COMPLEMENTARITY AND CONTRARIETY in Glycomics
Majid Ali, M.D.
III. Complexities of Structure
IV. Complexities of Function
V. Cellular Glucose Oxidation
VI. Carbohydrates and Generation of Reactive Oxygen Species
VII. The Too-Much/Too-Little Sugar Dilemma
VIII. Rapid Hyperglycemic-Hypoglycemic Shifts
IX. Glucose Toxicity and Hexosamine Pathways
X. Sugars and Prions
XI. Sugars and the Inflammatory Response
XII. Sugars, Adhesion Molecules, and Infections
XIII. Anti-adhesive Therapies
XIV. Sugars and Cancer
XV. Of Sugars, Chimp Brains, and the Human Intellect
XVI. Sugars Protect Proteins
XVII. Carbohydrate Signaling
From a teleologic perspective alone—in light of aspects of molecular complementarity and contrariety presented in earlier chapters— carbohydrates (sugars) would be expected to play crucial roles in nearly all facets of the web and kaleidoscope of human biology. Indeed, carbohydrates are highly sophisticated and efficient players in the “cellular information technology.” Thus, it is surprising that in clinical medicine, carbohydrates are usually dismissed as mere fuel substances for the body. Even in that limited view, the enormous significance of rapid hyperglycemic/hypoglycemic shifts (“sugar roller coasters”) is seldom fully appreciated by clinicians. The complexities of carbohydrate structure and functions have been recognized for decades. Structurally, carbohydrates are far more varied than proteins and nucleic acids, with complex and branching configurations rather than the simple boxcar linkages among amino acids and nucleotides. They dangle from nearly all structural and functional proteins as well as many lipids, thus altering their structures and functions. The structural complexity of carbohydrates accounts for their remarkably broad—and as yet poorly understood—range of functional diversity. That is also the principal reason why carbohydrate research is considered “the Rodney Dangerfield of pharmaceutical research.” It gets no respect. Fortunately, that is changing now. The central roles of carbohydrates in cellular proliferation, differentiation, and demise are increasingly recognized. Additional critical roles of sugars involve cell recognition systems, immunologic surveillance—including folding, quality control, and assembly of peptide-loaded histocompatibility complex—and molecular decoy systems by which microbes and cancer cells use sugar groups on their surfaces to slip past the immune cells. In this chapter, I focus on those aspects of carbohydrate metabolism and cell recognition systems that illuminate or expand the major themes of this volume: molecular complementarity, molecular contrariety, and the essential oxidative-dysoxygenative nature of the health/dis-ease/disease continuum.
Carbohydrates are ubiquitous in cells. Both inside and outside the cells, sugars pave the way for proteins and lipids.1-7 Within the cells, carbohydrate modifications of proteins and fats are pivotal events that modulate their structural and functional integrity. In the extracellular milieu, they provide key components of the cellular reconnaissance and surveillance systems involved in the health/dis-ease/disease continuum, as well as in evoking host responses in infectious, metabolic, ecologic, immunologic, degeneratory, and neoplastic disorders. In recent decades, there have been intense, albeit marginally successful, attempts by the pharmaceutical industry to develop “carbohydrate drugs,” even though heparin, a sugar, has been extensively used as the drug of choice for many coagulative disorders for decades. Still, the carbohydrate chemistry has not had the same press as those of proteins and lipids. That may change. Consider the following:
The additional complexity and variety generated within the proteome by carbohydrates will provide further challenges beyond the one-to-one correlation of genetic sequence and protein sequence. We have already heard the word “glycomics” whispered by guests at the ball.8
The above quote is taken from a recent special issue of Science devoted to the newer understanding of carbohydrates in cellular information transfer as well as essential modifications of lipids and proteins that impart structural and functional integrity to glycolipids and glycoproteins. To underscore how recent has been the emergence of sugar chemistry as a specific field in pharmacology and medicine, it may be pointed out that the term glycobiology was introduced as recently as 1988 by Raymond Dwek, an Oxford University carbohydrate chemist. The term glycomics is a sugar enthusiast’s answer to the champions of proteomics.
III. COMPLEXITIES OF STRUCTURE:
The Letters of the Carbohydrate Language
Carbohydrate literally means “hydrate of carbon.” However, carbohydrates (sugars) do not contain individual water molecules. Rather, they are polyhydroxy aldehydes and polyhydroxy ketones. Carbohydrates are so designated because the empirical formulas of most of them can be expressed as Cx(H2O)y. The molecular formula of glucose is C6H12O6 (A in Figure 1). In solution, the aldehyde group of glucose at carbon 1 reacts with hydroxyl group at carbon 5 to form two cyclic structures designated á-D-glucose and â-D-glucose (B in Figure 1). The -OH group on the carbon 1 in the former is axial to the ring, whereas it is equatorial to the ring in the latter. Based on structure, carbohydrates are also divided into two broad classes: aromatic compounds that contain a ring (benzene or related) structure; and aliphatic compounds that do not contain such structures. In general, aromatic carbohydrates are more stable and resilient than their aliphatic counterparts. Thus, it does not come as a surprise that all vitamins except ascorbic acid have an aromatic configuration. This makes teleologic sense, since vitamins not only facilitate biochemical reactions, but also serve as potent redox restorative, oxystatic, and defense molecules, especially as far as their roles as antioxidants. Most hormones are also aromatic. On the negative side, many pesticides (such as DDT) and many other synthetic chemicals (PCBs, PBBs, and related compounds) are also aromatic and thus stable and resilient. They have long half-lives and cause long-term toxicity.
The extraordinary structural complexities of carbohydrates are created by the nature of the process by which monosaccharides are aligned to form disaccharides, oligosaccharides, and polysaccharides.9-12 For example, two identical monosaccharides can bond to produce 11 different disaccharides. Beyond that, monosaccharides create branching structures. Thus, a small number of monosaccharides can unite to create a staggering range of possible configurations to form various carbohydrate compounds. That is in sharp contrast to the case of two amino acids that can form only one dipeptide, or two nucleotides that can generate only one nucleic acid configuration. That is so because amino acids and nucleic acids connect to each other in only one way. To present two widely divergent scenarios, four different nucleotides can generate only 24 distinct tetranucleotides, whereas four different monosaccharides can be aligned in 35,560 unique tetrasaccharides.10 The “letters of the carbohydrate language” evidently are far more versatile than those in protein or nucleic acid worlds.
Figure 1. Schematic (A) and Cyclic (B) Structure of Glucose
Diagram from 725 (25.16) and 726 (25.17)
Most mammalian proteins contain sugar moieties and hence should be properly called glycoconjugates. There are two main types: glycoproteins and proteoglycans. In general, glycoproteins are highly branched and contain short oligosaccharide (glycan) chains without repeating sequences. By contrast, proteoglycans comprise long, linear, and unbranched glycans and do not contain repeating disaccharides. Glycoproteins include most of the molecules that serve as biomembrane receptors, hormones, and mediators of inflammatory and healing responses.
Glycolipids—also called glycosphingolipids— are lipids containing covalently bound sugars. (Sphingolipids are a group of amphipathic, polar lipids.) Glycolipids are divided into four groups: cerebrosides, sulfatides, globosides, and gangliosides. All contain a polar head group composed of sugars attached to ceramide by a glycosidic bond. Glycolipids—and glycoproteins—are not synthesized by template mechanisms as are nucleic acids and proteins. Rather, these compounds are generated by activities of enzymes—predominantly of glycotransferases—which are expressed or activated differentially during development, differentiation, and demise of cells.
IV. COMPLEXITIES OF FUNCTION
Though accounting for only about one percent of the total body mass, sugars play extraordinarily diverse roles. The structural complexity of sugars is the bane of carbohydrate chemists, who like to conduct molecular dissections. It is a boon to cells as they participate in the web and kaleidoscope of human biology. The many roles of sugars in cellular energetics—for immediate, intermediate, and long-term energy needs—are generally well understood. Less known are the roles of carbohydrates in transfer of cellular intelligence. In biologic systems, recognition of cells, as well as invading microbes, is attributed to complementarity of surface structures that encode information on one entity that is readily deciphered by the other. In modern times, Emil Fischer, the German chemist, is credited with the lock-and-key hypothesis put forth to explain specificity of interactions among enzymes and their substrates. Paul Ehrlich, the German immunologist, extended that concept of surface complementarity to describe specificity of antigen-antibody reactions. Frank Lillie, the American investigator, invoked that complementarity to explain recognition between sperm and egg. Details of the evolution of those concepts are given in the immunology section of the second volume of this textbook.
The earlier indications of the existence of the intelligence functions of sugars came from several lines of evidence, including the following: (1) injections of polysaccharides into animals lead to production of antibodies; (2) major ABO blood types are determined by sugars; (3) the influenza virus binds to erythrocytes through sialic acid, a sugar commonly present in cell membranes; (4) carbohydrates contained in glycoproteins and glycolipids form a “sugary coat” for the cells that is essential for various cellular intelligence functions; (5) embryonal cells of various types in mixtures can sort themselves by carbohydrate-dependent cell recognition systems; and (6) sugars bind selectively, rapidly, and reversibly to a large family of proteins called lectins. In all of the above situations, carbohydrates are strategically positioned to provide a rich diversity of complementarity. For example, embryonal retinal and hepatic cells in a mixture can rapidly separate into zones composed of their respective clusters. To cite other examples, monosaccharide, oligosaccharides, and polysaccharides provide distinctive recognition characteristics to lectins, a large family of proteins that also participate in the core of cellular recognition systems. Some aspects of lectins are discussed in the chapter on complementarity and contrariety of proteins.
After decades of glossing over the contribution of sugars in creating and maintaining such complementarity, sugar polymers are now recognized as especially well designed for carrying information. Some readers might be surprised to read that, per unit weight, carbohydrates can carry more information than proteins or nucleic acids.
All cells, as mentioned above, carry sugary coats composed of a very large number of glycoconjugates (glycolipids and glycoproteins). More significantly, the repertoire of such carbohydrate moieties increases in specific ways as cells develop and differentiate in health and undergo degeneration and decay in disease. Sialic acid—a sugar ubiquitous on cell membranes—is among the well characterized molecules that play critical roles in the cell recognition systems of the body. For example, the array of carbohydrate moieties on the surface of malignant cells are strikingly different that those on the surface of non-neoplastic cells. Some aspects of this subject are presented in the chapter entitled “Complementarity and Contrariety in Lipidomics.”
V. CELLULAR GLUCOSE OXIDATION
Cellular glucose oxidation begins with cytosolic glycolysis that generates NADH and pyruvate. NADH so produced can either reduce pyruvate to lactate or donate reducing equivalents to the mitochondrial electron transport chain. NADH destined for mitochondria is transported through two shuttles: (1) the glycerol phosphate shuttle; and (2) the malate-aspartate shuttle.79 Mitochondrial NADH (along with FADH2) provides energy for ATP production by the electron transport chain through oxidative phosphorylation. Pyruvate reduced to lactate leaves the cells and is carried to the liver, where it is oxidized through the tricarboxylic acid cycle to generate four molecules of NADH, one molecule of FADH2, carbon dioxide, and water.
Pyruvate serves as the substrate for gluconeogenesis. Most of pyruvate generated in cytosol, however, is transported into mitochondria, where it is oxidized through the tricarboxylic acid cycle to four molecules of NADH, one molecule of FADH2, carbon dioxide, and water. Transfer of cytosolic NADH into mitochondria is primarily conducted by the malate-aspartate shuttle.* Mitochondrial NADH and FADH2 provide energy for ATP production by the electron transport chain through oxidative phosphorylation.
Electron flow in the mitochondrial electron transport chain involves the following five complexes:80
Complex I: NADH:ubiquinone complex;
Complex II: Succinate:ubiquinone
Complex III: ubiquinone:cytochrome c
Complex IV: cytochrome c: cytochrome c oxidase ATP synthase; and
Complex V: ATP synthase.
* The malate shuttle involving the malate-citrate antiporter provides the mechanism by which 2-carbon units of substrate are transferred from the mitochondria to the cytosol for synthesis of fatty acids. Acetyl CoA is the primary molecule required for synthesis of fatty acids in the cytosol. It is a product of mitochondrial metabolism that does not traverse well the mitochondrial membrane, hence the value of the malate shuttle.
The first four enzyme complexes are associated with the inner mitochondrial membranes. Cytochrome c and the mobile carrier ubiquinone are other important molecular species associated with that location.80 Electron transport begins with donation of electrons by cytosolic and mitochondrial NADH to NADH: ubiquinone oxidoreductase (complex I). The complex next transfers its electrons to ubiquinone, which also receives other electrons from a family of FADH2-containing dehydrogenase, including succinate:ubiquinone oxidoreductase (complex II) and glycerol-3-phosphate dehydrogenase. Ubiquinone, in turn, donates its electrons to ubiquinone:cytochrome c oxidoreductase (complex III) by ubisemiquinone radical-generating Q cycle.81 The next phase in electron transport involves cytochrome c; cytochrome c oxidase (complex IV).
VI. CARBOHYDRATES AND GENERATION OF REACTIVE OXYGEN SPECIES
Superoxide generation occurs at two sites in the inner mitochondrial membrane: (1) NADH dehydrogenase at complex I; and (2) at the interface between ubiquinone and complex III.82 A proton gradient is generated by electron transfer through complexes I, III, and IV that drives ATP synthase (complex V). As that gradient increases, there is a concomitant increase in the electrochemical potential difference produced by it. That potential, in turn, prolongs the life of ubiquinone and other superoxide-generating electron transport intermediates. Beyond a certain threshold, that potential markedly increases superoxide production. Thus, excess intracellular generation of reactive oxygen species (ROS) is driven by the proton electrochemical gradient generated by the mitochondrial electron transport chain. Overexpression of uncoupling protein-1 (a specific uncoupler of oxidative phosphorylation that is capable of collapsing the proton electrochemical gradient) reduces ROS production.83 ROS generation is prevented by overexpression of manganese superoxide dismutase (Mn-SOD), the major mitochondrial antioxidant enzyme system, in gene transfer experiments.84
Hyperglycemia causes excessive production of reactive oxygen species (ROS). The primary source of the substrate for increased ROS production in hyperglycemia is the tricarboxylic cycle.85 The main mechanism of that is the proton electrochemical gradient generated by the mitochondrial electron transport chain described above. Those aspects of redox dynamics in hyperglycemia were first established by studies with bovine aortic endothelial cells. The blockade of the malate-aspartate shuttle in those cells with aminooxyacetate does not affect hyperglycemia-induced excess production of reactive oxygen species (ROS). However, blockade of glycolysis-derived pyruvate transported into mitochondria by 4-hydroxy cyanocinnamic acid abolishes the generation of such ROS.
Advanced glycation end products (AGEs) are produced in excess in hyperglycemia. 86 That process is initiated and perpetuated by mitochondrial superoxide85 and involves increased production of AGE-forming methylglyoxal derived from fragmentation of glyceraldehyde-3-phosphate. The enzyme facilitating that reaction is glyceraldehyde-3-phosphate dehydrogenase. That enzyme is reversibly inhibited by ROS. Further evidence is drawn from the demonstration that excess production of methylglyoxal is blocked by Mn-SOD, as well as by UCP1.85
In addition to glucose, other readily oxidizable substrates, such as Amadori adducts, reactive carbonyl and dicarbonyl compounds, and polyunsaturated fatty acids are increased in diabetes. Excess of these substrates leads to increased non-enzymatic oxidative pathways.87 Those changes are generally associated with decreased levels of ascorbate and glutathione. Furthermore, other products of glycoxidation and lipoxidation are also increased.
The above brief review of glucose-related and glucose-triggered phenomena is presented to firmly establish the relatedness of glucose metabolism to redox homeostasis. That relatedness, as becomes evident from later sections of this article, forms the core of the oxidative insulin dysfunction model. Those dynamics are the core issues of insulin pathophysiology in the context of the proposed model.
VII. THE TOO-MUCH/
TOO-LITTLE SUGAR DILEMMA
I first recognized the “too-much/too-little sugar dilemma” during my work with patients with fatigue/fibromyalgia complex and those with diabetes mellitus. On the too-little side, most patients with fatigue/fibromyalgia complex experienced troublesome symptoms of hypoglycemia. On the too-much glucose side, the potential of hyperglycemia to cause oxidative coagulopathy became clear to me during high-resolution (x15,000) phase-contrast microscopy of freshly prepared peripheral blood smears of diabetic subjects with poor control of hypoglycemia.88-90
At a fundamental level, glucose is the principal source of energy for the human cell. The redox dynamics provide the pathways for extracting energy from glucose. From a teleologic perspective, glucose metabolism and redox equilibrium must be intricately related in all of their aspects. Glucose metabolism generates reactive oxygen species (ROS), reactive nitrogen species, and oxidative products of glycation.50 Not unexpectedly, hyperglycemia is accompanied by excess ROS production. Not enough glucose means cellular starvation and consequent oxidosis. Too much glucose also causes oxidosis in many ways. Thus is created the too-much/too-little-glucose dilemma. Close regulation of glucose in the blood as well as the intracellular compartment is a metabolic high-wire balancing act. Oxidosis created by glucose dysregulation puts under increasing stress all hormonal pathways involved in glucose regulation. It also affects myriad molecular dynamics that regulate: (1) glucose traffic at cell membranes; (2) delivery to mitochondria of NADH and pyruvate produced by cytosolic glycolysis; and (3) the transport to the liver of reduced pyruvate (lactate) to serve as a substrate for gluconeogenesis.
The teleologic consideration suggests that hyperglycemia-induced excess generation of ROS must influence—and be influenced by—endothelial nitric oxide dynamics. That, indeed, is true.74 ROS lowers nitric oxide levels in diabetes. Seemingly, nitric oxide chemistry pitches in to counter regional oxidosis. Two recognized mechanisms are involved in the relationship between nitric oxide and hyperglycemia-induced generation of ROS. First, hyperglycemia-induced excess production of sorbitol (which is potentiated by nitric oxide) is blocked by manganese superoxide dismutase. Second, overproduction of mitochondrial superoxide enhances the activity of enzyme aldose reductase and stimulates the production of sorbitol by that enzyme. The activity of aldose reductase is reversibly downregulated by nitric oxide modification of a cysteine residue in the enzyme’s active site.92
Adipocytes appear to autoregulate their responsiveness to insulin in many ways. Specifically, such cells secrete free fatty acids and a large number of metabolically active polypeptides, including leptin, adipsin, Acrp30/AdipoQ, and tumor necrosis factor-á(TNF-á), that affect insulin metabolism in different ways.76-80 All those pathways also influence and, in turn, are influenced by local redox dynamics.
A clear understanding of the relationship between the symptoms of hypoglycemia and the rate of change in the blood sugar level is essential for understanding the glucose-insulin-adrenaline dynamics. Sharp rises in the blood sugar level evoke sharp responses from the beta cells of the pancreas that release insulin. Sudden bursts of insulin cause a sudden release of adrenaline and its cousins, the adrenergic molecules. What are generally considered to be symptoms of hypoglycemia are, by and large, symptoms of brisk adrenergic responses. A rapid insulin response induces a brisk adrenergic response. From a clinical standpoint, prevention of hypoglycemic symptoms requires a focus on the metabolic events that occur two to three hours before the development of symptoms.
VIII. RAPID HYPERGLYCEMIC-HYPOGLYCEMIC SHIFTS:
The Sugar Roller Coasters
Below, I reproduce some text from The Butterfly and Life Span Nutrition101 to illustrate this sequence of events. An eight-year-old girl has a blood sugar level of 100 mg/dl (or 1,000 mg in one liter of blood). Since she has a total circulating blood volume of about five liters, the total quantity of glucose in her circulating blood is 5,000 mg or five grams. A teaspoonful of sugar holds four grams of sugar. Suppose this girl drinks on an empty stomach a can of soda containing eight to ten teaspoons full of sugar. This means that girl pours six to eight times as much sugar into her blood as exists at any time. Such a large bolus of sugar creates a tide of glucose that evokes a brisk insulin response which, in turn, triggers an adrenergic surge. Similar molecular roller coasters are caused when she drinks a 12-ounce glass of commercial orange juice. How is the sugar molecular roller coaster initiated? With sugar overload. How is the sugar molecular roller coaster perpetuated? By withdrawal symptoms. “Highs” in the blood sugar levels are followed by the “lows” that create biologic demands for yet more sugar. Sugar craving is another name for sugar addiction. An American child at the turn of the century consumed between five and ten pounds of sugar per year. His counterpart today ingests 150-175 pounds. How many thousands of molecular roller coasters does that come to? The numbers add up. This is the essence of the hypoglycemia problem. How does our sugar industry respond to all this? They keep physicians on their payroll to publish absurd studies showing that our children are not hurt by sugar. This is the simple truth behind the hypoglycemia controversy.
Insulin triggers a cascade of myriad oxidative events. And so does adrenaline. Hypoglycemia is oxidizing, as is hyperglycemia. One of the mechanics by which high intracellular concentration of glucose causes cellular toxicity is the hexosamine pathway, briefly described below.
IX. GLUCOSE TOXICITY AND HEXOSAMINE PATHWAYS
Glycolysis is the splitting of a molecule of glucose to form two molecules of pyruvic acid. It involves ten steps. The net reaction per molecule of glucose is expressed below:
—– 2 pyruvic acid+2ATP+4H
In the above conversion, only two net moles of ATP are formed for each mole of glucose, producing 24,000 calories of stored energy. However during the ten successive steps of the above conversion, a total of 56,000 calories are lost as heat energy, giving an overall efficiency for ATP formation of 43 percent. It may be added here that only an equivalent number of ATP (two moles) is generated in the citric acid cycle. Clearly, the major portion of final ATP (nearly 90 percent) is generated during subsequent oxidation of hydrogen atoms released during earlier stages of glucose breakdown.
The concentration of intracellular glucose-6-phosphate is responsive to both glycogen synthesis and glucose transport, since it is an intermediary between those two processes. Since the activity of glycogen synthase is decreased in type 2 diabetes, the intracellular concentration of glucose-6-phosphate is higher than in normal subjects. Furthermore, insulin-stimulated increase in the concentration of glucose-6-phosphate is blunted in the disease.102 Similarly, intracellular glucose is a metabolic intermediary between glucose transport and hexokinase activity and its concentration is determined by the relative strengths of the processes. Metabolic studies of these aspects of glucose metabolism suggest that impaired insulin-stimulated glucose transport is responsible for the diminished rate of insulin-stimulated glycogen synthesis in the muscle tissue in type 2 diabetes.102
Some aspects of the complementarity and contrariety of influences of glucose on insulin function may be attributed to the hexosamine pathway.103 This pathway provides an alternative to glycolysis at the level of fructose-6-phosphate and involves the enzyme fructose-6-phosphate amidotransferase and production of glucosamine-6-phosphate and other hexosamine products.103 Glucosamine inhibits insulin-stimulated glucose uptake as well as GLUT-4 translocation.104,105 Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice results in insulin resistance (failure of insulin-induced glucose uptake in muscle).106 Activity of glutamine:fructose-6-phosphate amidotransferase is also increased in the skeletal muscle of diabetic patients.107
X. SUGARS AND PRIONS
The sugar enthusiasts are staking loftier claims: Without modification by sugars, they assert, proteins and lipids are not what they seem to be. Now there is another hard blow to those who subscribe to the simplistic notion that recognition of individual genes and proteins will one day eradicate human disease.
In the early 1980s, a simple idea was advanced to explain a cluster of strange diseases: Prions are aberrant proteins that can replicate without RNA or DNA to cause infectious diseases. It was considered a biological blasphemy of the first order. In 1997, Stanley Prusiner of the University of California was awarded the Nobel Prize for his heretic notion of prion replication without the intervention of RNA or DNA. Then followed another equally heretical notion: the differences between prion types (strains) are largely determined by the number and types of carbohydrates attached to the aberrant proteins. That also caused much consternation among the protein enthusiasts. However, evidence to support that glycosylation theory rapidly accumulated.9 The three best-known examples of prion diseases are Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (“mad cow disease”). It was found that prions isolated from the brain of a patient with the classical form of Creutzfeldt-Jakob disease differed from those from the patient with variant Creutzfeldt-Jakob disease primarily in their sugar pattern.
XI. SUGARS AND THE INFLAMMATORY RESPONSE
Selectins are carbohydrate-binding proteins expressed by endothelial cells and blood platelets. These proteins are synthesized in response to signaling compounds (cytokines) produced by injured cells. Some selectins bind to sialyl Lewis x (SLeX), a specific type of sugar on the surface of leukocytes, cull those cells out of circulation, and facilitate their adhesion to and passage across the endothelium cells to evoke beneficial as well as harmful responses in subendothelial tissues. In mice, inhibition of binding of L selectin, one subgroup of selectins, to SLeX sugar moiety results in attenuated inflammatory response. However, intensive follow-up efforts to develop drugs to block L selectin binding and control ongoing and destructive inflammatory responses in autoimmune disorders in humans have failed to produce favorable clinical outcome. Another subgroup of selectins called P selectin is expressed both on endothelial cells and platelets. Pharmacologic blockade of this selectin has also been attempted to suppress both ischemic-reperfusion vascular injury and inflammatory response in autoimmune disorders. There is no evidence that
such blockade will yield good long-term results either. It is noteworthy in this context that the production of signaling cytokines that bind to selectins and initiate the inflammatory cascades (which the drugs are hoped to block) is triggered and regulated by oxidative-dysoxygenative events that begin with erythrocytes and the circulating plasma.
Concurrently with the research in prion pathogenicity, interest was kindled in defining the roles of sugars in pathogenicity of viruses and other microbial species. It is known that minor modification of the structure and/or interference with the function of sugar moieties of viral glycoproteins can lead to significant changes in viral replication, especially as regards hepatitis B and C viruses. The therapeutic potential of such findings is the possibility of gumming up glycoprotein-processing enzymes in the endoplasmic reticulum of cells infected with those viruses. Specifically, the compound N-nonyl deoxynojirimycin disrupts glycoprocessing in the liver so that M protein of the viral envelope cannot be constructed. Again, it is not clear what the long-term benefits and risks of such endoplasmic blockade might be. The use of other carbohydrate drugs has also been investigated to interrupt the viral life cycles in other ways. Some agents that block the ability of flu virus to exit infected cells by binding to viral neuraminidase have shown some early promise.
Another glycobiological approach to infectious and inflammatory disorders is based on the hope that pharmacologic agents can be developed to block the very synthesis of sugars that serve as the key recognition molecules for mediators of inflammation. For example, inhibitors of several glycosyl transferase enzymes are being investigated for that purpose.
It was predictable that experience with biological effects of glycosylation of proteins would with time also ignite interest in the study of how sugars might also alter the structural and functional characteristics of lipids. To cite a specific example, the compound NB-DNJ, also called Vevesca, was shown to be effective at maintaining healthy phospholipids in patients with lipid storage diseases.10
XII. SUGARS, ADHESION MOLECULES,
Many strains of Escherichia coli and other microbial species adhere to epithelial cells. Such adhesion to erythrocytes is designated hemagglutination and is the basis of the diagnostic hemagglutination test. The recognition of the role of sugars in such binding came from studies that showed that monosaccharide mannose and some other similar sugars inhibit hemagglutination. Escherichia coli also exhibits a special affinity for the tissues of the urinary tract, but is usually not involved in upper respiratory infections. Those patterns of microbial affinity for specific tissues is called tissue specificity and is a generalized phenomenon. By contrast, group A streptococci often colonize skin and the respiratory tract, but seldom the urinary tract tissues. Microbial specificity also varies not only among species but also between individual cultures belonging to the same species, depending upon their age, health, and genetic make-up. The molecular basis of microbial adhesion is highly specific binding of their surface lectins to complementary sugars on the surface of host cells. That union is a requisite for subsequent infection of the host.
Urothelial cells of individuals whose erythrocytes lack P blood-group substance do not bind to the P-fimbriated E. coli. Not unexpectedly, such persons are much less susceptible to urinary infections with E. coli than those who carry P substance. However, when urothelial cells are first coated with the synthetic glycolipid containing galabiose, the microbes readily bind to them. The K99 strain of E. coli causes diarrhea in farm animals, but not in humans. That microbial species binds specifically to a glycolipid containing N-glycolylneuraminic acid, which is a special type of sialic acid. That glycolipid is not present in human cells which instead contain N-acetylneuraminic acid, a nonbinding analogue of sialic acid. Thus, a minor difference between two highly similar sugars—the replacement of an acetyl group by a glycolyl group—radically alters the ability of the microbial species to cause clinical disease.
XIII. ANTI-ADHESIVE THERAPIES
Because sugars play crucial roles in recognition systems of bacteria, the potential use of sugars as molecular decoys with antimicrobial effects is under intense scrutiny. However, the use of sugars to intercept bacteria before infections is not new. As early as 1979, experiments were conducted with injection of mannose-specific strains of E. coli and methyl alpha mannoside—a sugar that inhibits bacterial in vitro adhesion to epithelial cells—to investigate the protective roles of sugars. The results showed that the presence of sugars reduced the colonization of the urinary tract by bacteria.(Sharon 1993). Similar experiments with other globotetraose and other sugars yielded similar results. Additional experiments showed that glycopeptides from the blood of cows protect newborn calves from lethal doses of E. coli. Injections of antibodies against mannose prevent mice from certain infections by mannose-specific E. coli.
XIV. SUGARS AND CANCER
The glycobiological dynamics of inflammation and infection are directly relevant to the subject of neoplasia, since host responses to tumor cells largely harness similar molecular pathways. Beyond that, cancers cells cleverly use some sugars as their protective shields against—molecular decoys to dodge—assault by the immune cells. For example, metastasizing tumor cells in the circulating blood seize P selectin on blood platelets and use those corpuscles as decoys. That is a partial explanation of the empirical benefits of heparin in patients with disseminated cancer, as well as of the experimental observation that heparin markedly reduces long-term organ colonization by tumor cells. It is noteworthy in this context that I observe advanced changes of oxidative coagulopathy in nearly all patients with widespread malignant tumors. Heparin arrests oxidative coagulopathy in many ways, as discussed later in the section dealing with that pathophysiologic entity.
XV. OF SUGARS, CHIMP BRAINS, AND HUMAN INTELLECT
What separates human intellect from the chimp mind? Some sugar enthusiasts proclaim that it is a particular variant of a sugar called sialic acid.
At the genomic level, humans differ from chimps by a paltry one to two percent. More fascinating than that for me is the claim that to date only one gene has been found that exists in chimps but not in humans. That gene codes for the enzyme CMP-sialic acid hydroxylase. By inserting oxygen, that gene converts a sialic acid variant called N-acetylneuraminic acid into another variant celled N-glycolylneuraminic acid. What makes that yet more interesting is that that gene is also absent in all other mammals studied so far. Evidently, what separates humans from chimps and all other mammals is the evolutionary advances in the brain structure and intellectual functions. The absence of that singular gene in humans has raised an interesting question: Could that gene have something to do with the apparent differences among the human intellect and the mind of chimps and other apes? Could such a circumstance be a result of some such interplay of evolutionary pressures and molecular biology?
Darwin, of course, was right about his notions of the impact of environment on living beings and nonliving things. But in emphasizing selection of the fittest as the operant mechanism, he seems not to have elaborated upon the matter of nature’s preoccupation with complementarity and contrariety. Enter sialic acid! Many pathogens gain entry into cells by latching onto sialic acid and spreading on the cell membranes. What would happen if that sialic acid were to be changed by some complementarian or contrarian role of the genetic dice? Could the resulting sialic acid variant not protect the host cell as well? Or could it become more sticky for the pathogens and facilitate their invasion of the cell? Could it make pathogens more virulent and host cells more vulnerable? To extend that line of reasoning, could the brain development have been altered by such changes in its immune response? Could changes in host defense of human brain cells triggered by such a genetic mutation—the loss of that “sialic acid gene”—have paved the way for the evolution of the human brain and developments of its myriad functions beyond where chimps and other apes could reach? That possibility has been recognized and is being actively investigated.11
XVI. SUGARS PROTECT PROTEINS
Sea monkeys (brine shrimp), along with myriad seeds, micro-organisms, and many arthropods, protect themselves from severe cold and drought conditions by several strategies. One such approach involves assuming a sugar-coated ‘glassy’ state. An excellent example is the arthropod family called tardigrades. The insects secrete a rock-hard coating of ‘candy’ that encases their cells and protects their delicate bodies from severe dehydration and other noxious environmental influences. Availability of water brings them back to life. The essential molecular phenomenon involved in the formation of the protective glassy state is protection of protein molecules by sugars.
Proteins generally have a complex structure with tertiary and quaternary folds that confer upon them specific biologic activities. Those structural folds are very vulnerable to the deforming influences of free radicals, chemical moieties, and physical elements such as temperature. True to its complementarian fixation, Nature has designed ingenious molecular mechanisms to shield vulnerable proteins. One of the most important mechanisms involves protection of protein molecules by their ‘sugar-daddies.’ It may be noted that structural protein molecules regularly subjected to the most intense mechanical stresses, such as proteins that form cartilage, ligaments, and bone, are heavily glycosylated. The widely reported clinical benefits of glucosamine and chondroitin in the treatment of arthritis among nutritionist-physicians may be viewed as just one reflection of the sugar-protein dynamics. Precisely how sugars stabilize protein molecules is not well understood. The variability and complexity observed so far is baffling. Notwithstanding, chemists in the pharmaceutical industry are intensely interested in using the sugar approach to stabilize therapeutic agents composed of proteins.12
XVII. CARBOHYDRATE SIGNALING
Carbohydrates play diverse and often seemingly contradictory roles in cellular signaling functions.13-15 The example of nitrate reductase, the first enzyme in nitrate assimilation, may be given to illustrate the existence of carbohydrate signaling at the very foundational levels of cellular energetics. This enzyme functions at the crossroad of the two basic energy-consuming pathways of nitrate assimilation and carbon fixation. Sunlight regulates nitrate reductase gene expression, as it does many of the carbon fixation genes. In the cytosol, this enzyme secures its reductant not from photosynthesis but from carbohydrate catabolism. It turns out that sucrose also assumes the function of sunlight in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. 13 However, sucrose by itself does not evoke the full expression of nitrate reductase genes in the plant. It may be mentioned here that both the light and sugar responses are mediated by a 2.7-kilobase region of 5′ flanking sequence of the nitrate reductase gene.
Higher plants carry sucrose-sensing (signaling) pathways that are essential for their existence as highly organized multicellular organisms. For example, crucial signaling is mediated by the proton-sucrose symporter in the key transport step involved in the resource distribution system of such plants.16 Sucrose symporter activity declines in plasma membrane vesicles isolated from leaves fed exogenous sucrose via the xylem transpiration stream. The transcriptional activity diminishes and/or there is a decrease in mRNA stability with a reduction in the level of symporter message. This pathway is independent of hexokinase as the sugar sensor.
Developmental glycobiology is a burgeoning field. Recent advances in technologies for studies in genomics and bioinformatics are revealing the existence of a broad range of complex, intertwined, and interacting pathways that control gene expression in response to changes in the ‘carbohydrate status’ of organisms. 17-19 Undoubtedly, such work will lead to precise delineation of carbohydrate signaling in pathways of cellular development, differentiation, dedifferentiation, and demise. Protein glycosylation is a crucial first step in regulating a wide range of cell signaling in cellular development, differentiation, and demise. Not unexpectedly, differential receptor glycosylation creates different biologic functionalities of proteins. A good illustrative case study is of a glycosyltransferase that modulates Notch signaling. The Notch gene encodes a receptor protein that mediates a broad range of cell fate decisions. Many clinicopathologic diseases have been linked with aberrant Notch signaling, including: leukemia (TAN-1), a congenital syndrome associated with axial skeletal defects (spondylocostal dysostosis); another congenital syndrome associated with stroke and dementia (CADASIL), and congenital syndrome associated with liver, cardiovascular, and skeletal defects. The interest in Notch signaling was first sparked by the identification of a novel glycosyltransferase, designated Fringe, that is essential for the growth and patterning of the Drosophila wing imaginal disc. Subsequent studies revealed that both Notch and its ligands are substrates for Fringe. In keeping with the theme of complementarity and contrariety in signaling, Fringe inhibits the activation of Notch by one ligand and potentiates the activation of Notch by another ligand.
These studies of carbohydrate signaling in health and disease were conducted in plants over 350 years after the experiments of van Helmont in 1648. 20 Not unexpectedly in view of Nature’s preoccupation with complementarity and contrariety, most nutrients are not only building blocks of organic matter, but also serve as signaling molecules or cofactors for such molecules. In modern terms, the notion that the presence of soil nutrients—or its lack— is sensed by plants is often attributed to the work of Brezeale during the early years of the last century. In 1906 he documented increased transport of nutrients in response to starvation.21 Since then, transporters for most macronutrients and micronutrients have been cloned. Examination of those cloned transporters have revealed the molecular basis for the regulation of uptake for many of the macronutrients (NH4+, NO3, K+, Ca2+, H2PO4, SO42, and Mg2+) and micronutrients (Cl, Zn, Mn2+, Fe3+, and Cu2+). It is now recognized that nutrient availability affects the transcription of the transporter gene. Direct application of such knowledge in clinical medicine has been very limited so far. However, it seems safe to predict that such plant studies will lead to similar studies in human tissues in the future.
Complementarity and contrariety in carbohydrate structure and function as well as cellular recognition systems dependent upon sugars are presented. The burgeoning field of glycomics carries a great potential of answering innumerable and critical questions about human biology. Cellular oxidative metabolism not only provides energy for life functions of cells, but also results in free radical generation, thus playing a critical role in redox homeostasis. Through modifications of lipids and proteins, sugars actively participate in the major homeostatic mechanisms of the body. Indeed, the future work in glycomics, in my view, will show that sugars are as intricately involved in cell development, differentiation, and demise as any other family of molecules in the body.
Carbohydrates serve myriad additional roles in the web and kaleidoscope of human biology, including key functions in recognition of cells by microbial species.
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