Conformational studies on blood group and related oligosccharides

Abstract

Complex carbohydrates have become the centre of much interest because it has been shown that they serve as determinants of specificity on cell surfaces. Human red blood cells contain a number of antigenic determinants on their surface. The ABH(O) and Lewis systems are the best known of these. Due to difficulties in isolating and purifying these antigens, detailed knowledge of the chemical structure of the ABH(O) and Lewis antigens was obtained using the glycoproteins present in secretions. These blood group substances, as they are known, carry similar antigens as those present on the blood red cell membrane of the individual.

Many approaches, such as enzymatic degradation, hapten inhibition studies, and partial acid hydrolysis and alkaline degradation, have been used in elucidating the structure of the carbohydrate portions of the blood group substances. In many of the hapten inhibition studies, oligosaccharides from human milk and the carbohydrate fragments obtained from hydrolysis of glycoproteins have been used as inhibitors. The use of specific antibodies, lectins, and enzymes in these studies has provided much knowledge as to the size and specificity of the combining site of these macromolecules. In no other system related to cell surface carbohydrates is there such detailed information. However, these studies have also led to the accumulation of much data which could not be rationalized.

Antibodies against chemically defined oligosaccharides have also been used as specific reagents for detection and quantitation of unknown oligosaccharide sequences on the surface of cells. Such antibodies have been raised to specific oligosaccharides coupled to proteins. This method suffers since it introduces antigenic sites not present in natural antigens and also destroys part of the natural structure of the oligosaccharide.

The application of conformational energy calculations in the field of carbohydrates has led to a number of interesting and important results. Hence, an attempt has been made to calculate theoretically the favored conformations of the blood group and related carbohydrates and to correlate their shapes to their biological and other properties.

This thesis contains five chapters. A brief review of the earlier work on the blood group and related oligosaccharides and a summary of recent developments have been presented in Chapter 1. A summary of the principles of conformational analysis is included, where the various potential energy functions are discussed.

Chapter 2 deals with the conformational analysis of the disaccharide fragments of the blood group carbohydrates. Conformational energy maps were constructed both with and without the inclusion of energy due to the exo-anomeric effect. The preferred conformations proposed from the energy calculations for the disaccharides are, in general, in agreement with NMR data. These studies suggest that the addition of energy due to the exo-anomeric effect to the total potential energy does not alter the positions of either the global or local minima but may slightly affect their relative energies.

The possible conformations of lacto-N-tetraose, lacto-N-neotetraose, related disaccharides, and other milk oligosaccharides have been computed theoretically and are discussed in Chapter 3. Lacto-N-tetraose favors a 'curved' conformation, while lacto-N-neotetraose favors an approximately 'straight' conformation. This difference in overall shape may explain the greater ability of lacto-N-neotetraose compared to lacto-N-tetraose to inhibit the cross-reaction of blood group Pi fractions with Type XIV pneumococcal antipolysaccharide. The disaccharides Gal(1-4)GlcNAc, Gal (1-3)GlcNAc, and lactose, which exhibit about equal inhibitory properties, favor conformations which are similar in overall shape. From these studies, it has been suggested that it is the overall shape of the molecule which is important for activity, rather than the residue or linkage present at the nonreducing end alone.

In Chapter 4, the possible conformations for the A, B, H(O), Lea, and Leb oligosaccharide moieties have been proposed and are discussed. It has been shown that the conformation of the common core fragment of these oligosaccharides is not significantly altered by the addition of L-fucose, galactose, and N-acetylgalactosamine residues at the nonreducing end to give the various determinants of blood group specificity. Using the most probable conformations arrived at from these calculations, correlations have been made between the shapes of these oligosaccharides and their biological properties. It has been suggested that those antibodies, lectins, and enzymes which cannot distinguish between type 1 and type 2 structures have small binding sites. The mode of binding of A1 and A2 antigens with their specific antibodies has been explained using a two-pocket model for the antibody binding site.

Possible conformations of some mannotetraoses and several milk oligosaccharides have been computed and are discussed in Chapter 5. Changes in the terminal residue at the reducing end (cyclic to acyclic form) of these molecules do not affect the favored conformations of the remaining oligosaccharide moiety. However, differences in the overall shape of the native and reduced forms of the mannotetraose, Man( 1-3)Man( 1-2)Man( 1-2)Man, are much less marked than between the native and reduced forms of lacto-N-tetraose. These differences are related to the effectiveness of the native forms as inhibitors of antibodies produced using synthetic antigens.

Thus, the present studies provide information about:

The favored conformations of various blood group and related carbohydrates,

The effect of the addition of various sugar residues such as galactose, N-acetylgalactosamine, and L-fucose to 'core' carbohydrate fragments, and

The conformational differences in type 1 and type 2 blood group structures.

These studies relate the overall shape of the molecule to its biological properties and rationalize most of the available experimental data (i.e., the interactions of these carbohydrates with antibodies, lectins, and enzymes). They also provide information as to how best to use the immune method for the identification of unknown oligosaccharides by specially prepared antibodies.