1H NMR in the structural and conformational analysis of oligosaccharides and glycoconjugates

Progress in Nuclear Magnetic Resonance Spectroscopy

EISEWIER

27 (1995) 445-474

‘H NMR in the structural and conformational analysis of oligosaccharides and glycoconjugates Elizabeth F. Hounsell University College London, Glycoprotein StructurejFunction Group, Depament of Biochemimy and Molecular Biologv, Darwin Building, Gower Street, London, WClE 6BT. UK

Received 12 September 1994

Contents 1. Introduction 2. Monosaccharides to oligosaccharides; ‘H NMR parameters and methods 2.1. Monosaccharide nomenclature and their NMR data 2.2. NMR methods for oligosaccharide analysis 3. Ofigosaccharide antigens and recognition determinants 3.1. Blood group and related structures 3.2. NMR of blood group ABH antigens 3.3. NMR of Ley Leb, Le’ and,Ley antigens 3.4. NMR of sialylated recognition determinants 4. O-Linked gfycoprotein chains 5. Proteogfycans 6. N-Linked glycoprotein chains 7. Mammalian gfycolipids 8. Mycobacterial and bacterial glyco(peptido)lipid conjugates Acknowledgement References

445 446 446 447 450 450 450 451 451 461 462 465 466 468 470 470

1. Introduction This review will attempt to draw together the large amount of ‘H NMR data now available on mono- to oligo-saccharides of importance in biomedicine. A unique feature of carbohydrate biochemistry is that a family of monosaccharides reoccurs in different situations, so that similar oligosaccharides can be found linked to proteins, lipids and within other natural products, such as, antibiotics, steroid conjugates, etc. Most serum, cell matrix and cell membrane proteins have attached oligosaccharide chains (glycoproteins and proteoglycans) and lipid or peptidolipid conjua gates abound as eukaryotic and prokaryotic glycolipids, glycophosphatidyl-inositol anchors, mycobacterial glycopeptidolipids (GPL) and bacterial polysaccharides linked to lipid A. The monosaccharides involved have a limited number of molecular weights but vary signi&urtly in the orientation of functional groups around the glycosidic ring and their linkage position and sequence in oligosaccharides. Due to this diversity and the general inability to crystallise such conjugates, NMR is the method of choice for their identification and conformational analysis. The reoccurrence of oligosaccharide sequences on different molecules means that an NMR fingerprint is a valuable tool in structural identification. Furthermore, the conformational information obtained for oligosaccharides from readily available sources can be used,predictively in other scenarios. 0079-6565/95/$29.00 @ 1995 Elsevier Science B.V. All rights reserved SSDI 0079-6565(95)01012-2

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E.F. Hounsell J Progress in Nuclear Magnetic Resonance Spectroscopy

27 (1995) 445-474

The areas which will be discussed herein are: oligosaccharides recognised by antibodies as tumour-associated and differentiation antigens; oligosaccharides specifically recognised by endogenous carbohydrate binding proteins (selectins) in many areas of cell and glycoconjugate trafficking; oligosaccharides having roles in the extracellular environment (as components of proteoglycans, mucins, etc.); oligosaccharides involved in cell growth regulation; oligosaccharide chains of proteins which affect protein folding, intracellular transport, conformation, stability, activity and antigenicity; and microbial glycoconjugates of importance in vaccination strategies. In the majority of these situations only ‘H or ‘H-detected 13C experiments are of sufficient sensitivity for NMR analysis. The exceptions, which will not be discussed further, are chemically synthesised oligosaccharides, repeating oligosaccharide units of bacterial polysaccharides, and plant polysaccharides.

2. Monosaccharides to oligosaccharides; ‘H NMR parameters and methods 2.1. Monosaccharide nomenclature and their NMR data

With respect to NMR parameters, monosaccharides can be classified into several groups. Although carbohydrate chemistry and biochemistry were named after the CsH1206 composition of glucose (Fig. 1) and its epimers allose, altrose, mannose, gulose, idose, galactose and talose, other compositions abound, the most common being: 2-deoxy, 2-aminosugars; 6-carboxylated uranic acids (e.g. iduronic acid abbreviated to IdoA); 6-deoxy-sugars (e.g. fucose and rhamnose); pentoses (e.g. ribose, deoxyribose, arabinose and xylose); sialic acids; phosphorylated and sulphated esters; and those having acyl substituents (such as methyl, acetyl, pyruvyl, lactyl, etc.). Table 1 gives representative ‘H chemical shifts and 3J a,~ coupling constants for monosaccharides discussed in this review as components of oligosaccharides in which each is linked to an adjacent monosaccharide via a glycosidic bond oriented either below (a) or above ( j?) the plane of the ring. Additional ‘H NMR data collected for reducing monosaccharides can be found in Refs. [l, 21. In mammalian glycoconjugates monosaccharides are predominantly hexopyranoses having a ‘C4 configuration as the L-sugars and 4C1 configuration as the D-sugars (Fig. 1). These give characteristic coupling constants (Table 1; Refs. [ 1, 2)) from which the ring geometry can be calculated. The major deviation from the chair form so far found occurs in chair-skew boat distortions which occurs in the IdoA residues of proteoglycans (Table 1). It is more difficult to define the ring geometry of furanohexoses and pentoses [3]. Ribose and deoxyribose are the most common representatives of pentoses because of their presence in RNA and DNA. Several skewed (envelope) forms are possible which may be important in DNA-protein interactions. The presence of the ring oxygen adjacent to the glycosidic bond in the monosaccharides discussed so far defines their unique anomeric properties which give distinct preferences for the orientation of the glycosidic linkage. Other related compounds called pseudo- or carba-sugars have different heteroatoms in the ring, commonly N or S, and occur for example in natural plant inhibitors of glycosylation reactions (glycosyltransferase inhibitors) such as castanospermine and nojiromycin, or in bacterial antibiotics. One last type of monosaccharide to be discussed, inositol, has no heteroatom (Fig. 1). This monosaccharide in phosphorylated form is an ubiquitous intracellular regulator and is also found in the glycosylphosphoinositol (GPI) lipids which anchor many different (glyco)proteins in the cell membrane. Monosaccharides are usually linked together via a condensation reaction between the oxygen at Cl and a hydroxyl group at positions other than Cl, but alternatives exist such as trehalose where two glucose residues are linked between Cl and Cl (Fig. 1). Another specific case are the sialic acids where Cl is designated as the C02H group and C2 as that bearing the glycosyl oxygen. The most common of these is N-acetylneuraminic acid (NeuAc), 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-Znonulosonic acid (Fig. 1). NMR chemical shifts and coupling constant data for NeuAc, and its different C7 to C9 glycerol side chain epimers, D-mannitol, D-ak&Ol, Dglucitol and allitol are given

E.F. Houn.wIIjProgress

in Nuclear Magnetic Resonance Spectroscopy 27 (1995) 445-474

-D-Glcp 4Cj conformation p

p

l

447

-D-Xylp

C4 conformation

CH20H

a -D-G&

a -L-Fucp

lnositol

a -L-Rhap

a-N-acetylneuraminic acid R=COCH3 a-N-glycolylneuraminic acid R=COCH20H

g&y@?_

d?p&-+

H

GalpI-4GlcNAcpl--N-acetyllactosamine Fig. 1. Some commonly

Glcal - 1aGlc a,a -Trehalose occurring

monosaccharides

and their ring conformation

in Ref. [4]. NeuAc occurs predominantly as the /?-anomer free in solution, but in glycoproteins and glycolipids (gangliosides) it is linked in the cc-form via the oxygen at C2 to the C8 or C9 hydroxyl group of the glycerol side chain of adjacent NeuAc residues or to the C6/C3 hydroxyl groups of hexopyranoses giving distinct NMR parameters [S, 61. The specificity of biosynthesis resides in the enzymes which control interconversion of one sugar to another in the metabolic pathways and the glycosyltransferases which catalyse the condensation reactions. Control is at the levels of substrate, acceptor and enzyme concentrations and is specific for monosaccharide type, linkage position (including branching) and anomeric configuration. There is one additional reaction now termed glycation, which is not enzyme catalysed and occurs in mammalian serum and tissues in the presence of excess glucose, e.g. in diabetes. This is a reductive amination procedure via Schiff base formation and Amadori rearrangement leading to sorbitol attached to e-NH2 groups of lysine in proteins (particularly albumin, haemoglobin, etc.). 2.2. Nh4R methods for oligosaccharide analysis The methodology for NMR analysis of oligosaccharides differs only little from that for other natural products, in particular proteins and DNA, but chemical shift assignment can be aided by

448

E.F. Howsell/ Progress in Nuclear Magnetic Resonance Spectroscofl27

Table 1 Representative ‘H chemical shifts and ‘J,,,

(1995) 445-474

coupling constants for some common monosaccharides

Chemical shifts’ (ppm from acetone at 2.225 ppm in D20 at 22-27°C) Proton

Monosaccharide

e-D-GtC-(1 -+ b-D-GfC-(1 -+ a-D-Man-(1 + /I-~-Man-(1 -+ a-D-Gal-(1 + /I-D-Gal-(1 -t fi-D-G~cNAc-(1 + a-D-GalNAc-(1 -+ /?-D-GalNAc-(1 -B a-L-Fuc-(1 -+ a-L-Rha-(1 --t fi-D-xyl-(1 -+ 3-0-Me-a-L-Fuc-(1 + 3-0-Me-a-L-Rha-(1 -+ 2,3-di-O-Me-a-t.-Rha-(1 3,6-di-O-Me-B-D-Glc-(1

+ -+

Hlb

H2

H3

H4

H5

H6

H6

CH,

NAc

5.1 4.4 4.9 4.7 5.2 4.5 4.7 5.2 4.7 5.1 4.9 4.5 4.8 5.0 5.1 4.7

3.56 3.31 3.98 4.04 3.84 3.52 3.75 4.24 3.96 3.69 4.06 3.27 3.70 4.24 3.94 3.34

3.72 3.51 3.83 3.63 3.90 3.67 3.56 3.92 3.87 3.90 3.80 3.43 3.40 3.59 3.52 3.31

3.42 3.41 3.70 3.58 4.02 3.92 3.48 4.00 3.92 3.79 3.46 3.61 3.52 3.41 3.51

3.77 3.45 3.70 3.37 4.34 3.71 3.45 4.07 3.65 4.1-4.9b 3.74

3.77 3.74 3.78 3.76 3.69 3.78 3.90 3.79 3.80 -

3.87 3.92 3.89 3.93 3.71 3.75 3.67 3.68 3.75 -

E

_

_

1.23 1.28

_

2.04 2.04 2.01 -

_

3.89 3.77 3.73 3.51

_ _ 3.66

_ _ 3.78

1.32 1.32 1.32 -

-

Coupling constants 3Ju,u(Hz) Monosaccharide

a-D-C&-(1

-+

/?-D-Glc-(1 -+ a-D-Man-(1 -+ /&D-Man-(1 + cc-D-Gal-(1 --, b-D-Gal-(1 --) a-~-GlcNS(6fS)-(l -+ fi-D-GIcNAc-(1 + a-D-GalNAc-(1 + a+Fuc-(1 -+ p-D-GIcA-(1 + a-L-Rha-(1 -) /!?-D-xyl-(1 -+ a-L-IdoA-(1 + ‘Cd 4C1 “So

Coupling constant J 1.2

J 1.3

J3.4

54.5

J 5.6

35.6

J 6,6

3.8 8.0 1.8 1.5 3.6 7.9 3.7 8.2 4.1 3.9 7.8 1.8 7.7 1.8 8.0 7.2

10.0 9.4 3.8 3.8 10.0 9.9 10.4 10.4 11.3 9.5 9.4 3.2 9.7 2.5 9.5 9.9

9.1 8.9 10.0 10.0 3.4 3.5 9.0 8.5 3.1 3.4 10.1 9.6 9.8 2.3 9.2 5.9

9.9 9.7 9.8 9.8 0.8 1.1 10.0 9.5 1.2 1.0 9.9 9.6 9.5,X 2.0 5.5 3.2

5.1 518 5.7 5.0 6.4 7.9 2.5 6.2 7.0 6.5

2.1 2.2 2.0 2.0 6.4 4.3 2.2 2.3 5.4

12.1 12.3 12.7 10.0 12.3 11.7 11.3 12.0 11.7

6.1 5.0 J.,

_

_ 12.95 5*r.5e.q

_

’These are averaged values for non-reducing terminal sugars linked by a glycosidic linkage to the adjacent monosaccharide. Signals for protons at the ring carbons are shifted downfield when linked by another monosaccharide at the hydroxyl group of that carbon. b These signals vary considerably more than other signals due to conformational features as discussed in Ref. [5] and shown in Table 2. ’HSax 3.29, H5eq 3.93.

E.F. HounselllF’rogress in Nuclear Magnetic Resonance Spctroscopy 27 (1995) 445-474

449

computer programmes designed specifically for oligosaccharides [7-l 11. Oligosaccharides have the disadvantage-of having a large number of signals in a narrow region of the spectrum between 4.2 and 3.5 ppm. Signals arising outside this region are useful starting points to trace connectivities around the glycosidic rings by either 1D difference spectroscopy or, more frequently 2D COSY, relayed COSY and TOCSY experiments. Because of the difficulty of assigning signals in the 4.2 - 3.5 ppm region, (pseudo) 3D and 4D homonuclear NMR can find particular applications for tracing specific signals [12]. Once the chemical shifts have been assigned, NOE data can be used to indicate through-space interactions across the glycosidic bonds leading to conformation and linkage information, although not always unequivocal. Long range heteronuclear scalar couplings ( 3Jc,u) through the glycosidic bond can provide additional corroborative proof of linkage position [13-161. These last two parameters have primarily been used to provide information on the dihedral angles 4, II/(Fig. 2) around the glycosidic bonds. 3.Jc,” coupling constants are acquired via HSQC or HMQC experiments. Homonuclear triple quantum filtered COSY experiments have proved useful for oligosaccharide studies for example to give unambiguous assignments of the C5 and C6 protons in hexopyranoses and hence to allow measurements of their coupling constants to establish the gauche-trans arrangement around the C5 to C6 bond (Fig. 2). There are many excellent reviews on these pulsed NMR methods that are currently used in natural product NMR analysis. The only major differences in the analysis of oligosaccharides compared with proteins or DNA, for example, are in the spectral width, relative complexity, solution flexibility and molecular size. With respect to the last, oligosaccharides can give either positive or negative NOE levels and therefore for tri- to deca-saccharides ROE experiments are often preferred over NOE experiments. Mixing times for these experiments are within the 3@1OOms range for lower to higher molecular weight samples. Some recent reviews on pulse methods are covered in the present progress series and there are also several books with chapters containing applications to oligosaccharides [17-191. Accessible journal reviews dealing more specifically with oligosaccharides and glycoconjugates are Refs. [20-26-J. Ref. [27] gives an overview of NMR and other physicochemical methods in the structural and conformational analysis of oligosaccharide determinants of glycoproteins. The majority of applications of ‘H NMR of oligosaccharides have used solutions in D20 at 22°C and compared chemical

0

e-m_

Fig. 2. The dihedral angles across oligosaccharide glycosidic bonds and the configurations around the C5-C6 bond. For example, the w rotomer population for Glc is normaliy around 60% gg (P = 180”) and 40% gt (P = - 60”).

450

E.F. Hounsell/ Progress in Nuclear Magnetic Resonance @ectroscopy 27 (1995) 445-474

shifts with respect to acetone at 2.225 ppm. An alternative strategy is to analyse acetylated oligosaccharides in organic solvents [28]. To gain more information about solution dynamics, studies of native oligosaccharides can be performed in Hz0 and water/acetone or water/methanol [29, 301, the latter at low temperatures ( - 5 to - 10X) to reduce the rate of hydroxyl group exchange with HzO. Discussed in this review is the progress made in ‘H NMR characterisation of the diverse array of oligosaccharides found in nature. This will cover aqueous solution studies of isolated oligosaccharide sequences which are recognition determinants for antibodies and carbohydrate binding proteins and the carbohydrate chains which bear them released from protein (N- and O-linked glycosylation) or lipid. NMR studies of glycopeptides, glycoproteins and lipid conjugates are also covered, the latter in organic solution or as micelles.

3. Oligosaccharide antigens and recognition determinants 3.1. Blood group and related structures The classic oligosaccharide recognition determinants are the blood group antigens bound by naturally occurring antibodies. These were originally discovered due to the cross-reactions that occur in human blood transfusion [31]. However, the oligosaccharides of the ABO system defined then, and structurally closely related oligosaccharides, occur on many tissues, not just red blood cells, and in other mammalian species. They also compose some of the capsular polysaccharide sequences of bacteria from whence it is proposed the anti-ABO antibodies arise in mammals due to a cross-reaction. Thus blood group A individuals having the sequence Fucal-2 [GalNAcal-3]Galfilon glycoproteins and glycolipids of cells and tissues have anti-B positive sera and therefore can not accept B positive red blood cells having the sequence Fucal-2[Galal-3]GaIpl-, and vice versa. Blood group 0 individuals have the precursor H-antigen, Fucal-2Gal/Il-, and both anti-A and anti-B serum antibodies. They are therefore universal donors of red blood cells but can only accept group 0 blood. The genetic basis of the blood group ABO system has recently been described in terms of minor base-pair changes in the genes encoding the blood group transferases which catalyse the addition of Galalor GalNAal-3 to the precursor [32]. There are many additional antigen and regulatory controls encompassed within the blood group system but these do not have significant implications for transfusion. These and related oligosaccharide structures (reviewed in Ref. [27]) have variable distributions in tissues which alter in differentiation and disease and hence are important targets for antisera, monoclonal antibody reagents and endogenous cell adhesion molecules (carbohydrate binding proteins; selectins). As such, many studies have employed NMR spectroscopy to determine the structures of oligosaccharides and to characterise the specificities of protein binding. Additionally, NMR chemical shifts and NOES have been interpreted in terms of conformational information in order to design inhibitors of binding. The present section highlights these conformational studies relating to proton chemical shift data given in Table 2. 3.2. NMR of blood group ABH antigens

In early NMR studies [33], NOE data were used to discriminate between possible conformers of the blood group H type 1 determinant Fucal-2Galfil-3GlcNAc/Il(sometimes also called Led). One of the most favoured conformers hasa&, $n Gal W,lO” and Fuc 6O”Jo”. This places the HS of the fucosyl group in close proximity (2.5 A) to the 03 of GlcNAc therefore accounting for the large specific deshielding for the H5 proton (Table 2). There followed a series or papers by Lemieux’s group [34-361 on detailed chemical shifts and conformational analysis of blood group antigens and studies of their recognition by lectins. The equivalent H type 2 sequence, Fucal-ZGalfll-4GlcNAc/Il-, was studied by Rosevear et al. [37], and Rao et al. [38] by NOE experiments giving Fuc 4n, $n, 55”,

E.F. Houmell/Progress in Nuclear Magnetic Resonance Spectroscopy 27 (1995) 445-474

451

0” &- lo” and Gal &,, $u 60”, 15”. The latter studies have now been extended to molecular dynamics

of blood group H and A oligosaccharides using the CHARMm force field with carbohydrate parameters [39] and studies on blood group A were revisited more recently [40] by NOE and T1 simulations together with Karplus analysis relating dihedral angles to 3Jc,u coupling constants from spectra of ’3C enriched oligosaccharides. 3.3. NMR of Le”, Leb, Le” and LeY antigens The early studies of Lemieux also encompassed analysis of the Lewis antigens Le” and Leb and related sequences called Le” and Ley. These began to be of great interest, in 1981 after the demonstration that the Le” structure, Galbl-4[Fucal-31 GlcNAcfil- was that recognised by a monoclonal (hybridoma) antibody as a specific differentiation antigen called stage specific embryonic antigen-l (SSEA-1) [41,42]. Studies on this sequence and its isomer Le”, Gal/?-3[Fucal-4]GlcNAc/Il-, began in earnest in attempts to understand the various specificities of monoclonal antibodies which could distinguish such closely related structures. These included studies on series of antibodies recognising putative tumour-associated antigens of mammary epithelia [43, 441 shown to discriminate between Le’, Le” and the difucosylated analogues LeY, Fucal-2Gal~l-4[Fucal-3]GlcNAc/?1and Leb, Fucal-2Galj?1-3[Fucal-4]GlcNAcjI-. Detailed ROESY experiments were used to examine conformational preferences in the tri- to octasaccharide molecular weight range and their scope and limitations have been discussed [44, 451. By far the most studied oligosaccharide sequence by NMR is the Le” trisaccharide. After its claim to fame as the first epitope recognised by a monoclonal antibody to be characterised, it was subsequently implicated in functional recognition in cell adhesion. Complete chemical shift assignment by 3D COSY-ROESY or TOCSY-ROESY methods [46], correlation of qualitative ROES with molecular modelling [47] and molecular dynamics and NOE simulations [48] do not significantly extend the conclusions previously drawn [33, 431. For this relatively rigid molecule with its Fuc residue stacked onto the Gal, the H5 proton is in close proximity to both 04 of GlcNAc and the ring oxygen of Gal and this gives it a deshielding of 0.6 ppm greater than for H5 in the blood group H Fuc (Table 2). This is also true for Le” where a similar conformation is proposed, but with H5 of Fuc near 03 of GlcNAc and the ring oxygen of Gal [49]. 3.4. NMR of sialylated recognition detenninants The sialylated analogues of Le” and Le” (SLe”, SLe”) present a greater challenge to conformational analysis as the C7-C9 glycerol side chain of the sialic acid (NeuAc) has the potential for flexibility, the ~2-3 linkage to Gal is relatively non-sterically hindered and the the carboxyl group at Cl will have significant water/salt effects. The rewards for this analysis are being sort worldwide as SLe” is the most useful so far of the carbohydrate based tumour antigens (under the name Ca 19.9) [49] and SLe” is one of the highest affinity ligands for cell adhesion molecules involved in inflammatory mechanisms (e.g. the sequence on neutrophils which binds to the endothelial cell selectin, ELAM-1) [SO]. Studies so far suggest that the Le” and Le” components are similar to the unsialylated forms, but that the NeuAca2-3Gal sequence has multiple low energy minima resulting in a series of solution conformers for SLe”/SLe” in fast exchange [Sl]. Other types of sialic acid containing recognition determinants of glycoproteins which have been studied in detail are the SD” antigen [52] with the structure NeuAcaZ-3[GalNAcj?l-4]Gal/?; sialic acid analogues of influenza virus neuraminidase and haemagglutinin ligands [53] and poly 2-8 linked NeuAc sequences of the adhesion molecule NCAM and certain glycolipids [54,55]. The NMR data for this latter sequence and conformational inferences have been corroborated from analysis of this same oligosaccharide sequence found in group B meningococcal and Escherichia coli Kl (colominic acid) capsular polysaccharides [54,55]. These studies have suggested that the polysaccharide exists in a helical conformation in solution for which the 4 (defined as 06-C2’-08-C8) and t/j (defined as C2’-OS-C8-C7) angles are in the ranges: 4 - 60-O”, $15-175” or @O-120”, $55-175”.

opl-3

7 al-4

bal-3

PI-3

al-3

B Aal-2Qpl-4

w

.pl-3

PI-40fil-3

5pe2B Aal-2npl-4.pl-3

Type la Aal-ZOpl-3

5.189 3.778 H3 3.665 H4 3.741 H5 4.293 CH, 1.233

3.810 4.219 1.234

5.297 3.78 3.69 3.82 4.87 1.280

H3 H5 CH,

Hl HZ H3 H4 1~5 CH,

Hl 5.349 HZ 4.317 CH, I .227

5.310 3.801

Hl H2

H2

Hl

Hl 5.028 H2 3 800 H3 3 895 H4 3792 H5 4.882 CH, I 180

Fuca (l-3/4)

Fuca

(l-2)

The NMR ‘H chemical shifts (ppm from DSS at 22°C in D,O) for fucosylated

Table 2

4546 3.85 393 4201 3 557 3.75 3 72

HI HZ H3 H4 H5 H6

4499 3 484 3.650 3.892 3.63 3 733

111 4.697 H3 4.026 H4 4.278

Hl HZ H3 H4 HS H6 H6

H4 3.894

Hl H2 H3 H5

Hl HZ H.3 114 H5 H6 H6 NAc

5.259 3.872 4.015 4.1 19

5.193 4.245 3.91 3.99 4.235 3.76-3.80 3.76-3.80 2.039

Hl HZ H3 H4 H5 H6 NAc

HI H2 H3 H4 H5 H6 H6 NAc

4.697 3.949 4.017 3.755 3.548 3918 2.032

4.624 3.81 3.986 3.529 3.49 3.89 3.78 2.056

GlcNAcB

NeuAca

?? , GalNAc; 0, Glc; 0, GlcNAc; A, NeuAc

Gala1 or GalNAca

A, Fuc; Cl, Gal;

4.647 3S80 3.846 3.880 3.679 3.7-3.8 3.7-3.8

HI 4.523

Hl 112 113 H4 H5 H6 H6

oligosaccharides;

HI 5.301 HZ 4.393 CH, 1.282 Hl (5.493 HI (5.435 CH, 1.295

5.034 3.813 3.911 3.806 4.853 1.292

Hl HZ H3 H4 H5 CH,

HI H2 H3 I14 H5 CH,

5.208 3.742 3.668 3.762 4.390 1.292

HI 5.028 H5 4.865 CH, 1.258

5.024 3.805 3.926 3.821 4.856 1.253

5.124 3 689 3902 3.789 4.831 I 177

HI 5.145 H5 4.341 CH, 1.272

5.151 3.755 3.683 3.730 4.340 1.270 ii1 HZ H3 H4 H5 Ctf,

Hl H2 H3 H4 H5 CH,

L@Aal-20Pl-3

PI-3 a al-4

tll HZ HB H4 H5 CH,

Le' op1-49p1-3 Aal-

4.663 3.604 3.806 3.862 3.576 3.737

4.459 3.491 3654 3.899 3.594 3 793 3 749

HI HZ H3 H4

111 HZ Ii3 114 II5 H6

4.550 3.854 3.774 4.203

4.712 3.820 3.879 4.166 3.538 3.755

HI 4.662 H2 3.58

111 tt2 H3 H4 H5 H6

HI HZ H3 H4 H5 H6 H6

HI HZ Ii3 H4 H5 NAc

HI HZ H3 H4 HS H6 NAc

5.197 4.250 3.922 3.994 4.257 2.041

5.218 4.172 3.960 3.970 4.308 3.740 2.023

4.601 3.830 4.129 3.730 3.506 2.052

4714 3.958 3.876 3.957 3.578 3.97-3.87 2 026

HI H2 113 Ii4 II5 H6 NAc

4.604 3.852 4.170 3.740 3.536 3.880 2.062

Ht 4.604 NAc 2.061

Hl tt2 H3 H4 H5 NAc

Ht 112 H3 H4 H5 H6 NAc

a2-3

SLC’ f p1-3

01-3 al-4

a

Table 2 (continued)

Hl H2 H3 H4

Hl 5.130 H5 4.839 CH, 1.170

4.517 3.523 4.081 3.933

HI 4.539 II3 4.032 II4 3.92

HI 5.002 H5 4.832 CH, 1.168

FucU (l-3/4)

FWX

(l-2)

Gala1 or GalNAcu

HZ 3.89 H3 3.83 H4 3.93 II5 3.58 116 3.94.0

Hl 4.729 113 4-098

GlcNAcB

H3a H3e H4 HS Nat

1.798 2.760 3.69 3.86 2.030

H3r 1.761 H3e 2.762

NeuAca

E.F. Hounsell/Progiess

in Nuclear Magnetic Resonance Spectroscopy

27 (1995) 445-474

455

Information on the conformational freedom of sialylated oligosaccharides is also being obtained from NMR studies of gangliosides, glycopeptides and mucin oligosaccharlde chains (Section 4). Table 3 shows the structure of the oligosaccharide sequences of the most common gangliosides, which by definition are sialylated glycolipids. The oligosaccharide is linked to a lipid tail often through the base sphingosine which defines the lipid ceramide, e.g. -0-CH2

I CH-NH-C(O)-(CH&CHJ I CH(OH)-CH = CH-(CH&CH, As these are not soluble in water, the majority of NMR studies have been carried out in DMSO-d6 (containing l-2% D20 as a spin lock and to optimise solubility and minimise micelle formation) Table 3 Structures of the common mammalian gangiiosides; 0, Glu; Cl, Gal; ?? , GalNAc; A, NeuAc Ganglioside

Structure p1-4op1-

GM3

f a2-3 pl-4Opla2-3 E 012-8

GD3

GM2

GD2

GM1

GMlb

GDla

GDlb

GTla

431-3

~1-4opl1 a2-3

q3l-3

p14op1a2-3 4 a2-8

OBl-341-3

p1_4op1! a2-3

p~-3mpl-30pb40pl-

f a2-6

1f31-3mw31a2-3

pl40p1-

a2-3

?pl-3mpl-3 ?

fw40pb

4 4 4 E

~2-3 a2-8

f31-3mpl-3 pbtopi-

a2-3

a2-3

a2-8

GTlb

i

plampl-3

pb40p1-

a2-3

a2-3 a2-8

gg~1-3?%i””

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E.F. HounsellJ Progress in Nuclear MagneticResonance Spectroscopy

27 (1995) 445-474

[X-59]. In contrast, the data for free oligosaccharides in most cases have been collected in DsO at 22°C (295 K) and related to the internal standard acetone, the methyl protons of which resonate at 2.225 ppm: this provides data which can usually be compared to within f 0.005 ppm from laboratory to laboratory for example for mucin oligosaccharides (Tables 4 and 5). Oligosaccharides can be released from glycolipids by ozonolysis for comparison of the data in DzO [58] or obtained by chemico-enzymatic synthesis [60]. More recently a high resolution NMR study of the ganglioside GDla has been carried out successfully as a 1: 40 dispersion in phospholipid micelles in aqueous solution. The exchange with bulk water is slowed down naturally rather than by means of a cryosolvent (e.g. acetone-water mixtures at low temperature) and Tip measurements have revealed a second low energy conformer distinct from those in previous studies, having an NOE from H8 of NeuAca2-3 linked to GalBl-3GalNAc and the CH, of the N-acetamido group of the internal GalNAc residue [61]. The first comprehensive studies of the conformational preferences of sialylated oligosaccharides were carried out on short glycopeptides isolated from the urine of a patient with aspartylglucosaminuria [62]: these have the sequence

These unique glycopeptides are based on the GlcNAcjl-N-Asn N-linked glycosylation sites which will be discussed further is Section 6. On the other hand, O-linked carbohydrate chains of glycoproteins usually have sialic acids linked within the core region, the most common structures being the following: f NeuAca2 6 f NeuAca2 - 3Gal/I-3GalNAc-0-Ser/“Ihr

.. .

Glycopeptides of this type have provided additional NMR and conformational data on sialylated recognition determinants [63-671 which have shown that there are several interactions between the oligosaccharide and peptide parts of these short O-linked chains. This was also evident from work on the importance of the NeuAc residues on the specificity of serum antibodies called anti-M and anti-N [68] which recognise short sequences of peptide in the presence of the oligosaccharide(s) shown above. Thus ‘M’ has been characterised as the sequence Ser-Ser*-Thr* . . . at the amino . . . , where the terminus of the erythrocyte molecule called glycophorin and ‘N’as Leu-Ser*-Thr* asterisk indicates the 0-glycosylation site. More recent papers [69-711 have explored the conformations of O-linked cores by NMR and molecular graphics. Many different types of structures are now known as discussed in Refs. [27, 72-741 and below.

4. O-linked glycoprotein chains

O-linked chains having GalNAc linked to Ser or Thr amino acids have classically been called mucin-type due to their abundance in these high molecular weight glycoproteins which line the bronchial, gastrointestinal and urogenital tracts, but are also present on serum and cell membrane proteins where they have distinct roles in protein conformation and recognition [27]. These chains are classified by the substitution pattern of monosaccharides linked to the GalNAc (discussed below) and are identified characteristically by NMR analysis after release from protein by a specific mild reduction (alkali-catalysed p-elimination) which gives the alditol GalNAcol at the reduced end of the oligosaccharides. The Galfll-3GalNAc sequence shown above is known as O-linked glycoprotein core 1 [27] (Table 4). The sialic acid found naturally in humans is always NeuAc, but NeuGc is found in tumour tissues (Fig. 1) and therefore it is important to be able to recognise the C5 acyl substitution which is easily achieved by NMR as the methylene protons of NHCOCHzOH (NeuGc) resonate at 4.12 ppm and the methyl protons of NHCOCHs (NeuAc) resonate at around 2.03 ppm

E.F. Hounselll Progress in Nuclear Magnetic Resonance Spectroscopy 27 (1995) 445-474

451

(with respect to acetone at 2.225 ppm at 22°C). NeuGc is a common constituent of non-human glycoproteins, for example the recently characterised oligosaccharides of bovine submaxillary mucin [72-741 (Table 5). Similar oligosaccharide sequences, but having a sialic acid analogue called KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid) where C5 is substituted by OH and not NHCOCH3 or NHCOCH,OH, have been characterised by NMR from the jelly coat of eggs of the newt Pleurodeles waltlii [75]. Structures include the Le”, LeY, and ALeY sequences based on a GlcNAc/?-3GalNAc core (core 2; see below). Oligosaccharides of fish eggs were also shown to have these structures and additional oligosaccharides [76] where the KDN was further substituted by Fucrl-3Fucal-4 sequences. (In mammalian glycoconjugates sialic acids are normally terminal residues either singly or in oligo/polysialic sequences discussed in Section 3.4) The O-linked chains present on mucins are particularly important in interactions due to the polyvalency of the chains and their structural diversity. Thus they are prime targets for high affinity binding of selectins and are recognised as tumour-associated antigens by monoclonal antibodies. The diversity of the neutral oligosaccharides of mucins as determined by ‘H NMR of released and reduced chains has previously been discussed [27] and chemical shift data given which distinguished reduced GalNAcol in cores designated as the linkage to GalNAc by Gal at C3 in the absence (core 1) or presence (core 2) of GlcNAc at C6 [77], by GlcNAc at C3 in the absence (core 3) or presence (core 4) of GlcNAc linked at C6 [78], GalNAc at C3 (core 5) [79], GlcNAc at C6 (core 6) 1803 and GalNAc at C6 (core 7) [81]. The diversity of different backbone sequences based on these cores and their ‘HNMR chemical shift assignments are shown in Tables 4 and 5 and exemplified by the oligosaccharides characterised from the glycoproteins of respiratory mucus of patients suffering from bronchiectasis where sixteen different oligosaccharides were characterised having core 1 or core 2 [82], nineteen oligosaccharides having core 3 or core 4 [83] and another eleven oligosaccharides having core 4 [84]. Further diversity of human mucin chains is provided by chain terminating sequences additional to the blood group ABO and Lewis type structures, e.g. GlcNAcal-4Gal [SS], and by the backbones having either long linear sequences of Gal-GlcNAc repeats [77] or the presence of branching at Gal residues by GlcNAc at C6 in the presence [77] or absence [86] of GlcNAc at C3. These sequences can also be sulphated [27]. Recent papers have given detailed chemical shift assignments of different sulphation patterns and have shown the extent of their structural diversity [87-891. The linear repeating backbone [-3Gal/I-rlGlcNAc/?-1, sequence can also be polysulphated to give the glycosaminoglycan keratan sulphate (KS). Two types of KS can be distingusihed by NMR [90-931; type I linked to protein via N-glycosylation (Man,GlcNAc, to Asn) and type II when linked via 0-glycosylation (GalNAc to Ser/Thr). The sulphation of both types can be shown to be at C6 of Gal and GlcNAc residues which gives a downfield A6 of 0.45 ppm for the protons at C6,0.16 ppm for H5 and 0.035 ppm for H4 [90]. Additional substitution with fucose and sialic acid can also be distinguished [90-931. Other glycosaminoglycan chains are linked to protein via an additional type of 0-glycosylation, Xylpl- to Ser, which has been characterised by NMR spectroscopy at the oligosaccharide level in the presence [94] and absence [95,96] of phosphate and linked to Ser or Ser-Gly. This linkage occurs in the proteoglycans heparan sulphate, heparin, chondroitin sulphate and dermatan sulphate discussed in Section 5. One additional glycosaminoglycan chain is that of hyaluronan which is usually discussed along with proteoglycans although it is not found linked to protein and is not sulphated (Table 6). However, it has similar roles and structural properties important in maintaining extracellular matrix architecture, as a lubricant and in angiogenesis (growth of blood capillaries and maintenance of an endothelial barrier), for example. NMR parameters for hyaluronan are discussed in Ref. [97].

5. Proteoglycans

Proteoglycans other than keratan sulphate are oligosaccharide-to-protein conjugates where oligosaccharides called glycosaminoglycans are made up of highly sulphated long repeats of uranic acids and aminosugars (Table 6). Probably the most studied by NMR is heparin, initially

.@I-9

ICI

0

?? p l-30

n

op1-3’

npl-30

Chemical

I

Core type

The ‘H NMR chemical

Table 4

shifts

4.391 4.069 3.468 4.277 3.931 2.966 4.285 3.989 4.238 2.045 3.810 3.718 4.395 3.888 3.680 3.749 3.647 3.647 2.060

H2 H3 HJ NAc

HI HI Hz H3 H4 HS H6 H6 NAc

4.289 3.996 3.546 4.145 3.65 2.037

4 392 4064 3 507 4 194 3.69 2.051

HZ H3 H4 HS H6 NAc

HZ H3 H4 HS H6 NAc

HZ H3 H4 1~5 H6 NAc

GalNAcol

shifts (ppm from DSS at 22°C in D,O) Gal4

for O-linked

HI 4.464 H4 3.900

HI 4.477 H4 3901

Gal’

core regions;

HI 4.538 NAc 2.064

HI 4.537 H6 3.931 NAc 2.066

4.604 3.584 3.950 2.085

HI 4.599 NAc 2.082

HI H3 H6 NAc

GlcNAc3

0, Gal; 0, GlcNAc;

GlcNAc6

0, GalNAc-ol;

HI H2 H3 H4 H5 n6 H6 NAc

5.103 4.235 3.921 4.043 4.075 3.791 3.768 2.049

GalNAc

NeuAc

?? , GalNAc; A, NeuAc, + NeuGc

NeuAcJ

Aa2-6

NeuAcl

*a2-3Uf31-3’

Aa2-q

?p?1-3’

0

AaZ-3opl-30

NeuAcl

\

Hal-60

VII

2.046 3.739 3.676 4.256 3.872 3.397 3.990

NAc

HI Hl

4.378 4.055 3.534 4.244 3.466 2.039 4.378 4.067 3.524 4.240 3.84 3.475 2.042

HZ H3 H4 H5 H6 H6 NAc

4.390 4.074 3 498 4 187 3 68.3 6J 2 046

HZ H3 H4 H5 H6 NAc

H2 H3 H4 H5 H6 NAc

H2 H3 H4 HS H6 3.565 H6 3 797 NAc 2.05s

8.841 3.379 4021 3.933

H2 4 242

H3 H4 H5 H6

H3a’ H3e NAc

1.699 2.723 2032

H3a’ I 800 H3e 2.774 NAc 2.032

1.692 2 726 3 6-3 7 2033

H3a 1 800 H3e 2 774 H4 3 6-3.7 NAc 2034

Hl 4.541 H34.117 H4 3.927

4.916 4.186 4.075 3.992 4.084 3 750 3 779 2046

H3a H3e H4 NAC

111 HZ H3 H4 H5 H6 H6 NAC

HI 4.477 H3 4.122 H4 3.901

HI 4 547 H3 4.122 H4 3.93 I

HI 4.583 H6 3.928 NAc 2.059

Cal, IV

hcNAc II

LacNAcI

Aa2-6

NeuAclI

shifts

ep1-30

pi-3:

??

?pl-4mp1-6, ?

4/l-3

• ~1-3.~1-3~f31-

300 l-30

?pl-4/1-3=pl?

.plY

Chemical

Core type

Table 4 (continued)

4.283 3.987 3.518 4.242 2.045

4.400 4.063 3.457 4.289 3.9-3.95 2.06s

HZ H3 H4 HS H6 NAc

HZ H3 H4 H5 NAc

4.292 4.002 3.538 4.153 2.037

4.401 4.051 3.497 4.168 2.047

4.264 3.988 3.585 4.195 3.480 2.034

H2 H3 f14 H5 NAc

H2 H3 H4 H5 NAc

HZ H3 H4 HS H6 NAc

GalNAcol

4.470 3.536 3.668 3.923

HI 4.472 H4 3.925

HI HZ H3 H4

HI 4.447 HZ 4.457 114 4.157

Hl 4.481 H4 3.927

Gal4

HI HZ H3 H4

4.464 3.561 3.673 3.897

H14.442 HZ 3.524 114 3.910

Hl 4.464 H4 4.127

HI 4.449 H4 3.921

Gal3

HI 4.562 H6 3.998 NAc 2.060

GlcNAc6 4.601 3.8S9 3.939 2 079

4.558 3.777 3.725 3.692 3.614 4.000 3.840 2.068 HI 4.597 H6 3.9S2 NAc 2.082

HI HZ H3 H4 H5 H6 H6 NAc

Hl 4.720 Hl 4.629 HI 4.650 NAc 2.027 NAc 2.081 NAc 2.076

2.042n.034

H 1 4.68814.70 1 H2 X3.912 H6 3.84I3.900 Nat

Hi HZ H6 NAc

GlcNA? GalNAc

H3e H4

&AC 2 03.4

1.699 2.734 3.6-3 7

H3a

NeuAc

$ m 5

E 1 e _= ?I

.a

1.3”

k2-6

NeuAc

V

?f31-4.pI-60 ?

?p1-3op1-3’ ?

?pl-4.f31-6 ?

op1-4.p1-3’

?pl-4Opl-6, ?

Gal V

GabN

\

0

0

3.723 3.671 4.247 3.843 3.375 4.027 3.932 3 707 2.044

3.624 3.637 4.283 3.998 3.520 4.245 4.237 3.905 3.701 2.043

HZ 4.393 H3 3.916 H4 3 583 NAc 2.041

Hl HI HZ H3 H4 H5 H6 H6 NAc

HI HI HZ H3 H4 HJ H5 H6 H6 NAc

Hl H2 H3 H4 HJ H6 H6

Hl HZ H3 H4

4.471 3.538 3.667 3.924 3.715 3.78 3.746

4.473 3.539 3.669 3.925

Hl 4.456 HZ 3.531 H3 3.63 3.7 H4 3.925 3.917

4.576 3.772 3.744 3.717 3.600 3.994 3.831 2.060

NAc

4.564 4.561 3.811 3.790 3.790 3.602 3.998 3.838 2.062

HI H2 H3 H4 H5 H6 H6

HI Hl HZ HZ H3 H5 H6 H6 NAc HI Hl HZ H3 H3 H6 H6 NAc NAc

4.624 4.649 3908 3.923 3.6-3.7 3.955 3.7-3.8 2.079 2.070

5.087 4.220 3.917 4.042 4.077 3.773.80 NAc 2.088

HI HZ H3 H4 HS H6

H3a 1.706 H3e 2.730 H4 3.63.7 NAc 2.034

B B $

t

w

B

i?

NeuGc

A al-5

analogues

0

0

’

4.268 3.98 4.222 3.493

HZ H3 HS H6 NAc

H3 H5 H6 NAc

H2 1.720

1.720 2.741 4.124

I13a 1.718 H3e 2 141 NGC 4.123

H3e 2.750 NGc 4.124

H3r

H3a H3e NCi

NeuGc

4.640

4.J37

by ‘H NMR

HI 4.597 H4 4226

(X,1.237

HI 5.116

Fuc3

oligosaccharide

172-741.

O-linked

HI 4.498

HI

Gal

acid containing

characterised

HI 4.613 H6 4.007 NAc 2.082

H6 4.046 NAc 2.067

HI

HI 4.615 NAc 2.081

GlcNAc

VI in Table 4 have also been partially

4.268 3.985 4.214 3.482 2.033

3.981 4.210 3.490 2.027

4.255

NAc 2.033

of NeuAc II and NeuAc

\

\ 0

?? a2-6

f al-3

PI-i

?? a2-6

\

H2 H3 HS H6

GalNAcol

shifts (ppm from DSS at 22°C in D,O) for the N-glycolylneuraminic 0, Gal; +, NeuGc; ?? , GlcNAc; ?? , GalNAc; A, Fuc

I-3’

?? a2-6

‘opl_4opl-3’

Marl-3

Aal-2DpI-4

Aal-2opl-&fl

Oligosaccharide

The ‘H NMR chemical mucins; 0, GalNAc-01;

Table 5

5.281

HI 5.358 H5 4.312 CH,I.252

H5 4.263 CH, I 274

III

HI 5.310 H5 4.223 CH, I .233

Fuc*

HI H2 H4 NAc

gland

5 183 4.23 3.995 2.039

GalNAc

chains of bovine submaxillary

w & 2 P 2: a 3 : 2 s

R z YS

Q 5.

;

m S’ 2 R E-

% _=

E.F. Howsell/ Progress in Nuclear Magnetic Resonance Spectroscopy

27 (1995) 445-474

463

Table 6 Generalised structures for glycosaminoglycans Glycosaminoglycan

Structure

Hyaluronan (HA)

-3GkNAcp MGlcAP I-

Chrondroitin sulphate(CS)

-3GaiNAcp 1-4GlcAPl-

I i4so; i6s0, IkrmaUtn sulphate (DS)

-3GalNAc~l_4IdoAal-

I 4so; Heparan sulphate (HS)

-4GlcNR’al-4GlcA/31-

I *3so; i6so; Heparin (HEP)

-QclcNR’a I -4IdoAa l-

I *3so; tiso;

I 2so;

’ R = AC or SO;. Heparan sulphate has, as a generalisation, undergone less de-N-acetylation which precedes N-sulphation, epimerisation and O-sulphation (in that order). Therefore HS is character&d by higher NAc, lower NSO; , less IdoA and less OSO; .

characterised as high molecular weight polymers, and more recently structurally dissected by analysis of its isolated oligosaccharides. Primary interest in this proteoglycan is its use as a blood anticoagulant for which uncharacterised polymeric forms of heparin have been used clinically for half a century [98]. The search for more homogeneous low molecular weight analogues with high activity has suggested that the polymer contains specific oligosaccharide sequences which have a high affinity for binding to proteins involved in the coagulation cascade, in particular antithrombin III. From this has arisen the concept that many different functional sequences may be present with specificity for anticoagulation, antiangiogenesis, smooth muscle cell proliferation and binding to acidic and basic growth factors (summarised in Ref. [99]). Molecular diversity for specific recognition arises in heparin by biosynthetic alterations in the basic sequence of repeating glucosamine (GlcNH2) and glucuronic acid (GlcA). The GlcA can undergo epimerisation at C6 to IdoA (compared to glucose and idose in Fig. 1 GlcA and IdoA have COzH at C6; inversion at C6 means that the linkage above the plane of the ring at Cl is a for IdoA and fl for Glc/GlcA). After epimerisation the originally N-acetylated GlcNH, can be de-N-acetylated and N- and 0-sulphated. The most abundant monosaccharide components so far found in heparin are IdoA f 2S, GlcNAc f 6S, GlcNS + 3s + 6s. Thus the naturally occurring heparin sequences with the highest anti-thrombin III binding affinity have been characterised and synthesised as [10&104]

In contrast a unique sequence for binding of heparin cofactor II could not be found on heparin but it was found on dermatan sulphate (Table 6 and see below) [ 1051 and two alternative sequences have been proposed for binding of acidic fibroblast growth factor [106, 1071.

464

E.F. Hounsell/Progress

in Nuclear Magnetic Resonance Spectroscopy 27 (1995) 445-474

Table I ‘H NMR assignments and chemical shift data (6 in ppm from acetone set at 2.225 ppm) for bovine lung heparin samples at 22°C Original’

Fraction

De-N-S

Re-N-AC

N/O-OAcAc De-O/N-S

Reduced

Iduronrte

1

6s

AS

Major

HI

5 199

5.227

’ bs

6s

5.191

5.193

‘6l.i 5.2

5.13

H2

4.327

4.360

4.30

4.327

ND

4.28

H3

4.191

4.190

4.22

3.998

NDb

4.21

H4

4.107

ND

ND

ND

ND

ND

CH,OH Minor

C5.S

2.95

HI

5.161

5.23

5.27

15.088 H2

4.32

4.28

4.38

H3

4.18

4.11

4.16

CH,OH

2.94

OS

OS

-0 OH 0 Major

0 OH

K

NH s

Es

r&l

NH

R

H

NH S

Hl

5.425’

5.423’

5.16

5.191

5.5

$42

Hz

3.273

3.424

3.092

3.380

3.4

3.1

H3

3.654

3.830

3.748

3.848

3.8

3.76

H4

3.769

ND

4.04

ND

ND

ND

H5

4.010

ND

ND

ND

ND

ND

2.052

CH,

2.284

CH&

2.261

Minor

HI

ND

5.48

5.43

5.213

5.6. 5.5

5.50

H2

ND

3.29

3.28

ND

ND

3.31

H3

ND

3.68

3.73

ND

ND

3.76

-

2.059

CH,

GlcNH,

2.035

2.064

2.061

2.035

HI

4.97

H2

4.19

2.033

* Commercial heparin and after de-N-sulphation (de-N-S), de-N-sulphation and re-N-acetylation (Re-N-AC) or N- and 0-acetylation (N/O-OAcOAc), totally de-sulphated (De-O/N-S) b ND, not determined c Distinct r3C chemical shifts determined by HMQC experiments.

E.F. Hounsell/ Progress in Nuclear Magnetic Resonance Spectroscopy

2 7 (I 995) 445-474

445

The variety of possible different recognition determinants of heparin has been glimpsed from detailed ‘H NMR studies of oligosaccharides isolated following enzyme digestion by heparinase and heparatinase. Chemical shift data reported include those for eight di- [108,109], six tetra- [99,101, 103-105,110-1121, five hexa- [99,101,103,105,112] and two octa-saccharides [99,112]. Additional diversity may also be present or induced by chemical methods. For example Desai et al. Cl133 have shown that alkali treatment can result in epimerisation of the uranic acid to a+galactopyranosyluronic acid. Many previous studies have addressed the ‘H NMR and ‘%NMR of oligomeric heparins before and after chemical alteration. Most recently studies which review the previous data have explored conformational properties and viral binding activities of oligomeric heparin preparations [114, 1151. A summary of some of the ‘H NMR chemical shifts found for bovine lung heparin proteoglycans is given in Table 7. When the preparations shown were tested for their activities in (i) inhibiting the replication of the AIDS virus HIV-l in vitro and (ii) inhibiting the binding of monoclonal antibodies to recombinant HIV gp120, the results revealed that N-desulphation reduces activity, which is largely restored on N-acetylation. Selective 0-desulphation also markedly reduces activity, whereas carboxyl reduction has little effect. Overall the results show that the anti-HIV activity of heparin does not depend simply on negative charge density. As with the other proteoglycan-binding (glyco) proteins discussed in this review, the specific interactions of anionic oligosaccharide functional groups with basic amino acid motifs of the interacting protein are being mapped [116, 1171. It is hoped that ligands will be designed at the oligosaccharide level for high affinity binding to specific protein sequences such as those in anti-thrombin III for improved anticoagulants and as antiviral agents and inhibitors of interactions in the extracellular matrix. One major series of studies Cl173 has shown that oligosaccharide can bind down to the physiologically useful level of 10-s M. In addition to heparin, other proteoglycans and sulphated oligosaccharides have anticoagulant and antiviral activites. Thus, the [-41doA2Sal-3GalNAc4Sal-]s sequence on dermatan sulphate (DS) has been character&d [118] as a high affinity binding site for heparin cofactor II in the clotting cascade. In the case of HIV gp120, DS and not heparin inhibits binding of the virus to its cell receptor molecule, CD4, whereas heparin inhibits HIV infection and antibody binding by interacting with a motif in an upstream region of gp120, the V3 loop [115]. The range of activities of differently sulphated polymers [116,119-1221 extends to many other sulphated polysaccharides for which detailed NMR data are being reported [119]. For example several recent studies have addressed the structural analysis of chondroitin sulphate (CS) oligosaccharides by NMR [120-1223, analogous to studies of heparin sequences and the structures and conformations of algal, plant and bacterial anionic polysaccharides such as the carrageenans and fucoidans are beginning to be re-examined by high resolution NMR studies [123, 1241. Such polymers are important in the food and materials industries and have proven or potential pharmaceutical applications. In mammals the proteoglycans make up the major part of the extracellular matrix. Resides these important roles, the sequences of CS, DS, heparin and heparan sulphate glycosaminoglycans also occur at cell surfaces linked via Xyl-Ser/‘Thr cores to membrane glycoproteins which may also have conventional N-linked protein chains (as discussed in the following) and O-linked chains linked via GalNAc-Ser/Thr (Section 4). 6. N-linked glycoproteinchains The first data sets for high field ‘H NMR of oligosaccharides were those collected to characterise the N-linked chains of glycoproteins which in mammals have the restricted core regions: f Fucal Manal\ f GlcNAcfll-4 Manal-3’

I 6 Manpl-4GlcNAc/?-4GlcNAc/?l-Asn

466

E.F. HounsellIProgress

in Nuclear Magnetic Resonance Spectroscopy

27 (1995) 445-474

These cores are further extended to give (a) oligomannose chains (three to six Mana residues linked to the Manx residues shown above),(b) hybrid structures (having Manu residues linked to one of the Manu shown above and Galal-4GlcNAc linked to the other Manx), and (c) complex chains (where both the Manx residues have Galal-4GlcNAc substituents at the 2, 4 and/or 6 positions giving di- to hexa-antennary branched structures). The Galpl-4GlcNAc sequences can be substituted by a variety of attached oligosaccharides (called glycoforms of N-linked glycosylation of proteins) many of which have already been introduced in the previous Sections in this article as blood group and related sequences and including sialylated, sulphated and phosphorylated structures. In addition to the use of ‘HNMR for the structural characterisation of N-linked chains isolated from glycoproteins, early studies concentrated on understanding the relative flexibility/ conformational space explored by the side chains (antennae) via detailed analysis of the branching Mana(l -6)[Mana(l-3)]Man trisaccharide [125-1341. These studies were highly instructive in showing the difficulties of interpreting NOE data in often highly flexible linkages from oligosaccharide-oligosaccharide structures. Molecular dynamics for 500 ps to 2 ns time steps has now been used to explore the cores of N-linked chains using the carbohydrate-tailored force fields in AMBER [133, 1341, TRIPOS/Sybyl with PIM parameters for carbohydrates [135-1373, GEGOP [138] and GLYCAM93 [ 139). ‘H NMR has been invaluable in structural analysis of the diverse glycosylation patterns occurring distal to the Mana sequences shown above. ‘HNMR chemical shift data documented include mannosed-sulphate and mannosed-phosphate residues diesterified to methyl groups together with a bisecting GlcNAc on Manctl-6 [140]; repeating Manal-2Manulsequences [141]; Fuc residues at C6 of internal GlcNAc and at C3 of backbone GlcNAc [142]; N-glycolyl sialylated biantennary chains [143, 1441; 4-O-acetyl-NeuAc [145]; partially mannosylated high mannose chains [146]; al-3 galactosylated Gal-GlcNAc sequence [147], also on sulphated, sialylated chains [148]; the NeuAca2-6GalNAc/I1-4GlcNAc~l-2Manul-3 sequence as one antenna [149]; polylactosamine, i.e. repeating [-3Galj?l-4GlcNAc/I-1, [150], and trisialylated diantennary chains [lSl]. NMR has also been used to trace the microheterogeneity of oligosaccharide chains, i.e. the variablility in perhipheral glycosylation, e.g. sialylation, fucosylation and the number of antennae [152-1581. Within the studies quoted above are collections of the ‘H NMR chemical shift data for various types of N-linked chains both for released, isolated oligosaccharides and for glycopeptides having single oligosaccharides and one or two amino acids. Glycoproteins on the other hand normally have more than one carbohydrate chain often including mixtures of both O- and N-glycosylation. NMR is finding increasing use in studies of large glycopeptides and glycoproteins [159-164-J. The consensus from these studies is that the rate of internal motion of the distal regions of N-linked chains can allow for quite sharp lines compared to globular protein domains. There is a decrease in the conformational mobility of protein and carbohydrate near the attachment site, but little difference in the chemical shifts of isolated and attached oligosaccharides except where chains interact with themselves or with the protein through space. The relative flexibility of the oligosaccharides together with the multiple antennae of N-linked chains are likely to be highly relevant to their interactions at the cell surface. O-linked chains on the other hand appear to have different conformational attributes which involve far more protein-oligosaccharide interactions. Glycolipids, discussed next, will have other unique structure/function relationships.

7. Mammalian

glycoiipids

Oligosaccharides linked to lipids in mammalain cell membranes are primarily attached to ceramide (see Section 3.4). These glycosphingolipids can be characterised into several groups (Fig. 3) of which one group has N-acetyllactosamine (lacto-series) backbones with similar structures to those already discussed in Sections 2, 3 and 5. Gangliosides are a subcategory of glycosphingolipids

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461

8) cd8ctoqkwdda

O,So-Gal~l-ICa

J

Of@-KS

hziAea* _308lp1 _lax

I Galal-4Gal~l-1Ca

b)

Ghm+ummida

Fig. 3. The families of glycosphingolipids

derived from glycosylceramides.

bearing sialic acids, the NMR data for some of which have already been described (Section 2, Table 3) and their conformational analysis has contributed to the characterisation of sialylated oligosaccharide recognition determinants. Like gangliosides, the globo-series of glycosphingolipids (Fig. 3) have oligosaccharide structures which are specific for glycolipids and so far not detected in glycosylated proteins. NMR analysis has primarily been carried out in DMSO-ds as solvent for intact glycolipids and in D,O for isolated or synthesised oligosaccharide fragments. Detailed chemical shift assignments and conformational information have been obtained for example for globoside I (blood group P antigen), GalNAcfi1-3Galal-4Gal/.Il-4Glc~l-1Cer, and globotrioside, Gala1-4Galj?l-4Glc~l-1Cer [165-1671, where the Galal-4GaljI-4 sequence is the antigen called blood group PK which is important as a bacterial receptor on cell membranes. Another important antigen based on this sequence is the Forssman antigen, GalNAcal3GalNAcfll- 3Galal-4Gal~1-4Glcj?-1Cer which has been studied by NMR, NOE and relaxation studies [168, 1691. The lacto-series glycosphingolipids have also been studied extensively by ‘H NMR in DMSO-d6. These have a variety of blood group and related sequences on polylactosamine backbones [170-1763 the shortest of which is called paragloboside, Gal~1~GlcNAcj?1-3Gal~l-4Glc~l1Cer. As with glycoproteins these are important tumour-associated antigens. Their endogenous function has been less easy to imagine than for glycoproteins, the oligosaccharides of which are accessible away from the surface membrane. However, it is likely that the unique position of the oligosaccharides of glycolipids embedded directly in the membrane have specific functions. These may only become apparent when studies of the effects of lipid on conformation and orientation are known. To this end studies have begun on characterising the lipid moiety in the context of oligosaccharides by NMR in solution (micelles) and in the solid phase [59, 61, 177-1791. One additional category of mammalian lipid-containing glycosylated molecules are the glycophosphoinositol (GPI) anchors which anchor proteins into cell membranes by an oligosaccharide core linked via inositol phosphate to lipid, the inositol phosphate-lipid bond being cleaved by phospholipase enzymes. Specific roles have been suggested for the GPI anchors such as quick release

468

E.F. Hounsell/Progress

in Nuclear Magnetic Resonance Spectroscopy 27 (1995) 445-474

of protein from the membrane by the phospholipases attached and released protein.

and a possible altered conformation PLP-D,PLP-C

Protein-CO,-PE

for the

site of action

I 6 Manal-2Manul-2Manal 6’ -1 GalNAc/?l-4Manal-4GlcNH#l-6Insl-P-Lipid 2 Phosphoethanolamine

(PE)

Features common to the GPI anchors of mammals and microorganisms are the characteristic GlcNH, and Manu in the core [180]. The glycan moiety of a brain and lymphocyte antigen, Thy-l, and trypanosome GPI anchor for example have been characterised extensively, the latter by NMR and molecular dynamics [181]. The inositol molecule itself has an essential role in intracellular signalling in its various phosphorylated forms characterised by ‘H NMR 11821.

8. Mycobacterial and bacterial glyco(peptido)lipid conjugates There is a wide range of monosaccharides and glycoconjugates which have been identified in microorganisms which are related to mammalian structures but are often unique. The GPI anchors are an example of glycoconjugates first identified in microorganisms (Leishmaniu and Trypunosoma) and since found as an important membrane constituent of mammalian cells. In mycobacteria, conjugates of peptide, lipid and oligosaccharide also occur, called glycopeptidolipids (GPLs; previously C-mycosides, Fig. 4 [183, 184]), but the peptide is of only limited size and the monosaccharides can be diverse acylated monosaccharides not found so far in mammals. NMR is uniquely suited to the identification of these novel monosaccharides which are also found as components of bacterial antibiotics. NMR can also be used to readily distinguish other glycoconjugates found in Mycobucterium species, i.e. the phenolic glycolipids [185-1871 and trehalose-containing lipooligosaccharides [188-1901. Lipid-oligosaccharide and peptide-oligosaccharide conjugates also occur on bacterial cell surfaces; the oligosaccharide capsular antigens are linked via the component lipid A (Fig. 4), detailed NMR data for which have recently become available from synthesised inner core lipo-oligosaccharide [191, 1921. The structures of the capsular polysaccharides are not discussed further here as they are a subject in their own right, but as mentioned earlier many have structures which cross-react with mammalian oligosaccharide sequences of glycolipids and glycoprotein. NMR has proved to be an essential tool in their structural and conformational characterisation. Progress in NMR studies of the mycobacterial components is now beginning to accelerate after the early pioneering work on their structural characterisation. The phenolic glycolipids have been character&d from four Mycobacterium species: that from M. leprue [185] contains 2,3-di-Omethylrhamnose, 3-U-methylrhamnose and 3,6-di-O-methylglucose in a-linkage (‘H NMR Hl chemical shifts at 5.4, 4.4 and 4.2 ppm in CDCl, with respect to CHCIS at 7.2 ppm, and the phenolic doublets at 6.92 and 7.14 ppm). The trisaccharide component of the glycolipid (Fig. 4) has been synthesised and analysed in CDCIJ, CD30D and DzO and the results from chemical shift and NOE studies in the three solvents used to provide conformational information correlated with the inferences from hard sphere exoanomeric (HSEA) molecular mechanics calculations [187]. The phenolic glycolipids of M. boois BCG were shown Cl863 to consist of different monosaccharides by ‘HNMR, i.e. 2-0-Me-a-L-Rhap

and a-L-Rhap-(1 + 3)-2-0-Me-a+Rhap.

E.F. HounselllProgress in Nuclear Magnetic Resonance Spectroscopy 27 (1995) 445-474

A.

469

Glycopeptidolipids Lipid-D-Phe-D-alloThr-D-Ala-L-alanimll

I P (Hex),,-Rhaa I-2Tsl(6d)

1, I Rhaa(3,4-Me)

Hex is various derivativesof Fuca, Rhaa, Glcfl, GlcNp, and GlcAP: Tal(6d) is 6deoxytalose.

a

Lipoligosacdwidcs Pw 116 3- M&hid-3 KNAcl3Fuca

l-4 Glcpl-w&p1-6

3-Me&l-3 RhaaltRhaal-2

[Manal,IRhral, Rhaal-3Glcfll-6

2-MeFuca I-3Rhaa 1-3

GlcPl-3Rhaal-3 KNAc is N-acylkanosamim C.

Inner core and lipoid-A-region of bacterial capsular polysaccharidcs (W Hepal

1 7 CP-Hepa1-3Hepa1-5Kdoa2-6GlcN(4P)~1-6GlcNalP

I Lipid

! Lipid

Hcp is L-g&ero-D-maww- hcptose and Kdo is 3deoxy-W 2-octulosonic acid (C8 analogue of KDN) Fig. 4. A summary of structures of conjugates character&d so far from mycobacteda. (A) Glycopeptidolipids; (B) Lipooligosaccharides; (C) The inner core and lipid-A region of bacterial capsular polysaccharides (CP). Glc, GlcA, GlcN, Xyl and Man residues are D-p; Rha, Tal and Fuc are L-P and * designates a l-carboxyethylidene

substitution across C4-C6. Other phenolic glycolipids so far characterised are the related structure from M. knmasii (non-pathogenic) and M. tuberculosis, i.e. 2-0-Me+0-Ac-a-L-Fucp-( 1 + 3)-2-O-Me-a-L-Rhap(1 + 3); 2,4-di-0-Me-a-L-Rhap, 2,6-dideoxy-4-O-Me-a-D-Arap; 2,4-di-0-Me-a-D-Manp, and 2,3,4All these glycolipids have been tri-0-Me-a-L-Fucp-(1 + 3)-a+Rhap-(1 + 3)-2-O-Me-a+Rhap. found to be antigenic and seem to be promising species markers, demonstrating the importance of their characteristation by methods capable of distinguishing subtle differences in D or L configuration (by NOE studies), linkage and acyl substitutions. Several Mycobacterium species have also been studied for their lipo-oligosaccharides. The structures found for these complex oligosaccharides together with the peptidoglycolipids (GPLs) of mycobacteria are detailed in a review by Brennan [190] and approximately summarised in Fig. 4. Thus the structures of mycobacteria dramatically demonstrate the potential diversity of oligosaccharides found in nature and NMR as an invaluable tool in their characterisation.

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Acknowledgement

The author wishes to thank Mrs. Gail Evans for her help in preparing the typescript.

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