyl groups of the galactose residues and attached through both O- and N-glycosidic bonds.

The total mass of the proteoglycan exceeds 2 10⁸ Da.

❖ Heparin (from Greek hepar – Liver) is a polydisperse polysaccharide of complex structure. Its molecular weight can reach up to 40 kDa, whereas for its low-molecular-weight form it is 10–15 kDa. It possesses a block structure comprising two types of blocks containing D-glucuronic and L-iduronic acids, respectively. The strongly acidic character of heparin is conferred by A large number of O- and N-sulfate groups. The polysaccharide chain is linked to the polypeptide chain via a D-xylopyranose residue connected by a β-O-glycosidic bond to a serine residue (Xylp-β1 —> Ser).

The principal biological property of heparin is its anticoagulant (blood-clotting Prevention) activity. It is widely used as a medication for the Treatment of various thromboses, myocardial infarction, and other diseases.

❖ Introduction/37.html">Bacterial Cell wall peptidoglycan (murein) is a structure that provides bacteria with external protection against the environment. It is the major component (50–80%) of Gram-positive bacterial cell walls. It represents a cross-linked polymer lattice in which polysaccharide chains, consisting of N-acetylglucosamine and its derivative N-acetylmuramic acid residues, are interconnected by short peptide chains.

Muramic acid is condensed with a tetrapeptide fragment via its carboxyl group. A pentaglycine bridge serves as the connecting link between two polysaccharide-peptide chains, attached on one side to the side-chain amino group of a Lysine residue and on the other to the carboxyl group of the C-terminal D-Alanine residue. In some bacteria, other diaminocarboxylic acids are found instead of lysine: the lysine homolog Ornithine (Н₂N(СН₂)зСH(NН₂)СООН) or meso-diaminopimelic acid.

✵ β-Lactam Antibiotics. During murein biosynthesis, glycopeptide chains with a D-alanyl-D-alanine sequence at the C-terminus are initially formed. Cross-linking (Condensation) of these chains is mediated by the enzyme transpeptidase and is accompanied by the Cleavage of the terminal D-alanine residue.

Peptidoglycan biosynthesis is blocked by A number of β-lactam antibiotics, including penicillin, which accounts for their antibacterial efficacy. β-Lactam antibiotics are structurally similar to the D-alanyl-D-alanine fragment and are capable of forming a stable complex with the enzyme, thereby inhibiting the biosynthetic process.

The widespread use of penicillin in clinical practice has led to The Emergence of antibiotic-resistant bacterial strains. Such resistance is associated with the production of penicillinase (β-lactamase) by these bacteria, an enzyme that cleaves the β-lactam ring to yield inactive penicilloic acid, followed by decarboxylation.

To combat such resistant microorganisms, antibiotics resistant to the action of β-lactamases are used—for instance, imipenem, which belongs to the carbapenem group—or a combination of a traditional antibiotic with β-lactamase inhibitors, such as clavulanic acid, is applied.

❖ Teichoic Acids (from Greek teichos meaning wall) account for up to 50% of the dry weight of gram-positive bacterial cell walls. They are extremely rare in the cell walls of gram-negative bacteria.

The molecular weight of these biopolymers reaches up to 2 million Da. Typically, teichoic acids are linked to murein via phosphodiester bonds involving the primary hydroxyl group of muramic acid residues. Other polymers, known as lipoteichoic acids, are linked via phosphodiester bonds to the C6 carbon atom of the carbohydrate residue in glycolipids. Through their lipid moiety, they are anchored in the membrane, while the polyglycerophosphate chain is located on its outer side or sometimes spans The cell wall to emerge on the cell surface.

Teichoic acids can function as bacterial antigens and, acting as polyanions, they influence cation exchange.

There are two main types: glyceroteichoic acids (glycerol-based) and riboteichoic acids, which are based on D-ribitol.

The hydroxyl groups of these polyols can be attached to sugar residues (usually glucose and glucosamine, less commonly galactose, mannose, rhamnose, galactosamine, and their derivatives), amino acids (D-alanine, L-lysine), or carboxylic acids (acetic and succinic acids). In some cases, mixed teichoic acids containing both glycero- and ribophosphate units have been identified.

In a number of bacteria, the sugar residues glycosylating the polyol are incorporated into the main polymer chain or are directly linked via a phosphodiester bond. Examples of such teichoic acid structures are given below.

❖ Lipopolysaccharides (LPS) are specific Components of the outer membrane of gram-negative bacteria. They are involved in bacterial interactions with the environment and determine toxic and antigenic properties. When animals are infected by bacteria, O-specific antibodies against the lipopolysaccharide are produced.

LPS consists of three main components: lipid A, which anchors the complex to the bacterial lipid membrane; an intermediate oligosaccharide core; and the O-specific polysaccharide, which is responsible for antigenic activity. The total molecular weight reaches up to 20 kDa.

✵ Lipid A is a glucosamine disaccharide with a β1 —> 6 linkage, containing phosphate groups at C1 and C4’ and acylated at the amino and hydroxyl groups at C3 and C3’. The most common acyl components are tetradecanoic (myristic), 3-hydroxytetradecanoyl, and 3-tetradecanoyloxytetradecanoic (3-myristoyloxymyristic) acids.

Lipid A is responsible for the toxic properties of LPS. This compound is a potent immunostimulant. Removal of the phosphate residue from the glycosidic center reduces toxicity while preserving immune activity.

✵ O-Specific polysaccharide. Its structure determines not only the species but also the strain affiliation of the bacterium. The repeating unit of the polysaccharide typically contains from 2 to 6 monosaccharides, including rare sugars such as amino sugars, deoxy derivatives, uronic acids, and combinations thereof. Some of them contain non-carbohydrate components: carboxylic and amino acid residues, amino alcohols, phosphoric acid residues, and the like. For example, the repeating unit of the O-specific polysaccharide of Shigella dysenteriae (serotype 9) includes acetic and pyruvic acid residues, with the latter forming a 4,6-O-ketal with a galactose residue.

❖ Antigen-antibody-based diagnostic agents. The structural properties of O-specific polysaccharides have been successfully applied to develop Diagnostics for infectious diseases. Specifically, scientists utilized the fact that, from the perspective of The Immune System, a natural polymer antigen and its synthetic counterpart—which is a conjugate of a core polymer with synthetic antigenic sites—are remarkably similar, triggering the production of identical antibodies.

Furthermore, in some cases, the strongest Immune Response is associated exclusively with a portion of the antigenic site known as the antigenic determinant. For instance, mycobacterial diseases such as tuberculosis and leprosy present strikingly similar clinical pictures in their early stages. The natural antigen of Mycobacterium leprae is a lipidophenol glycosylated with a trisaccharide, where the 3,6-di-O-methyl ether of D-glucose acts as the determinant antigen, serving as the basis for the design of the synthetic antigen.

When an Organism is infected, antibodies directed against the antigenic sites of the pathogens appear in the bloodstream, and these are precisely what diagnostic assays are designed to detect. Synthetic antigens are placed into the wells of a polystyrene microplate, and the patient's blood plasma is added. If specific antibodies are present in the plasma, they bind to the antigenic determinants.

Next, this antigen-antibody complex is labeled using specialized biochemical Reagents, and a multiscan spectrophotometer is employed to measure the optical density, allowing for the calculation of antibody concentrations.

Control Assignments

Test 4 (Sample Variant)

1. Determine the configuration of the following monosaccharides:

2. Draw the Haworth projection for the structure of β-L-mannopyranose.

3. This monosaccharide is:

A) α-D-pyranose; B) β-D-pyranose; C) α-L-pyranose; D) β-L-pyranose; E) α-D-furanose; F) β-D-furanose; G) α-L-furanose; H) β-L-furanose.

4. Match the corresponding pairs:

A. murein; B. muramic acid; C. lipopolysaccharide; D. O-specific polysaccharide; E. lipid A.

I. glucosamine monosaccharide derivative; II. structure responsible for the antigenic activity of LPS; III. lipophilic disaccharide derivative of glucosamine; IV. specific component of the outer membrane of Gram-negative bacteria; V. peptidoglycan of bacterial cell walls.

5. The STRUCTURE OF THE repeating unit [—> 4)-α-D-GalA-(1 —>] corresponds to:

A. Cellulose; B. hemicellulose; C. agarose; D. amylose; E. amylopectin; F. agaropectin; G. pectin; H. inulin; I. Chitin; J. chitosan.

6. Determine the group of Glycosides to which rutin belongs and identify the plant in which it is found.

A. anthocyanins; B. glucosinolates; C. saponins; D. cardiac glycosides; E. Flavonoids; F. cyanogenic glycosides.

I. dahlia (Dahlia variabilis); II. black mustard; III. lily of the valley; IV. bitter almond; V. licorice; VI. Japanese pagoda tree (Styphnolobium japonicum).

7. Choose the correct name for the presented compounds

1. trehalose; 2. sucrose; 3. lactose; 4. maltose; 5. melezitose; 6. neuraminic acid; 7. indican; 8. arbutin; 9. dhurrin; 10. sinigrin; 11. cochineal; 12. syringin