Chapter 11 C

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Part C

Interactions of Carbohydrateswith Biomolecules Investigatedby NMR Techniques and Applications

NMR Spectroscopy of Glycoconjugates. Edited by Jess Jimnez-Barbero, Thomas PetersCopyright 2002 Wiley-VCH Verlag GmbH & Co. KGaA

ISBNs: 3-527-30414-2 (Hardback); 3-527-60071-X (Electronic)

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11.1

Introduction

Leukocytes and other migrating cells present a complex language of glycoconju-gates to communicate with their environment [1]. On their journeys through our

bodies they selectively adhere to various other cell types or to the extracellular ma-

trix. The glycoconjugates are involved in the decision on adhesion or repulsion be-

tween the cells. The glycocalix forms the front line of cell-cell recognition. Transi-

ent yet selective ligand-receptor recognition leads to movement under a controlled

speed and direction, the fine-tuning of velocity being steered by gradually multi-

plying weak singular interactions to tighter polyvalent contacts [2]. Hakomori de-

scribed the glycosphingolipid-based cell-adhesion and repulsion [3]. This cell-re-

cognition process does not involve proteins, thus making the homotype binding

between oligosaccharides of the cell membrane fundamentally different fromother cell-recognition processes. The weakness of carbohydratecarbohydrate inter-

actions does not mean that these are of minor biological importance compared

with other high affinity complexations. Transience is an asset of this biological re-

cognition event, not a liability [4].

Soon after the protein-independent cell-adhesion was described, the question of

structural details was approached by NMR spectroscopy [5]. Unexpectedly, the carbo-

hydrates showed no calcium affinity in the NMR titrations. The oligosaccharides had

been investigated in isotropic solution without their ceramide aglyca and conse-

quently without the orientating effect of an underlying membrane. Lacking such pre-

organization, no homophilic recognition was observed between the carbohydrates.On a different scale, the hooks of a zipper or of a Velcro fastener need tethering

to a supporting material to constructively interact with the other surface. Ob-

viously, the binding is absent in an isotropic mixture of hooks and loops which

are not supported by a scaffold. Can we expect to observe carbohydrate carbohy-

drate interactions in isotropic solution? Preorganization as a requirement of the

carbohydrate-carbohydrate interaction became obvious with the identification of

glycosphingolipid microdomains [6]. In the beginning dismissed as isolation arti-

facts, microdomains and membrane rafts are now widely accepted, and numerous

studies are currently investigating their composition and diffusion properties or

275

11

NMR Analysis of Carbohydrate Carbohydrate InteractionsArmin Geyer

NMR Spectroscopy of Glycoconjugates. Edited by Jess Jimnez-Barbero, Thomas PetersCopyright 2002 Wiley-VCH Verlag GmbH & Co. KGaA

ISBNs: 3-527-30414-2 (Hardback); 3-527-60071-X (Electronic)

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even observing them directly by atomic force microscopy [7]. Yet the molecular

surfaces of the cooperative carbohydratecarbohydrate binding which stabilizes

the microdomains remain the subject of speculation. The average diameters of

microdomains are in the region of 20 nm, and aggregates of such size cannot be

studied directly by high-resolution NMR. A second obstacle for solution NMR is

the necessity for pre-orienting the carbohydrates at a phase barrier the mem-brane or on a self-organizing synthetic scaffold [8]. Ultra-low affinities of the in-

dividual carbohydratecarbohydrate interactions combined with high off-rates of

complexation, fast exchange on the NMR time scale, and the averaging of reso-

nance signal sets make up the third group of problems. Finally, the conforma-

tions and relative orientations of monomers within homophilic complexes are

more difficult to analyze by NMR than those within heterophilic complexes,

which yield separate NMR resonance signals.

On the other hand, only NMR spectroscopy can directly observe a complex in

solution, analyzing the complementary molecular surfaces of the interacting spe-

cies. Quantification of average interproton distances from NOE data identifies hy-drophobic contact sites. Together with 3J couplings, chemical shifts, and relaxation

data, the relative orientation of interacting species can be studied in solution.

Based on the knowledge of the contact sites between glycosphingolipids, the over-

all structure of microdomains will be better understood.

The dominating theme of this chapter is the NMR analysis of homophilic inter-

actions between carbohydrates and the development of strategies to analyze weak

binding and cooperative effects on membranes.

11.2Membrane Models for High-Resolution NMR

Synthetic membrane models like micelles and liposomes can mimic the dense

packing of sugar head groups on biological membranes. Tumbling rates decrease

with increasing sizes of the membrane fragments, and excessive line-broadening

is one consequence. Wideline 2H NMR spectra of selectively deuterium-labeled

gangliosides on liposomes formed from 1-palmitoyl-2-oleoyl phosphatidylcholine

were acquired [9]. Micellar aggregates of natural glycosphingolipids are too large

for NMR analysis [10]. High-resolution spectra are obtained from glycolipids on

micelle-forming synthetic detergents like sodium dodecylsulfate (SDS-D25) [11].Uncharged micelles of similar size are obtained from dodecylphosphatidylcholine

(DPC-D38). This environment leads to a line-broadening of less than 3 Hz for the

carbohydrate chains of micelle-anchored glycolipids. Carbohydrate-enriched mi-

celles are obtained from (semi-)synthetic glycolipids bearing only one lipid chain

[12]. Specific carbohydrate carbohydrate interactions have not yet been observed

in the micellar environment; a reason for this could be the highly curved nature

of the micelle surface.

Planar phospholipid bilayer fragments, so-called bicelles, with diameters be-

tween 10 and 100 nm, are obtained from dimyristoyl phosphatidylcholine and var-

11 NMR Analysis of Carbohydrate Carbohydrate Interactions276

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ious detergents in the magnetic field of the NMR spectrometer [13]. The alkyl

chains of the glycolipids can insert into the bilayers, but the resulting severe line-

broadening effects restrict conformational analyses to 13C-labeled glycolipids [14].

Furthermore, such liquid crystalline membrane models tolerate only minoramounts of glycolipids, high dilution of carbohydrates being one plausible reason

for the absence of carbohydratecarbohydrate interactions in these membrane

models. Nevertheless, several complex gangliosides were investigated, and possi-

ble influences of the membranes on the carbohydrate head groups were analyzed.

Although the membrane environment restricts the accessible conformational

space of the carbohydrate head group, specific interactions of sialic acids or other

sugars with the membrane surface were not observed [15]. The orientation of the

sugars toward the solvent can be visualized in the form of a rotation cone of the

carbohydrate head group (Fig. 11.1 [16]). Detailed structural analysis of mem-

brane-anchored amphiphiles will profit from further developments of highly sol-vated membrane models for NMR studies.

11.3

Carbohydrate Carbohydrate Affinities

The affinities between uncharged carbohydrates are generally weak [17]. A 0.1 M

solution of methyl-b-d-ribopyranoside leads to a 13C NMR chemical shift variation

ofDd=+0.15 ppm for the anomeric carbons of b-cyclodextrin, too small to quanti-

11.3 Carbohydrate Carbohydrate Affinities 277

Fig. 11.1 Glycosphingolipids perform an axi-ally symmetric rotation about the bilayer nor-mal. Tilting and precession about this axis re-sult in a cone-shaped overall movement, theceramide primary alcohol group acting as aflexible joint between carbohydrate moietyand the membrane. Common structural fea-tures of the Lewis glycosphingolipids are asingle lactose unit (black hexagons) to whichat least one Gal-GlcNAc disaccharide (grayhexagons) is bound. The dimeric Lewis anti-

gens bear at the most three Fuc units (whitehexagons), which stabilize a secondary struc-ture. Oligosaccharide backbones of alternat-ing b(1-3), b(1-4) glycosidic bonds of the Gal-GlcNAc disaccharide units (left, LeX and LeY

series) form extended average conformations,while backbones of only b(1-3) glycosidicbonds (right, Lea and Leb series) result inbent structures which consequently rotate ina wider cone (Details in [16]).

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fy the ratio of free to complexed ligand at various titration steps [18]. A stronger

signal response is required to quantify weak binding affinities. Other sensitive

NMR parameters like paramagnetic shifts, diffusion rates, or transfer NOEs can

be measured. General NMR aspects of weak complexation have been discussed by

Gemmecker [19].

Although several metal complexes of mono- and disaccharides are known [20],there is no metal mediated di- or even oligomerization of simple sugars. Protic or-

ganic solvents increase the metal ion affinities of sugars, and, by this, emphasize

the structure dependence of complexation. Yet, weak complexes still require a

large excess of metal ions, which then occupy secondary binding sites and render

the separation of selective complexation from non-specific binding by NMR more

difficult. The suspected interaction sites of the LewisX (LeX) pentasaccharide

(Galb1-4[Fuca1-3]GlcNacb1-3Galb1-4Glcb1-O, where Gal = d-galactose, Fuc = l-fu-

cose, GlcNAc= d-N-acetylglucosamine, Glc = d-glucose) were studied by Henry et

al. in organic solvents [21]. The metal-binding sites were identified with moderate

precision by distance triangulation methods in the vicinity of Fuc and lactose, buta homotype carbohydratecarbohydrate binding was not observed.

11.4

LewisX Glycosphingolipids

A large fraction of the cell-surface glycosphingolipids present LeX as the terminal

trisaccharide unit [22]. Draber showed that calcium-dependent homophilic recog-

nition between LeX-LeX sphingolipids on intact cells was neutralized only by solu-

ble LeX

, but not by other sugars or membrane component [23]. Thus, the carbohy-drate head group mediated the interaction, and the composition of the ceramide

membrane anchor controlling the phase transition temperature is of minor re-

levance.

The branched LeX trisaccharide (Galb1-4[Fuca1-3]GlcNacb1-O) is stabilized by

hydrophobic stacking between the a-side of Fuc and the b-side of Gal. This well-

defined solution conformation, with only very little averaging about the glycosidic

torsions, made LeX an attractive NMR target [24]. The LeX pentasaccharide with

an additional lactose unit is the smallest Lewis glycosphingolipid found on cellu-

lar surfaces. Fatty acids and sphingosine of varying chain lengths are found in the

aglyconic ceramides. Microheterogeneity is also observed for the carbohydratehead group, and effective coupling and protecting strategies are necessary to

make homogeneous glycosphingolipids and derivatives accessible for NMR stud-

ies [25].

The LeX-LeX interaction is shown schematically in Fig. 11.2 a. As mentioned

above, there is no calcium complexation between LeX tri- or pentasaccharides in

solution (Fig. 11.2 b). The equilibrium is far on the side of the uncomplexed

monomers. Phase barriers are, in principle, no problem for solution NMR, but

the addition of calcium ions to liposome-anchored LeX glycolipids leads to im-

mediate precipitation. The formation of microdomains requires a dynamic equilib-


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rium between free and complexed carbohydrates within one sample. This can be

managed by a mixture of soluble LeX and membrane-anchored LeX glycolipids

(Fig. 11.2c [26]). The soluble LeX shows a small but detectable membrane affinity

to liposomes which are decorated with LeX glycolipids. Soluble LeX binds transi-

ently to the membrane, and consequently its average molecular tumbling rate

slows down. As a result, NOESY cross signals with higher positive values (more

negative NOE) are observed. The strong correlation between NOE intensity and

tumbling rate in the region of sign inversion of the NOE leads to a detectable ex-perimental signal even for a carbohydratecarbohydrate affinity in the M1 region.

The LeX trisaccharide was identified as the minimal structural unit for the homo-

philic interaction, but no structural details are obtained with this method.

11.4 LewisX Glycosphingolipids 279

Fig. 11.2 (a) Schematic representation of amembrane-anchored LeX pentasaccharide inequilibrium with a glycosphingolipid microdo-main as postulated to occur on cellular mem-branes. Microdomains are in dynamic equilib-rium with the monomeric sphingolipids. Thecone model in Fig. 11.1 is valid for mono-meric sphingolipids; their mobility is re-

stricted within the microdomains. (b) The or-ientating effect of the membrane is absent in

an isotropic solution of LeX trisaccharides.The calcium ion complexation is disfavoredand the equilibrium shifts to the left. (c) Onlyweak binding is observed for synthetic glycoli-pids consisting of an LeX trisaccharide linkedto an SDS micelle by a triethylene spacer(Details in [27]). (d) Two LeX trisaccharideslinked by an adequate spacer are thought to

bind calcium ions strongly enough to makethe complex accessible for NMR studies.

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11.5

Covalent Tethering of LeX Units [27]

If, in the case of the glycosphingolipids, the job of the membrane is confined to

the preorganization of the carbohydrate head groups, a covalent spacer between

the supposed recognition domains should effectively mimic this arrangement.Two trisaccharides binding one calcium ion is a reasonable assumption about sto-

chiometry for the bulky sugars involved. The complex formation should be more

favorable, with the equilibrium shifting to the right (Fig. 11.2 d). The synthetic

model restricts the number of interacting oligosaccharide moieties. An NMR anal-

ysis then can identify the relative orientations of the trisaccharide units in the

complex to define the complementary molecular surfaces within the homophilic

interaction.

The GlcNAc primary hydroxyl groups of the LeX methyl glycoside 1 are con-

nected via a methylene group in the bis-trisaccharide 2. 2 retains sufficient relative


Fig. 11.3 1H NMR spectra of2 between 0and 15.1 equivalents of calcium chloride inmethanolD4. Considerable chemical shift varia-tions are observed in the NMR titration. For

example, the exocyclic methylene protons ofGlcNAc migrate in opposite directions. 2c-H,3c-H, and 1c-H form a higher order spin sys-tem.

"

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mobility of the two trisaccharide units to permit either parallel or antiparallel di-

merization. The bis-lactoside 4 served as a model for non-specific binding of cal-

cium [28]. The NMR titrations of sugars 14 and calcium chloride were per-

formed in methanol. The 1H NMR spectra of 2 with increasing calcium concen-

tration are shown in Fig. 11.3. The 1H resonance signals were assigned, and sev-


Fig. 11.4 The 1H NMR chemical shifts [Hz]of1 (top) and of2 (below) in methanol areplotted against the amount of calcium chlo-ride added. Only non-specific calcium bindingis observed for 1, the resonance signals grad-ually migrating to lower fields (higher ppm).The large chemical shift variation with only afew equivalents of calcium chloride is due toa structural change of2. Both, high-field and

low-field shifts are thus observed. Calciumcomplexation causes a high-field shift for 1c-H which is inverted at higher calcium levelsbecause of the non-selective binding of cal-cium to 2. Therefore, all curves except forthe solvent resonance show a double expo-nential dependence on the calcium concentra-tion.

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eral well-separated signals could be used for the quantification of the calcium-

binding affinity of2.

A plot of the chemical shifts in Hz of three selected resonances and the solvent

methanol against the equivalents of calcium chloride added shows the strong cal-

cium dependence (Fig. 11.4). Only for 2, a large chemical shift variation with only

a few equivalents of calcium ions is observed. The affinity constant is defined by

the ratio of complex to free components [Eq. (1), where ROH is the sugar].

Ka ROH Ca2

ROH Ca2m

ROH Ca2

ROH Ca2 ROH1

The bound fraction (m was determined between 0% and 100%, and Eq. (2) was

then analyzed graphically.

m Ca2

K1a Ca2

m

Ca2 Ka mKa 2

The binding affinities Ka were determined from Scatchard plots (Fig. 11.5) [29].

The abscissa shows the bound fraction (m), and the ordinate shows the ratio of

11.5 Covalent Tethering of LeX Units [27] 283

Fig. 11.5 Scatchard plots of1 (&), 2 (n), 3(*) and 4 (l). The fraction of complexedcarbohydrate (m) is plotted against the ratio ofm/I (I=concentration of uncomplexed calciumions). The affinity constant Ka [M

1]is ob-tained from the y-axis by linear extrapolationto m = 0. Only compound 2 yields a straightline with an affinity constant of Ka=55 M

1.

Secondary binding sites are populated at highconcentrations of calcium ions, and a re-duced slope is observed at m>0.7. Weak affi-nities below Ka = 10 M

1 are slightly overesti-mated because full complexation (m= 1) is notreached without secondary effects playing adominant role.

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free calcium to the bound fraction m. The affinity constant is obtained by linear ex-

trapolation to zero complexation (m=0). The higher the determined value at the or-

dinate, the better the binding. Only 2 shows a straight line with cooperative cal-

cium binding of both trisaccharide moieties.

The affinity constant is approximately the product of the monomeric affinity

constants, equivalent to the summation of monomeric free energies of binding.


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11.5 Covalent Tethering of LeX Units [27] 285

Fig. 11.6 Compensated ROESY (600 MHz, 4kHz spin lock, 200 ms mixing time, CD3OD,300 K) of2 in the presence of 15 equivalentsof calcium chloride. Rotating frame NOEs,which are only visible in the presence of cal-cium chloride, are indicated by gray boxes.Other cross signals than those indicated donot change their intensities relative to theROESY of 2 without calcium chloride. Thecross signal intensities correspond to averageinterproton distances of between 1.8 and 4 .The chair conformation of pyranose rings is

characterized by the

3

JH,H coupling constants.Several intraglycosidic NOEs, like Fuc-H4 toH4, served as calibration distances. Importantfor the relative spatial orientations of the pyr-anosidic rings are the transglycosidic NOEsbetween CH groups flanking the glycosidicbond: here, for example, the one from Fuc-H1to GlcNAc-H3. The most predictive NOEs arebetween proton pairs separated by manybonds but close in space, so-called long-range NOEs, for example Fuc-H5 to Gal-H2.

3

Fig. 11.7 Solution conformation of the cal-cium complex of2. Top: Ten snapshots from a

molecular dynamics simulation including ROE-derived distance restraints. The rotational mo-bility about the covalent tether was not re-stricted, and the experimental NOEs were in-cluded as weak additional restraints. The exo-cyclic torsion of GlcNAc (a and a') populatesthe gauche-trans rotamer in a and the gauche-

gauche rotamer in a'. The methylene spacerlinking the two GlcNAc units is shown in green.

Rings a and a' are coplanar and assume across-shaped relative orientation. Both trisac-

charide moieties are in fast conformationalexchange, and the molecular dynamics simu-lation visualizes only one of the exchangingidentical conformations. Bottom: The positionof the calcium ion is tentatively assigned atthe hydrophilic contact site of the trisacchar-ide moieties in the averaged conformation of2. SCHAKAL (E. Keller, University of Freiburg,Germany).

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The affinity is strong enough to become detectable in water with Ka= 8 M1. No

enhancement was observed for lactosides 3 and 4: they show no cooperative bind-

ing of calcium ions. A large excess of calcium was used to determine the end

points of titration. Non-specific calcium binding dominates above 20 equivalents

of calcium chloride, visible as a kink in the Scatchard plot of 2. Based on this

complex of moderate calcium affinity, the actual goal of this NMR investigationwas approached: the identification of the relative orientation of two interacting

LeX oligosaccharides. The structural differences between free and complexed 2

were studied by rotating-frame NOESY-spectra [30]. Under the experimental condi-

tions short mixing time of 200 ms the volume integral of each cross signal

correlates to a single interproton distance (two-spin approximation). The offset-

corrected cross peak intensities are then calibrated according to the r6-depen-

dence of the NOE with the assumption of isotropic tumbling of 2 (Fig. 11.6) [31].

Fixed intraglycosidic ROEs served as reference ROEs. Without calcium ions, 2 ex-

hibits the same NOE intensities as those detected for the monomeric LeX-trisac-

charide 1. Thus, the methylene spacer allows independent mobility of the trisac-


Fig. 11.8 Two LeX pentasaccharides (lactosesare shown with white pyranose rings and LeX

trisaccharides are shown with filled hexagons)are sketched with the relative orientation ofthe LeX trisaccharide headgroups as deter-mined in the calcium ion complex of 2. The Yshapes symbolize the branched LeX pentasac-charides shown on the left; arrows point to-

wards the membrane surface. Trans-homophi-lic contacts between glycolipids on differentmembranes and cis-homophilic contacts be-tween glycolipids on the same membrane canaggregate to polyvalent cell-cell contacts. Bothtypes of LeX dimers are formed via a singlemolecular surface.

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charide units. In the presence of calcium ions, additional ROEs are observed

(boxes in Fig. 11.6). For example, an ROE between H2 of the GlcNAc and the H2

of Gal is evident. These new short interproton distances identify hydrophobic con-

tacts between the two LeX-trisaccharide moieties in fast exchange. The average in-

terglycosidic distances were included as restraints in a molecular dynamics simu-

lation. They are in agreement with a single structure characterized by a stackinginteraction between the two LeX moieties. The two trisaccharides lie on top of

each other and are approximately rotated by 908 against each other, resulting in a

crossed relative orientation (Fig. 11.7). A cation binding site is formed by a hydro-

philic pocket which is surrounded by the Fuc-2O, Fuc-3O, and Gal-6O together

with Gal-2O and Gal-3O of the second trisaccharide moiety.

From the cross-shaped relative orientation of the trisaccharide units, a cross-

shaped relative orientation of pairs of LeX-sphingolipids on the cell membrane

was proposed. Fig. 11.8 shows how glycolipids can interact to stabilize a cell-cell

contact. The cross-shaped dimerization allows for cis-homophilic and for trans-

homophilic binding without resorting to different recognition phenomena. Thus,one complementary molecular surface is sufficient to stabilize side-by-side con-

tacts within the same membrane and additionally the head-to-head contacts be-

tween glycolipids from different membranes. A principle which is discussed for

other cell-adhesion molecules, too [32].

Although it is potentially difficult to extrapolate conclusions beyond the environ-

ment where the experiments were carried out, NMR spectroscopy identifies a self-

complementary molecular surface of the LeX antigen. The synthetic models of nat-

ural glycoconjugates can mimic the weak homophilic interactions, which are ex-

pected to occur in a similar way at cellular surfaces. NMR spectroscopy yields de-

tailed structural information about the conformational aspects of cellular interac-tions.

11.6 References 287

11.6

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