Carbohydrate Structure

How does carbohydrate structure determine its biochemical function?

• Carbohydrates, also called glycans, can be divided into three major groups: (1) simple sugars, which consist of monosaccharides, disaccharides, and oligosaccharides (monosaccharides and disaccharides are described in Chapter 12); (2) polysaccharides, which consist of glucose homopolymers or disaccharide heteropolymers in which one of the two sugars is a hexosamine; and (3) glycoconjugates of proteins or lipids with covalently attached glycans.

• Glycoconjugates contain combinations of modified and unmodified monosaccharides, which are covalently attached to proteins and lipids as branched and unbranched structures. Deciphering glycoconjugate structures is difficult.

• Glycosyltransferases covalently link glycans to proteins and lipids, whereas glycosidases remove glycans through hydrolysis reactions. Enzymes that modify glycans are some of the most highly conserved proteins in eukaryotes.

• Oligosaccharides range in size from ~3 to ~20 branched and unbranched sugar residues. Human milk contains lactose-derived oligosaccharides, which can function as prebiotics or as soluble glycan decoys. Eating foods high in sucrose-based raffinose oligosaccharides can cause digestion problems because mammals, but not intestinal bacteria, lack the enzyme α-galactosidase.

• The most abundant carbohydrate on Earth is cellulose, a homopolymeric molecule that accounts for the majority of biomolecules found in plant cell walls. It consists of up to 1000 repeating glucose disaccharides called cellobiose, which are linked by β(1→4) glycosidic bonds. Most organisms cannot metabolize cellobiose because they lack the enzyme cellulase, a β-1,4 glycosidase.

• Chitin is a linear hexosamine polysaccharide consisting of repeating GlcNAc units linked by β(1→4) glycosidic bonds. Chitin provides many types of insects and crustaceans with an excellent biomaterial for building a strong exoskeleton.

• Glycosaminoglycans contain hexosamine polysaccharides that are covalently attached to proteins to form a class of glycoconjugates called proteoglycans. Chondroitin sulfate, heparan sulfate, and keratan sulfate are common repeating hexosamine disaccharides found in glycosaminoglycans and are produced by most cell types.

• Starch and glycogen are glucose homopolymers used as quick sources of metabolic energy in plants and animals, respectively. Starch is generated during daylight hours by plants, using energy from photosynthesis, whereas glycogen stores in animals are synthesized from carbohydrates and proteins in the diet.

• Plants synthesize two forms of starch: amylose and amylopectin. Amylose is a linear polysaccharide containing ~100 glucose units linked by α(1→4) glycosidic bonds. Amylopectin is an α(1→6)-branched glucose polymer.

• Starch granules are stored in plant organelles called amyloplasts. Amyloplasts are a type of plant plastid similar to chloroplasts, although amyloplasts do not carry out photosynthesis. Amyloplasts are found in potato tubers and seeds of leguminous plants such as beans.

• The presence of α(1→6) glycosidic bonds in amylopectin and glycogen creates branch points that greatly increase the number of nonreducing ends. Because glucose units can only be metabolized from the nonreducing ends of polysaccharides, a higher number of α(1→6) glycosidic bonds makes it possible to retrieve glucose more efficiently.

• Glycogen contains about three times as many α(1→6) glycosidic bonds per 100 glucose residues as amylopectin. Glycogen, therefore, has many more nonreducing ends to facilitate the release of glucose as an energy source.

What are the molecular determinants of human ABO blood types?

• Glycan modification of proteins takes place within the lumen of the endoplasmic reticulum compartment of the cell, whereas glycolipids are primarily generated in the Golgi apparatus.

• Proteoglycans are protein glycoconjugates that consist mostly of carbohydrate with only a small protein component. Peptidoglycans are proteoglycans that constitute the bacterial cell wall and contain peptide linkers of D and L amino acids between adjacent polysaccharide strands.

• Lectins are glycan binding proteins that mediate two classes of glycoconjugate binding interactions: (1) intrinsic glycoconjugate binding between glycans and lectins on human cells, and (2) extrinsic glycoconjugate binding between glycans and lectins on human cells and pathogen cells.

• Glycan attachments to glycoproteins occur through either the amide nitrogen atom of asparagine, leading to the generation of N-linked oligosaccharides, or the oxygen atom of serine or threonine residues, resulting in O-linked oligosaccharides.

• The most common N-glycosidic bond in glycoproteins is between asparagine and GlcNAc, whereas the most common monosaccharide used to create the O-glycosidic bond is GalNAc.

• Glycosyltransferases are enzymes that use nucleotide sugars as the carbohydrate donor and sequentially add sugar residues to extend the glycan group. A large number of glycosyltransferases are required to build the variety of glycan structures found in nature.

• Genetic differences in the expression and activity of glycosyltransferases account for immunologic incompatibility between individuals. For example, the ABO blood groups in humans are determined by the presence or absence of A-type and B-type glycosyltransferases.

• The GTA (A-type blood) and GTB (B-type blood) glycosyltransferase enzyme variants differ by only a few amino acids, yet they account for the differential addition of GalNAc or Gal to the O-antigen blood group glycan on erythrocyte membrane proteins and lipids.

• Individuals that inherit only one functional copy of the GTA gene have A-type blood, whereas individuals that inherit only one functional copy of the GTB gene have B-type blood. Individuals that inherit one copy each of the GTA and GTB genes have AB-type blood, which is an example of codominant inheritance. Individuals lacking functional copies of both the GTA and GTB genes have O-type blood.

• ABO blood types are very important in blood transfusions because of the presence of antibodies in plasma that bind to glycoconjugate antigens. If blood plasma in the recipient contains antibodies that bind to glycoconjugate antigens present on red blood cells (RBCs) from the donor, then the transfusion will be lethal to the patient due to massive clotting.

• There are two common types of transfusions, (1) RBC transfusions to replace oxygen-transporting functions of blood, and (2) plasma transfusions used to treat infections and other diseases. Whole-blood transfusions using unpurified blood are uncommon.

• Individuals with O-type blood are considered universal RBC donors because their RBCs do not contain either the A-type or B-type glycoconjugates, whereas AB individuals are considered universal plasma donors because their plasma does not contain antibodies that recognize either A-type or B-type glycoconjugates.

• Rhesus factor (Rh factor) is an RBC structural protein that serves as a marker of blood-type compatibility. Individuals who are Rh+ can obtain RBC transfusions from both Rh+ and Rh− individuals but cannot obtain plasma transfusions from Rh− individuals. Individuals who are Rh− can obtain RBCs only from Rh− individuals but can obtain plasma transfusions from Rh+ and Rh− individuals.

• Heparin is a highly sulfated glycosaminoglycan produced by immune cells. It is distinct from heparan sulfate, which is a hexosamine produced by all cells and found in glycosaminoglycans that function in interstitial fluid.

What is the molecular basis for methicillin-resistant bacterial infections?

• Bacterial cell walls are made of peptidoglycans, which are proteoglycans consisting of multiple strands of hexosamine polysaccharide chains. The chains consist of repeating units of a β(1→4)-linked MurNAc-GlcNAc disaccharide, which are tethered together by linkages between short oligopeptides.

• Bacteria can be divided into two broad groups on the basis of physical differences in the peptidoglycan layer (thick or thin) and whether or not they have an outer membrane. Gram-positive bacteria have a thick peptidoglycan cell wall and no outer membrane, whereas Gram-negative bacteria have a thin peptidoglycan cell wall surrounded by an outer membrane.

• The Gram test is done by staining bacteria with crystal violet dye, which is trapped inside the thick peptidoglycan wall of Gram-positive bacteria after an ethanol wash. In contrast, Gram-negative bacteria do not retain the crystal violet dye after the ethanol wash because the peptidoglycan wall is too thin to trap the dye.

• The antibiotic penicillin blocks bacterial cell wall biosynthesis by inhibiting the enzyme transpeptidase, which is required to form the oligopeptide linkages between hexosamine polysaccharide chains. Without transpeptidase to support cell wall biosynthesis during cell division, the penicillin-treated bacteria die.

• Some bacteria are resistant to penicillin because they produce an enzyme called β-lactamase, which hydrolyzes the β-lactam ring in penicillin to inactivate it. This type of penicillin resistance has been overcome by methicillin, an antibiotic that blocks transpeptidase activity without being a substrate for β-lactamase.

• MRSA is a methicillin-resistant strain of Staphylococcus aureus that expresses a variant transpeptidase enzyme that does not bind methicillin. The methicillin-resistant transpeptidase gene was obtained from another bacterial species through a process called lateral gene transfer.

What methods are used for determing the complex structures of glycoconjugates?

• Two research objectives of glycobiology research are (1) identification of glycan group structures on purified glycoproteins by using liquid chromatography and mass spectrometry and (2) applications of high-throughput, array-based screening assays to identify biologically relevant glycan binding interactions.

• Structural characterization of glycans on glycoconjugates is technically challenging because of variable bonding arrangements between many different sugars, the presence of sugar stereoisomers with identical masses, and the fact that glycan structures can differ in subtle ways between identical classes of glycoconjugates.

• High-performance liquid chromatography (HPLC), in combination with glycosylase cleavage, can provide information about the arrangement of sugars in a highly purified glycan fraction by using standard HPLC equipment. In contrast, mass spectrometry, which requires mass analyzers, identifies common glycans present in a mixed glycan sample on the basis of predicted mass-to-charge ratios.

• The fluorescent dye 2-aminobenzamide (2-AB) is covalently attached to glycan groups prior to analysis of glycan structures by HPLC. The glycans labeled with 2-AB are subjected to stepwise treatment with sugar-specific glycosylases to yield related glycan structures that can be separated and identified by HPLC.

• Mass spectrometry is used either as the primary analytical method for glycan characterization or as a complementary technique to augment elution data derived from HPLC. By comparing the observed masses obtained from mass spectrometry to predicted masses of common N-linked glycan groups, specific peaks in the spectrum can be assigned to glycan groups.

• Glycan identification by mass spectrometry can be performed using matrix-assisted laser desorption/ionization (MALDI) and time of flight (TOF) to calculate mass-to-charge ratios.

• Lectin and glycan arrays provide platforms to screen large numbers of samples for specific binding interactions by use of fluorescently labeled molecules. Two basic types of arrays have been developed for screening purposes: (1) protein arrays, which contain covalently attached lectin proteins or antibodies; and (2) glycan arrays, which contain covalently attached glycoproteins with intact glycan groups or chemically synthesized glycan groups.

• Lectin arrays contain a grid of lectin proteins that are covalently attached to a solid support and used to detect glycan groups in an experimental sample. By using sample material that is fluorescently labeled, a pattern of binding interactions can be obtained indicating the presence or absence of glycans in the sample.

• Antibody arrays are also useful tools in glycobiology research. The antibodies can either bind to specific glycan groups or bind to the protein moiety of glycoproteins. When glycan binding antibodies are used to detect fluorescently labeled glycoproteins in an experimental sample, the antibody array functions in much the same way as a lectin array. Antibodies that recognize the protein component of a glycoprotein can be used to investigate glycan modifications on glycoproteins.

• Glycan arrays are used to analyze the cellular glycome under various conditions using fractionated cell extracts or to detect lectins on the surface of pathogenic and nonpathogenic bacteria. Glycan arrays can be constructed using HPLC fractions of cell extracts containing glycoprotein mixtures that are covalently linked to solid surfaces or using chemically synthesized glycans of known structure.