Theoretical studies on the conformation of glycosaminoglycans heparin and heparan sulfate

Abstract

Heparin and, to a lesser extent, heparan sulfate are biologically important because of their anticoagulant and anti-lipaemic (fat-clearing) properties. They are composed of alternating uronic acid ( -L-iduronic acid or -D-glucuronic acid) and -N-acetyl-D-glucosamine joined through (1 4) glycosidic linkages. They differ in the relative amounts of the uronic acid moiety and also in the number of sulfate groups. These differences, which lead to different extents of anticoagulant and other activities, have not been understood in terms of their conformation.

Recently, some attempts have been made to study the three-dimensional structure or conformation of these molecules by X-ray fibre diffraction, but the results have not led to an unequivocal solution. Hence, conformational analysis has been carried out to propose the probable conformation for heparin, heparan sulfate, the neutral oligosaccharide, and the protein fragments at the site of peptide-carbohydrate linkage.

This thesis consists of seven chapters: Chapter 1 gives a brief description of the types of glycosaminoglycans and also the basic principles of conformational analysis, namely, the concept of the hard-sphere model and the form of different potential functions which take into account various types of interactions.

It is well-known that the conformation of monosaccharides is of paramount importance not only in understanding their physical and chemical properties but also in working out the preferred conformations of polysaccharides, since the former forms the building blocks of the latter. Among the two structural units, the conformation of -N-acetyl-D-glucosamine has been worked out both theoretically and experimentally. Such information is not available for the uronic acids. Hence, the potential energies of uronic acids and their methyl derivatives have been computed for ideal and distorted forms in the ¹C(1) and ¹C(4) conformations, and these calculations are dealt with in Chapter 2. Minimization of the energies of these molecules has been studied by tilting the axially oriented substituents suitably. Considerable release of strain is achieved when tilts of about 1.5-2° and 3-3.5° respectively are given to the axial -OH, -OCH , and -COOH groups involved in the Hassel-Ottar effect, and a tilt of about 2° is given to syn-axial -OH groups. The calculated free energy values, after adding a value of 0.55 and 0.97 kcal/mol for the anomeric effect of -OH and -OCH groups respectively, explain the favoured conformations assigned experimentally by NMR studies. The present data indicate that all the uronic acids studied favour ¹C(1) conformation except -D-altropyranuronic acid and -D-idopyranuronic acid, which may exist in ¹C(4)-¹C(1) equilibrium in solution. Similarly, all methyl pyranosiduronic acids exist exclusively in ¹C(1) conformations except methyl -L-altropyranosiduronic acid and methyl -D-idopyranosiduronic acid, which may exist in ¹C(4)-¹C(1) equilibrium in solution.

Chapter 3 deals with the conformational studies of heparin. From X-ray diffraction studies on well-oriented fibres of sodium and calcium salts of heparin from different sources, Atkins and coworkers suggested that heparin exists in three distinct conformations with tetrasaccharide periodicities of 16.5 Å, 17.3 Å, and 16.8 Å, respectively. From model-building studies, they proposed a model for the molecular shape of the sodium salt of heparin by assuming a ¹C(4) conformation for uronic acid moieties and a ¹C(1) conformation for -N-acetyl-D-glucosamine, invoking an approximate two-fold axis with a covalent repeat approximating to a disaccharide periodicity. The increase in the tetrasaccharide periodicity from 16.5 Å to 17.3 Å has been implied to be due to the change in the ring conformation of uronic acid moieties in different salts. The present study reveals that these models are found to be unsatisfactory from stereochemical criteria. Hence, from energy calculations, an alternative model has been proposed consistent with the observed tetrasaccharide periodicities. In these models, -N-acetyl-D-glucosamine residues exist in ¹C(1) form and one of the uronides in ¹C(1) and the other in ¹C(4) conformation. The observed differences in the fibre repeat of calcium and sodium salts of heparin from different sources have been explained by a change in the rotational angles rather than a change in the ring conformation. This model is also independent of the small changes in the relative proportion of the uronic acid moieties observed in heparin obtained from different sources.

The conformational studies of heparan sulfate have been described in Chapter 4. From sodium salt, a tetrasaccharide repeat of 18.6 Å has been reported in the solid state. The basic structure is a 2 helix with a disaccharide as a structural repeating unit corresponding to helical parameters n = 2 and h = 9.3 Å. From stereochemical criteria, probable conformations have been proposed with all sugar moieties in ¹C(1) conformation. These studies also suggest that the low tetrasaccharide repeat of 16.8 Å observed for calcium salt of heparan sulfate could not be explained by assuming 50% of uronides in ¹C(4) conformation as in heparin, since glucuronic acid, which constitutes about 70% of the total uronic acid content, favours only ¹C(1) conformation in heparan sulfate. These studies also reveal that the probable conformations of heparin and heparan sulfate differ mainly in the pyranose ring forms of uronic acid moieties; hence in the orientations of the -COOH and -SO groups.

Conformational studies of the neutral oligosaccharide fragment, which links the glycosaminoglycan with core protein, described in Chapter 5, suggest that this fragment favours extended conformations. Perhaps the extended nature of this fragment is required for separating the charged groups of glycosaminoglycans from those of the protein core. Except in one case (Glu-Gly-Ser(CHO)-Gly), not much is known about the amino acid sequence of the protein segment at the site of peptide-carbohydrate linkage in proteoglycans. On the contrary, a great deal of information is available about the amino acid sequence of a number of glycoproteins in the neighbourhood of peptide-carbohydrate linkage. Hence the prediction of the secondary structure of about 80 glycoproteins and glycopeptides which contain Asn, Asp, Ser, and Thr linked sugars has been carried out by using the methods of Chou and Fasman to obtain information about the stereochemical requirement at the site of peptide-carbohydrate linkage, and these results are described in Chapter 6. In most cases, the amino acid sequence at the site of sugar linkage assumes a -turn whether the sugar is linked to Asn, Asp, Ser, or Thr. The present analysis also suggests that the nature of the amino acid residue [Asn(CHO)-X-Ser/Thr, Asp(CHO)-X-Ser/Thr, Ser(CHO)-X-Y and Thr(CHO)-X-Y] plays a major role in deciding whether all or part of the amino acid residues in these sequences are involved in the -turn. In aspartic acid-linked sugars, the triplet sequence Asp(CHO)-X-Ser/Thr is conserved (like in Asn(CHO)-X-Ser/Thr) and bending begins with aspartic acid, thus involving the complete triplet sequence in the formation of the -turn. In proteins which contain Ser(CHO)-X-Y and Thr(CHO)-X-Y sequences, the amino acid residue preceding the above sequences is generally either Pro or Gly. These results are in disagreement with the “code sequence” Thr/Ser(CHO)-O-C-Y-Pro proposed for the linkage of sugar to threonine or serine from earlier studies.

In Chapter 7, a detailed conformational analysis of the amino acid sequence (Glu-Gly-Ser(CHO)-Gly) which occurs in proteoglycans has been carried out, and Type Ib of Ramachandran’s classification of -bend has lower energy compared to other bend types. The present study also suggests that the formation of a 4 1 backbone hydrogen bond is not a necessary condition for a stable bend conformation. These studies also reveal that the sugar residues can be linked to serine in Type Ib bend without steric strain. It thus seems that in native proteoglycan, these bends occur alternatively after every ten and thirty-five residue sequences along the protein chain.