British Medical Bulletin (1978) Vol. 34, No. 1, pp. 25-41
CHEMICAL ASPECTS OF MUCUS / R Clamp, A Allen, R A Gibbons & G P Roberts
CHEMICAL ASPECTS OF MUCUS J R CLAMP MD PhD FRIC MRCP Department of Medicine, University of Bristol
A ALLEN DPhil Department of Physiology, University of Newcastle upon Tyne
R A GIBBONS DSc Agricultural Research Council Institute for Research on Animal Diseases, Compton, Newbury, Berkshire
G P ROBERTS BSc PhD University Department of Surgery, Welsh National School of Medicine, Cardiff
GENERAL CONSIDERATIONS
flat but puckered, giving rise to a number of conformations of which the so-called "chair" form is most important. Secondly, and more important, therelationshipof each substituent to the over-all plane of the ring is disregarded. An attempt has been made to illustrate this in fig. 1. Although glucose does not occur in mucus glycoproteins, it is a convenient monosaccharide for purposes of illustration. In fig. la, glucose is drawn in the Haworth convention and all the bulky substituents (the hydroxymethyl group of C-6 and the
J R CLAMP
As discussed in the present Bulletin by Reid & Clamp (1978), mucus glycoproteins are high-molecular-weight proteins possessing many hundreds, if not thousands, of covalently attached oligosaccharide units. The units contain on average about 8-10 monosaccharide residues of five different types. These are as follows, with the systematic name where different given in parentheses: L-fucose (6-deoxy-L-galactose); D-galactose; Nacetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose); Nacetyl-D-galactosamine (2-acetamido-2-deoxy-r>galactose); and sialic acid. Sialic acid is the general name given to any substituted neuraminic acid. In humans the only important sialic acid is //-acetylneuraminic acid (5-acetamido-3,5-
FIG. I. Three different representations of the cyclic (pyranose) form of glucose ((J-D-glucopyranose) CH2OH HO HO
OH
&\feoxy-v>-glycero-T>-galacto-nomi\oson\c acid), although in
animals a number of other sialic acids occur, including Nglycollylneuraminic acid and various Orsubstituted derivatives (Schauer et al. 1974). In the following discussion and in this number of the Bulletin generally the trivial names are used and the configurational sign (D or L) has been omitted unless there is a possibility of confusion. Because of the reactivity of the reducing group, monosaccharides readily cyclize to form oxygen-containing ring structures. Although five-membered rings, known as "furanoses", do occur in nature, only the six-membered "pyranose" rings are important; and all the monosaccharides of mucus glycoproteins are pyranoses. The pyranose forms of monosaccharides are usually represented by the Haworth convention. In this convention the pyranose ring is shown as a flat, lozenge-shaped structure with the substituents on each carbon atom drawn at right angles to the plane of the ring. Although simple and easily understood, the convention suffers from considerable disadvantages. Firstly, the pyranose ring is not
OH
a : the Haworth representation b: the chalr-IIke confbrmational representation c: a modified Haworth representation All the bulky substituents (hydroxyl and hydroxymethyl groups) In ^-O-glucopyranose are equatorial. For clarity, all the hydrogen substituents on the ring have been omitted
25 Vol. 34 No. 1
CH 2 0H
CHEMICAL ASPECTS OF MUCUS
/ R Clamp, A Allen, R A Gibbons &GP Roberts FIG. 2. Haworth representation (left-hand side) and chair-like conformational representation (right-hand side) of the monosaccharides present in mucus glycoproteins
hydroxyl groups) appear to lie alternately above and below the plane of the ring. Figure 1 b is an attempt to illustrate the threedimensional structure of glucose in the chair form. In this diagram one can see that a substituent may lie in the general plane of the ring (equatorial) or be at right angles to that plane (axial). This may be clearer in fig. lc, which is really a modified Haworth representation, and here it is apparent that all the bulky substituents are equatorial. Whether groups, particularly those involved in linkages, are axial or equatorial is important in model-building techniques for the determination of polysaccharide structure. The linkage between two axial groups (diaxial) has a very different effect upon carbohydrate structure from that between two equatorial groups (diequatorial) (see Morris & Rees, 1978). After cyclization, the "reducing group" of a monosaccharide, which is C-l in the hexoses and C-2 in neuraminic acid, becomes the "potential reducing group". The process of cyclization creates a new asymmetrical carbon atom and for historical reasons the two isomers (anomers) are known as a and p. The a form has the same configuration as the reference carbon so that in D-hexopyranoses the a hydroxyl group is below the plane of the ring whereas the p hydroxyl group is above it. Figure 1, therefore, shows P-D-glucopyranose. For an L sugar, on the other hand, the converse would be true— as for L-fucopyranose (fig. 2). Figure 2 shows all the monosaccharides of interest in mucus glycoproteins in the Haworth convention Geft-hand side) and in the chair-like confonnation (right-hand side). Monosaccharides when joined together are capable of forming a large number of different oligosaccharide units. This is because a hexose, such as galactose, in the pyranose form can be substituted at one or more of the positions 2, 3,4 or 6. In addition, the substituting monosaccharide or monosaccharides can form a or p linkages. Thus, even when the sequence of sugars is known, a typical oligosaccharide unit can exist in many thousands of different structures. An additional problem is the degree of heterogeneity that occurs among the oligosaccharide units of an apparently homogeneous glycoprotein preparation. This is particularly important in the terminal monosaccharides, which may be absent in some of the units. This type of heterogeneity is often called "peripheral heterogeneity" or "microheterogeneity". For all these reasons, the determination of oligosaccharide structure is time consuming and requires a variety of methods. Ignoring for the moment the problem of anomeric configuration, these methods may be subdivided into:
CH20H
(3-Galactose CHjOH
OH CHzOHn
oH
HO
NHCOCH3 a-V-Acetylgalactosamine
NH.
c'o O H CH3
CH?0H
NHCOCH3 -/V-Acstylglucosarpine
CHiCOHN t/OH
\J
OH o-/V-Acetylnouraminic acid For clarity, all the hydrogen substituents on the ring have been omitted. In the Haworth representation of a-N-acetylneuramlnic add, R stands for CH(OH)-CH(OH}-CH2(OH)
that, in the original oligosaccharide, galactose was substituted at position 4. Periodate oxidation is also used to detect positions of substitution. This technique depends upon the presence of at least two free hydroxyl groups on adjacent carbon atoms. Wherever this occurs periodate cleaves between the two carbon atoms, oxidizing the hydroxyl groups to aldehyde, and in the process one mole of oxidant is reduced to iodate. In the example above, galactose would be oxidized between positions 2 and 3. If three adjacent hydroxyl groups are free, for example at positions 2, 3 and 4, two moles of oxidant are reduced and C-3 is released as formic acid. Thus it can be seen that the minimum requirement for any hexose to survive oxidation is that it be substituted at position 3. The introduction of a reduction step after oxidation, with the subsequent identification of the products, gives further structural information. In addition, the glycosidic linkage of an oxidized and reduced monosaccharide is very labile and can be cleaved under mild conditions, uncovering a further
i. those that indicate the positions of substitution of monosaccharides. Such methods include methylation studies, periodate oxidation techniques and methods of incomplete cleavage yielding disaccharides and larger fragments; ii. those that indicate the sequence of monosaccharide residues. Such methods include certain periodate oxidation techniques (for example Smith degradation: see following paragraph), the use of glycosidases and again methods of incomplete cleavage. Methylation studies are used to identify the positions of substitution in the monosaccharides. Chemical methods attach a methyl group to each free hydroxyl group and, after hydrolysis, the various methylated sugars are separated and identified. For example, if one found a trimethylgalactose derivative with methyl groups on positions 2, 3 and 6, one could assume 26
Br. Med. Bull. 1978
CHEMICAL ASPECTS OF MUCUS / R Clamp, A Allen, R A hydroxyl group. The cycle of oxidation, reduction and mild hydrolysis can be repeated, and this process is known as "Smith degradation". Glycosidases, or more correctly exoglycosidases, are specific not only for a particular monosaccharide in a terminal position but also for the anomeric configuration, that is whether the sugar is attached by an a or p linkage. If, for example, P-galactosidase is active against an oligosaccharide, then galactose must be in a terminal position, (J linked to some other sugar. Used sequentially, therefore, glycosidases can give valuable information about the order and configurational linkages of the monosaccharide residues. A number of methods have been devised partially to degrade the oligosaccharide units of glycoproteins and produce a reasonable yield of disaccharides and larger fragments. The structures of the saccharide fragments are of course easier to establish than the structure of the original, much larger, oligosaccharide unit. The identification of the fragments will enable a partial structure to be proposed, or even a complete structure if sufficient overlapping sequences are found. By convention the structures of oligosaccharides are usually represented in an abbreviated form. In a way similar to that used for amino acids, the monosaccharides are shortened to the first three letters of the name, except for glucose which is represented by "Glc" (to avoid confusion with "Glu" representing glutamic acid). JV-Acetylglucosamine, A^acetylgalactosamine and ./V-acetylneuraminic acid are abbreviated to GlcNAc, GalNAc and NeuNAc, respectively. Thus
Gibbons&GPRoberts
amino acid is converted to the corresponding
CHEMICAL ASPECTS OF MUCUS / R Clamp, A Allen, R A Gibbons & G P Roberts
CHEMICAL ASPECTS OF MUCUS J R CLAMP MD PhD FRIC MRCP Department of Medicine, University of Bristol
A ALLEN DPhil Department of Physiology, University of Newcastle upon Tyne
R A GIBBONS DSc Agricultural Research Council Institute for Research on Animal Diseases, Compton, Newbury, Berkshire
G P ROBERTS BSc PhD University Department of Surgery, Welsh National School of Medicine, Cardiff
GENERAL CONSIDERATIONS
flat but puckered, giving rise to a number of conformations of which the so-called "chair" form is most important. Secondly, and more important, therelationshipof each substituent to the over-all plane of the ring is disregarded. An attempt has been made to illustrate this in fig. 1. Although glucose does not occur in mucus glycoproteins, it is a convenient monosaccharide for purposes of illustration. In fig. la, glucose is drawn in the Haworth convention and all the bulky substituents (the hydroxymethyl group of C-6 and the
J R CLAMP
As discussed in the present Bulletin by Reid & Clamp (1978), mucus glycoproteins are high-molecular-weight proteins possessing many hundreds, if not thousands, of covalently attached oligosaccharide units. The units contain on average about 8-10 monosaccharide residues of five different types. These are as follows, with the systematic name where different given in parentheses: L-fucose (6-deoxy-L-galactose); D-galactose; Nacetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose); Nacetyl-D-galactosamine (2-acetamido-2-deoxy-r>galactose); and sialic acid. Sialic acid is the general name given to any substituted neuraminic acid. In humans the only important sialic acid is //-acetylneuraminic acid (5-acetamido-3,5-
FIG. I. Three different representations of the cyclic (pyranose) form of glucose ((J-D-glucopyranose) CH2OH HO HO
OH
&\feoxy-v>-glycero-T>-galacto-nomi\oson\c acid), although in
animals a number of other sialic acids occur, including Nglycollylneuraminic acid and various Orsubstituted derivatives (Schauer et al. 1974). In the following discussion and in this number of the Bulletin generally the trivial names are used and the configurational sign (D or L) has been omitted unless there is a possibility of confusion. Because of the reactivity of the reducing group, monosaccharides readily cyclize to form oxygen-containing ring structures. Although five-membered rings, known as "furanoses", do occur in nature, only the six-membered "pyranose" rings are important; and all the monosaccharides of mucus glycoproteins are pyranoses. The pyranose forms of monosaccharides are usually represented by the Haworth convention. In this convention the pyranose ring is shown as a flat, lozenge-shaped structure with the substituents on each carbon atom drawn at right angles to the plane of the ring. Although simple and easily understood, the convention suffers from considerable disadvantages. Firstly, the pyranose ring is not
OH
a : the Haworth representation b: the chalr-IIke confbrmational representation c: a modified Haworth representation All the bulky substituents (hydroxyl and hydroxymethyl groups) In ^-O-glucopyranose are equatorial. For clarity, all the hydrogen substituents on the ring have been omitted
25 Vol. 34 No. 1
CH 2 0H
CHEMICAL ASPECTS OF MUCUS
/ R Clamp, A Allen, R A Gibbons &GP Roberts FIG. 2. Haworth representation (left-hand side) and chair-like conformational representation (right-hand side) of the monosaccharides present in mucus glycoproteins
hydroxyl groups) appear to lie alternately above and below the plane of the ring. Figure 1 b is an attempt to illustrate the threedimensional structure of glucose in the chair form. In this diagram one can see that a substituent may lie in the general plane of the ring (equatorial) or be at right angles to that plane (axial). This may be clearer in fig. lc, which is really a modified Haworth representation, and here it is apparent that all the bulky substituents are equatorial. Whether groups, particularly those involved in linkages, are axial or equatorial is important in model-building techniques for the determination of polysaccharide structure. The linkage between two axial groups (diaxial) has a very different effect upon carbohydrate structure from that between two equatorial groups (diequatorial) (see Morris & Rees, 1978). After cyclization, the "reducing group" of a monosaccharide, which is C-l in the hexoses and C-2 in neuraminic acid, becomes the "potential reducing group". The process of cyclization creates a new asymmetrical carbon atom and for historical reasons the two isomers (anomers) are known as a and p. The a form has the same configuration as the reference carbon so that in D-hexopyranoses the a hydroxyl group is below the plane of the ring whereas the p hydroxyl group is above it. Figure 1, therefore, shows P-D-glucopyranose. For an L sugar, on the other hand, the converse would be true— as for L-fucopyranose (fig. 2). Figure 2 shows all the monosaccharides of interest in mucus glycoproteins in the Haworth convention Geft-hand side) and in the chair-like confonnation (right-hand side). Monosaccharides when joined together are capable of forming a large number of different oligosaccharide units. This is because a hexose, such as galactose, in the pyranose form can be substituted at one or more of the positions 2, 3,4 or 6. In addition, the substituting monosaccharide or monosaccharides can form a or p linkages. Thus, even when the sequence of sugars is known, a typical oligosaccharide unit can exist in many thousands of different structures. An additional problem is the degree of heterogeneity that occurs among the oligosaccharide units of an apparently homogeneous glycoprotein preparation. This is particularly important in the terminal monosaccharides, which may be absent in some of the units. This type of heterogeneity is often called "peripheral heterogeneity" or "microheterogeneity". For all these reasons, the determination of oligosaccharide structure is time consuming and requires a variety of methods. Ignoring for the moment the problem of anomeric configuration, these methods may be subdivided into:
CH20H
(3-Galactose CHjOH
OH CHzOHn
oH
HO
NHCOCH3 a-V-Acetylgalactosamine
NH.
c'o O H CH3
CH?0H
NHCOCH3 -/V-Acstylglucosarpine
CHiCOHN t/OH
\J
OH o-/V-Acetylnouraminic acid For clarity, all the hydrogen substituents on the ring have been omitted. In the Haworth representation of a-N-acetylneuramlnic add, R stands for CH(OH)-CH(OH}-CH2(OH)
that, in the original oligosaccharide, galactose was substituted at position 4. Periodate oxidation is also used to detect positions of substitution. This technique depends upon the presence of at least two free hydroxyl groups on adjacent carbon atoms. Wherever this occurs periodate cleaves between the two carbon atoms, oxidizing the hydroxyl groups to aldehyde, and in the process one mole of oxidant is reduced to iodate. In the example above, galactose would be oxidized between positions 2 and 3. If three adjacent hydroxyl groups are free, for example at positions 2, 3 and 4, two moles of oxidant are reduced and C-3 is released as formic acid. Thus it can be seen that the minimum requirement for any hexose to survive oxidation is that it be substituted at position 3. The introduction of a reduction step after oxidation, with the subsequent identification of the products, gives further structural information. In addition, the glycosidic linkage of an oxidized and reduced monosaccharide is very labile and can be cleaved under mild conditions, uncovering a further
i. those that indicate the positions of substitution of monosaccharides. Such methods include methylation studies, periodate oxidation techniques and methods of incomplete cleavage yielding disaccharides and larger fragments; ii. those that indicate the sequence of monosaccharide residues. Such methods include certain periodate oxidation techniques (for example Smith degradation: see following paragraph), the use of glycosidases and again methods of incomplete cleavage. Methylation studies are used to identify the positions of substitution in the monosaccharides. Chemical methods attach a methyl group to each free hydroxyl group and, after hydrolysis, the various methylated sugars are separated and identified. For example, if one found a trimethylgalactose derivative with methyl groups on positions 2, 3 and 6, one could assume 26
Br. Med. Bull. 1978
CHEMICAL ASPECTS OF MUCUS / R Clamp, A Allen, R A hydroxyl group. The cycle of oxidation, reduction and mild hydrolysis can be repeated, and this process is known as "Smith degradation". Glycosidases, or more correctly exoglycosidases, are specific not only for a particular monosaccharide in a terminal position but also for the anomeric configuration, that is whether the sugar is attached by an a or p linkage. If, for example, P-galactosidase is active against an oligosaccharide, then galactose must be in a terminal position, (J linked to some other sugar. Used sequentially, therefore, glycosidases can give valuable information about the order and configurational linkages of the monosaccharide residues. A number of methods have been devised partially to degrade the oligosaccharide units of glycoproteins and produce a reasonable yield of disaccharides and larger fragments. The structures of the saccharide fragments are of course easier to establish than the structure of the original, much larger, oligosaccharide unit. The identification of the fragments will enable a partial structure to be proposed, or even a complete structure if sufficient overlapping sequences are found. By convention the structures of oligosaccharides are usually represented in an abbreviated form. In a way similar to that used for amino acids, the monosaccharides are shortened to the first three letters of the name, except for glucose which is represented by "Glc" (to avoid confusion with "Glu" representing glutamic acid). JV-Acetylglucosamine, A^acetylgalactosamine and ./V-acetylneuraminic acid are abbreviated to GlcNAc, GalNAc and NeuNAc, respectively. Thus
Gibbons&GPRoberts
amino acid is converted to the corresponding
