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RELATIONSHIP OF STRUCTURAL CARBOHYDRATES TO BLOOD GROUP ANTIGENS

Carbohydrates are the fuel of life, being the main source of energy for living organisms and the central pathway of energy storage and supply for most cells. They are the major products through which the energy of the sun is harnessed and converted into a form that can be utilized by living organisms. According to rough estimates, more than 100 billion tons of carbohydrates are formed each year on the earth from carbon dioxide and water by the process of photosynthesis. Polymers of glucose, such as the starches and the glycogens, are the mediums for the storage of energy in plants and animals respectively. Coal, peat and petroleum were probably formed from carbohydrates by microbiological and chemical processes.

Carbohydrates comprise only about 1 percent of the human body; proteins comprise 15 percent, fatty substances 15 percent and inorganic substances 5 percent (the rest being water). Nevertheless, carbohydrates are important constituents of the human diet, accounting for a high percentage of the calories consumed. Thus some 40 percent of the calorie intake of Americans (and some 50 percent of that of Britons and Israelis) is in the form of carbohydrates: glucose, fructose, lactose (milk sugar, a disaccharide of glucose and galactose), sucrose and starch.

Sucrose is a major food sugar. Its world production rose from eight million tons in 1900 to nearly 88 million in 1977. No other human food has shown an increase in production on this order in the same period. The amount of sucrose produced by a country is an index of its average income. In the richer countries, such as the U.S., Britain, Australia and Sweden, the annual consumption is between 40 and 50 kilograms of sucrose per person, whereas in the poorer ones, such as India, Pakistan and China, it is five kilograms or less. It has often been suggested that the high sucrose diet may have detrimental effects on the health of people in developed countries, being responsible to some extent for the increase in such diseases as diabetes, obesity and dental cavities.

Carbohydrates are the raw materials for industries of great economic importance, such as wood pulp and paper, textile fibers and pharmaceuticals. The principal industrial carbohydrate is undoubtedly cellulose: its worldwide use is estimated at 800 million tons per year. Polysaccharides with gelling properties, such as agar, pectic acid and carrageenans, are important in the food and cosmetic industries.

The four major classes of compounds essential to life are nucleic acids, proteins, lipids and carbohydrates. Over the past 30 years the first three classes have received much attention from chemists and biologists, whereas during most of that time the carbohydrates were largely neglected, partly in the belief that their chemistry and biology had been fully worked out. In the past decade, however, research on carbohydrates has been revived and is now expanding rapidly. As a result of many new developments carbohydrate research is today broad and diverse.

The study of carbohydrates and their derivatives has greatly enriched chemistry, particularly with respect to the role of molecular shape and conformation in chemical reactions. Recent carbohydrate investigations have played a decisive role in the characterization of various antibiotics and anti-tumor agents. Such studies have led to the discovery of new biosynthetic reactions and enzymatic control mechanisms and are contributing significantly to the understanding of many fundamental biological processes, for example the interaction of cells with their environment and with other cells. As a result revolutionary new methods for combating bacterial and viral infections and for targeting drugs on diseased cells and organs are being envisioned. Carbohydrate research has also provided a basis for recognizing the enzyme deficiency underlying several genetic disorders and has led to the hope that they can be treated effectively. A common theme behind many of the recent findings, which is also a powerful driving force in carbohydrate research, is the realization that monosaccharides (the basic units of carbohydrates) can serve, as nucleotides and amino acids do, as code words in the molecular language of life, so that the specificity of many natural compounds is written in monosaccharides.

FIGURE 1: Common carbohydrate linkages

Carbohydrates are sugars or (like starch and cellulose) chains of sugars. To most people sugar is the common household foodstuff, which to the chemist is sucrose. Chemically the molecule of sucrose consists of two monosaccharides, or simple sugars, glucose and fructose, that are hooked together; it is thus a disaccharide. More than 20 different monosaccharides have been found in nature, all of which are chemically related to glucose or fructose. As a rule they are black crystalline solids that dissolve readily in water. Some of them have not been obtained in amounts sufficient for testing their sweetness, but the are still called sugars, as are the monosaccharides that are found to be not sweet.

Glucose is the best-known monosaccharide; indeed, it has probably been investigated more thoroughly than any other organic compound. It was undoubtedly known to the ancients because of its occurrence in granulated honey and wine. References to grape sugar, which is glucose, are to be found in Moorish writings of the 12th century. In 1747 the German pharmacist Andreas Marggraf, whose isolation of pure sucrose from sugar beets is an example of the chemical art of the time at its best, wrote of isolating from raisins "eine Art Zucker" (a type of sugar) different from cane sugar; it was what is now called glucose. The action of acids on starch was shown to produce a sweet syrup from which a crystalline sugar was isolated by Constantine Kirchoff in 1811. Later workers established that the sugar in grapes is identical with the sugar found in honey, in the urine of diabetics and in acid hydrolysates of starch and cellulose. The French chemist Jean Baptiste Andre Dumas gave it the name glucose in 1838. The structure of glucose and of several other monosaccharides, including fructose, galactose and mannose, was established by about 1900, mainly by the brilliant work of the German chemist Emil Fischer, who thereby laid the foundations of carbohydrate chemistry.


Water of Carbon

The name carbohydrate was originally assigned to compounds thought to be hydrates of carbon, that is, to consist of carbon, hydrogen and oxygen in the general formula (CH20). Indeed, glucose and other simple sugars such as galactose, mannose and fructose do have the general formula C61-11206. They are typical hexose monosaccharides, meaning that they have six carbon atoms. With the accumulation of more data the definition has been modified and broadened to encompass numerous compounds with little or no resemblance to the original "water of carbon." Carbohydrates now include polyhydroxy aldehydes, ketones, alcohols, acids and amines, their simple derivatives and the products formed by the condensation of these different compounds through glycosidic linkages (essentially oxygen bridges) into oligomers (oligosaccharides) and polymers (polysaccharides).

Much of the current interest in carbohydrates is focused on such substances as glycoproteins and glycolipids, complex carbohydrates in which sugars are linked respectively to proteins and lipids. They are termed glycoconjugates. It should also be noted that in the excitement about nucleic acids a simple fact is being forgotten: they too are complex carbohydrates, since monosaccharides are among their major constituents (ribose in RNA and deoxyribose in DNA).

Carbohydrates are the most abundant group of biological compounds on the earth, and the most abundant carbohydrate is cellulose, a polymer of glucose; it is the major structural material of plants. Another abundant carbohydrate is chitin, a polymer of acetylglucosamine; it is the major organic component of the exoskeleton of arthropods such as insects, crabs and lobsters which make up the largest class of organisms, comprising some 900,000 species (more than are found in all other families and classes together). It has been estimated that millions of tons of chitin are formed yearly by a single species of crab!

Mannose Galactose Glucose Ribose

FIGURE 2: Common monosaccharides

Carbohydrates Defined (CHO)

Most follow the (CH2O)n formula. Primarily 6 carbon (above) and 5 carbon sugars occur as monosaccharides (ex. glucose), disaccharides (ex. lactose), and polysaccharides (starch). Fiber: Structural carbohydrates or cell wall components, chains of sugars connected by beta linkages. Insoluble fibers are structural carbohydrates or cell wall components; chains of sugars connected by beta linkages. Soluble fibers (pectins, gums, and mucilages, which make oatmeal or apple sauce gooey) are not structural components per se, and microbes, not pancreatic enzymes, break the linkages.

CHO digestion

Pancreatic enzymes can break the alpha linkage, but not the beta linkages. The exception is lactose, which is a beta linked sugar.

Monosaccharides cross the intestinal lining, with glucose and the sodium ion, Na, co-absorbed. This is why we give salt and sugar to treat diarrhea.

CHO in the body

Glucose is used as fuel by many tissues. Excess glucose is stored as the polysaccharide glycogen in liver and muscle, but storage is limited. Excess glucose is converted to fat when glycogen stores are replete.

Five carbon sugars are a component of DNA and RNA.

CHO provides carbon chains for biosynthesis

Fiber affects gastrointestinal (GI) motility and can alter absorption of other nutrients.


Unusual Saccharides

One result of the introduction of the powerful new techniques was the discovery of many new saccharides, both simple and complex. In recent years the number of rare sugars isolated from natural sources has increased rapidly. They have provided the carbohydrate chemist with new and challenging problems of structural determination and synthesis. I shall illustrate this state of affairs with examples from an area in which I have been active, the amino sugars: sugars in which one or more hydroxyls are replaced by an amino group.

In 1875 a young physician named George Ledderhose was working during the summer semester in the laboratory of Friedrich Wohler in Gottingen when Ledderhose's uncle, Felix Hoppe-Seyler, a noted physiological chemist, invited him to dinner. At his uncle's suggestion he took the remains of the lobster they had eaten back to the laboratory, where he found that the claws and the shell dissolved in hot concentrated hydrochloric acid and that on evaporation the solution yielded characteristic crystals. He soon identified the crystalline compound as a new nitrogen-containing sugar, which he named glycosamine.

During the next 20 years much evidence was gathered to indicate that the new sugar has a structure derived by the replacement of the hydroxyl group attached to carbon No. 2 in the glucose molecule by an amino group. With the synthesis, which was still not definitive, of the amino sugar by Emil Fischer and H. Leuchs in 1903 the problem of its structure appeared to have been solved. The structure of glucosamine was unequivocally established, however, only in 1939, when Norman Haworth achieved an unambiguous synthesis that proved Fischer was correct in assigning the "gluco" structure to the amino sugar. A second amino sugar, galactosamine, was isolated in 1914 by P. A. Levene and Frederick B. La Forge at the Rockefeller Institute for Medical Research from acid hydrolysates of cartilage, tendon and aorta, but its structure was firmly established only in 1945, again attesting to the enormous difficulties such substances present. At the time, that was thought to be the end of the amino-sugar story.


Biological Markers

Until recently it was not recognized that nature can employ sugars for the synthesis of highly specific compounds that can act as carriers of biological information. This capability arises from the fact that a large number of structures can be formed from a small number of monomers. In other words, monosaccharides can serve as letters in a vocabulary of biological specificity, where the words are formed by variations in the nature of the sugars present, the type of linkage and the presence or absence of branch points. It is now known that the specificity of many natural polymers is written in terms of sugars, not amino acids or nucleotides. This idea is not entirely novel, but it has only recently become well established.

The central dogma of molecular biology limits the downstream flow of genetic information to proteins. Progress from the last two decades of research on cellular glycoconjugates justifies adding the enzymatic production of glycan antennae with information-bearing determinants to this famous and basic pathway.

In the 1920's it was still believed that the specific information in biological polymers was carried only by proteins. Between 1925 and 1937 Oswald T. Avery of the Rockefeller Institute, together with Michael Heidelberger and Walther F. Goebel, demonstrated that pure polysaccharides can carry specific immunological messages as antigens: substances that stimulate the production of an antibody specific to themselves. Thus the highly purified Type III pneumococcus "specific soluble substance" was an antigen even though it did not have any of the properties of a protein. This substance was shown to be polysaccharide, consisting of repeating units of cellobiuronic acid (a disaccharide of glucose and glucuronic acid).

It is well established that carbohydrates are ideally suited for the formation of specificity determinants that can be recognized by complementary structures, which presumably are carbohydrate-binding proteins, on other cells or molecules. An impressive variety of regulatory processes including cell growth and apoptosis, folding and routing of glycoproteins and cell adhesion/migration have been unraveled and found to be mediated or modulated by specific protein (lectin)-carbohydrate interactions. Currently, the potential for medical applications in anti-adhesion therapy or drug targeting is one of the major driving forces fueling progress in glycoscience.

The first indication that sugars serve as specificity determinants came from the discovery in 1941 by George K. Hirst in New York and by Ronald Hare in Toronto that the influenza virus caused red blood cells to agglutinate, or clump. The molecular basis of this phenomenon was for a time obscure. Mainly as a result of the efforts of Alhed Gottschalk in Australia, it was shown that the influenza virus binds to the red blood cell through sialic acid units on the cell surface. If the sialic acid is removed from the cell surface by the enzyme neuraminidase, the influenza virus will no longer bind to the cell.

The role of carbohydrates in recognition has been best demonstrated in the control of the lifetime of glycoproteins in the circulatory system and their uptake into the liver and of the uptake of lysosomal enzymes by cells. As often happens, these exciting discoveries originated with an unexpected observation, this one made in 1966 by G. Gilbert Ashwell of the National Institute of Arthritis, Metabolism, and Digestive Diseases and by Anatol G. Morell of the Albert Einstein College of Medicine in the course of an effort to understand the biological role of ceruloplasmin, a copper-transport protein found in the blood serum of man and other animals. When Ashwell and Morell removed sialic acid from rabbit ceruloplasmin and reinjected the modified ceruloplasmin into the animals, it almost completely disappeared from the circulatory system within 15 minutes. This was in striking contrast to the native glycoprotein, almost all of which remained in circulation after the same length of time.

Galactose hence serves as a recognition marker that determines the survival time of many serum glycoproteins in the circulatory system of man, the rabbit and the mouse. In bird and reptile species the recognition marker appears to be primarily acetylglucosamine. Clearance systems in which fucose and mannose are the markers have also been found.

A particularly interesting marker is mannose-6-phosphate, a sugar derivative that has recently been shown to act mainly in directing the intracellular traffic of glycoprotein enzymes normally present in lysosomes. This finding had its origins in Neufeld's discovery that the enzyme deficiencies in cells from patients afflicted by mucopolysaccharidoses such as Hurler's and Hunter's syndromes can be corrected by providing the cells with the missing enzymes. In 1974 she showed further that uptake into the cells depended on the presence on the enzymes of a carbohydrate recognition marker. In 1977 William S. Sly of the Washington University School of Medicine and Arnold Kaplan of the Saint Louis University School of Medicine identified the recognition marker as a phosphorylated sugar unit: mannose-6-phosphate. The function of the marker is apparently to prevent the secretion of the enzymes from the cells and to direct them into the lysosomes. When the enzymes are supplied from the outside, it is this recognition signal that promotes their binding to the cell surface; without binding they cannot enter the cells and reach the lysosomes.

By the covalent (electron-sharing) attachment of carbohydrates to proteins or by a modification of the sugars in glycoproteins it may thereby be possible to control the proteins' lifetime in the circulation and to direct them to the liver and perhaps also to other organs, as well as into lysosomes. Such techniques will have far-reaching uses for enzyme replacement therapy in cases of genetic disease and also for delivering drugs accurately into target organs and cells.


Other Biological Roles

Sugars on cell surfaces also appear to determine the life span of circulating cells and their distribution in the body. This role was originally demonstrated in 1964 by Bertram M. Gesner and Victor Ginsburg of the National Institute of Arthritis, Metabolism, and Digestive Diseases. They found that radioactively labeled rat lymphocytes migrated to the spleen when they were re-injected into the animal. If before re-injection the sugar fucose was removed from the surface of the cells by treatment with a specific glycosidase, the lymphocytes migrated to the liver instead, as if the fucose on the lymphocytes served as a "ZIP" code directing them where to go.

Old red blood cells have less sialic acid on their surface than young ones, and so it has been postulated that the decrease of sialic acid is the sign responsible for the removal of the older red blood cells from the circulatory system. This hypothesis seemed to be further substantiated by the finding that when red blood cells are taken out of the circulation, and when the sialic acid is removed from their surface and they are re-injected into the blood, their life span is extremely short; only a couple of days out of the normal lifetime of 120. In spite of these striking correlations there is considerable doubt whether the removal of sialic acid and the exposure of galactose units on the surface of the red blood cell are responsible for the removal of senescent red cells from the blood under physiological conditions in vivo.


Blood Group Antigens as Structural Carbohydrates

The ABO blood-group system was first described by Karl Landsteiner of the Rockefeller Institute in 1900, but it was not until 1953 that Walter Morgan and Winifred Watkins of the Lister Institute demonstrated that the specificity of the major blood types is determined by sugars. For example, the difference between the blood types A and B lies in a single sugar unit that sticks out from the end of a carbohydrate chain of a glycoprotein or glycolipid on the surface of the red blood cell. In blood type A the determinant is acetylgalactosamine, in blood type B it is galactose. The two monosaccharides differ by only a small group of atoms, but that little difference is sometimes a matter of life and death, since using the wrong type of blood in a transfusion can have fatal results.

FIGURE 3: Structure and production of ABO antigens


The blood group antigens have been dismissed by some researchers as merely 'icing on the cake' of glycoprotein structures. The fact that there are no lethal mutations and individuals have been described lacking ABO, H and Lewis antigens seems to lend weight to the argument. Research suggests that these antigens do indeed have function and argues that blood group antigens play important roles in modulation of protein activity, infection and cancer.

Glycoconj J 1997 Feb;14(2):159-73

The enzymatic removal by specific glycosidases of alpha-linked acetyl galactosamine from type A red blood cells or of alpha-linked galactose from type B red blood cells will convert both into type O cells. An effective conversion can, for example, be carried out by purified alpha-galactosidase from coffee beans or soybeans, as was demonstrated in our laboratory by Noam Harpaz and Harold Flowers. Such a conversion may be useful clinically when type 0 cells of rare subtypes are needed for transfusion.

The sugars that determine the specificity of substances in the ABO blood group are distributed in the biological world in forms similar to those found in human beings. The substances are therefore, also present in different mammals. Hence the red blood cells of the dog, the pig and the rabbit are invariably of type B and in some cases may also belong to type A. The ABO blood-group substances are present in birds and amphibians and even in plants and bacteria.

Tamio Yamakawa of the University of Tokyo suggested that dogs may possess a blood-group system specified by the sialic acid in red-blood-cell glycolipids. Whereas all European dogs so far examined have glycolipids that incorporate acetylneuraminic acid, Yamakawa and his co-workers have shown that representative Japanese dogs such as the Kishu and Shiba breeds often have glycolyneuraminic acid instead and that this occurrence is genetically determined. Akita and Hokkaido dogs from northern Japan seem to be exceptional in having only acetylneuraminic acid in their red-blood-cell glycolipids. The origin of the Japanese dog is still controversial, but since the glyconeuraminic acid glycolipid is inherited as a dominant trait, the findings suggest that the origins of the Akita and Hokkaido breeds are different from those of other Japanese dogs and that the Akita and Hokkaido breeds are related to European dogs.

Some blood group antigen-bearing proteins function as major transport channels within the erythrocyte membrane; these include the anion transporter (band 3: Diego and Wright antigens), the water channel (aquaporin: Colton antigens), and the urea transporter (Kidd antigens). At least two erythrocyte blood group antigen proteins have complement regulatory functions: the complement receptor type 1 (CR1, CD35: Knops antigens) and decay accelerating factor (DAF, CD55: Cromer antigens). Some blood group antigens reside on proteins with known receptor functions, such as the chemokine receptor (Duffy) and the hyaluronan receptor (Indian). The Cartwright antigens reside on an enzyme, acetylcholinesterase, and the Kell antigens reside on a protein that belongs to the CALLA-related family of neutral metalloproteinases. Finally, some blood group antigens reside on proteins that serve crucial structural functions necessary to normal erythrocyte lifespan and morphology. These proteins include band 3, glycophorins C/D (bearing the Gerbich antigens), and the Rh proteins. Both oligosaccharide and protein blood group antigens may act as receptors for bacterial, viral, and parasitic infectious agents.

Curr Opin Hematol 1996 Nov;3(6):473-9

The expression of blood group antigens was ubiquitously upregulated in the endothelial cells of fetal organs. In the process of their differentiation to endothelial naive embryonic mesenchymal cells expressed cytoplasmic ABH antigens. They were assumed as products of the activation of the respective genes. ABH antigen expression was considered as suggestive evidence for the assumption that blood group antigens could serve as early immunomorphologic markers of endothelial differentiation of mesenchymal cells, thus specifying the location of future blood vessels. Extending the conceptual framework of blood group antigens' significance we consider them as being possibly involved in the process of fetal morphogenesis.

Folia Med (Plovdiv) 1997;39(2):5-9

Blood-group antigens have been developed as a self-declaration mechanism in higher organisms, since blood cells carry different DNA from that of germ-line cells, and their selfishness must be strictly limited. If not, symbiosis between somatic DNA and germ-line DNA cannot be maintained since blood cells can express autonomy programmed within themselves. For the sake of maintenance of symbiosis, this self-declaration is not limited to blood cells and all somatic cells need a self-plural declaration mechanism such as blood-group antigens. Differentiation and development including induction and inhibition also depend on the self-declaration--recognition mechanism.

Med Hypotheses 1996 Mar;46(3):290-4

Although there are probably over a thousand publications on the associations of blood groups and disease, many are based totally on statistical analyses. Most of the earlier studies have been controversial, because they were small studies and/or had inadequate controls and/or had been analyzed incorrectly. Nevertheless, it is difficult to argue with the general pattern that emerges from the large body of statistical data on malignancy, coagulation and infection. Recent findings in membrane chemistry, rumor immunology and infectious disease (especially relating to bacterial receptors), add a scientific rationale for some of these findings, and there is an increasing rationale for some of the earlier statistical findings. Some of the more recent findings on parasitic/bacterial/viral receptors, the hematological abnormalities seen when high frequency blood group antigens are missing, and the association with immunologically important proteins are most convincing and suggest that blood group antigens do sometimes play an important biological role; this role may relate directly, or often be completely unrelated, to the red cell.

Pubbl Stn Zool Napoli II 1996;18(3):321-44


Use of Blood Group Glycoconjugates in Nanomedicine

In Robert A. Freitas' Nanomedicine, he writes:

Medical nanorobots can probably distinguish all of these cell types by surface chemical assay using chemosensor pads. Antigenic specificities exist for species (xenotype), organ, tissue or cell type for almost all cells, possibly involving as many as 104 distinct antigens.

In the case of red blood cells (RBCs), antigens in the Rh, Kell, Duffy, and Kidd blood group systems are found exclusively on the plasma membranes of erythrocytes and have not been detected on platelets, lymphocytes, granulocytes, in plasma, or in other body secretions such as saliva, milk, or amniotic fluid. Thus detection of any member of this four antigen set establishes a unique marker for red cell identification. MNSs and Lutheran antigens are also limited to erythrocytes with two exceptions: GPA glycoprotein (MN activity) is also found on renal capillary endothelium, and Lublike glycoprotein appears on kidney endothelial cells and liver hepatocytes. In contrast, ABH antigens are found on many non-RBC tissue cells such as kidney and salivary glands. In young embryos ABH can be found on all endothelial and epithelial cells except those of the central nervous system. ABH, Lewis, I and P blood group antigens are found on platelets and lymphocytes, at least in part due to adsorption from the plasma onto the cell membrane. Granulocytes have I antigen but no ABH.

ABO is the best known blood group system. Erythrocytes are typed as A, B, AB, or O(H), the latter indicating a lack of expression of either A or B. The H antigen is the precursor of A and B and is found on all red cell surfaces (up to ~1.7 x 106 antigens/RBC, or ~18,000/micron2) except those of patients with the rare Oh Bombay or H null phenotype. Because H is a precursor of A and B, type O erythrocytes have more H antigen than A or B erythrocytes, which in turn have more H antigen than AB erythrocytes (which express both A and B antigens). The number of A and B antigens on the red cell surface ranges from 12 x 106 (~10,00020,000/micron2); in 75% of Type A individuals, "double length" A antigens are also present (~500/micron2). MNSs factor antigens range from ~27005400/micron2, Rh factor antigens ~100300/micron2, Lewis factor antigens ~30/micron2, and so forth. Again assuming a ~(300 nm)2 chemotactic sensor pad, a nanorobot searching for a particular set of ~30 blood group antigens (Nspec = 30) requires t meas ~ 0.03 sec to make the self/nonself determination for a particular red cell membrane it has encountered. A nanorobot seeking to determine the complete blood group type of the membrane (e.g., in mapping mode) must in the worst case search all 254 known blood antigen types (Nspec = 254), requiring at most t meas ~ 2 sec.

Direct detection of blood group antigens, or of antibodies to blood group antigens in body fluids, permits at least partial self-recognition by blood borne nanorobots without the need for any direct cell contact, which may be useful in establishing theater protocols. ABH, Lewis, I and P blood group antigens are found in blood plasma, and serum IgMclass antibodies associated with the carbohydrate antigens of the ABO, Lewis, and P blood group systems are almost universal. AntiM and antiN are common, antiSda is found in 12% of normal people, and antiVw or antiWra is found in ~1% of patients.960 In persons who previously have been pregnant or transfused, 0.160.56% have anti D (Rh group) and 0.140.60% have anti E and anti C (Rh system), anti K and anti Fya, and several other antibodies in serum. Body secretions contain ABH, I and Lewis antigen but no P system antigens; Sda antigen is found in most body secretions, with the greatest concentration in the urine.