Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances

ARCHIVESOF BIOCHEMISTRY AND BIOPHYSICS Vol. 210, No. 1, August, pp. 405-411, 1981

Immunochemistry of the Lewis-Blood-Group System: Proton Nuclear Magnetic Resonance Study of Plasmatic Lewis-Blood-Group-Active Glycosphingolipids and Related Substancesir JANUS2 DABROWSKI,* PETER HANFLAND,? URSULA DABROWSKIS

HEINZ EGGE,$ AND

*Max-Plmck-lnstitut fiir Medizinische Forschung, D-6900 Heidelberg, tlnstitut fiir Experimentelle Htimatologie und Bluttrangfusonswesen der Universit(it Bonn, D-5800 Bonn, and SPhysiobgischChemisches Institut der Universitiit Bonn, D-5.900Bonn, West Germany Received March 2, 1981 The Lea-, Leb-, and H-type 1 (LedH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by ‘H nuclear magnetic resonance at 360 MHz in dimethyl-& sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Gala1 -+ 3GlcNAc) and type 2 (Gal@ - 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.

The primary goal of this work was to independently elucidate the structure of the substances I-IV (for formulae, see Table I) described in the two preceding papers (1, 2). On the other hand, this objective was considered a part of a broader program of exploiting the potential of NMR spectroscopy for complete structure determinations of glycosphingolipids without resorting to data obtained by other methods. The results of our comparative study on several structurally related glycosphingolipids (3,4) revealed regular differences in chemical shifts related to (a) the sugar composition, (b) the anomeric configuration, (c) the sequence, and (d) the sites of glycosidic linkage. Another valuable source 1This work was supported by a grant from the Deutsche Forschungsgemeinschaft. *This paper is part of a series on the immunochemistry of the Lewis-blood-group system.

of structural information was the nuclear Overhauser effect (4, 5). Although the regularities just mentioned are applicable in part to substances I-IV, the specific structural features of their highly crowded molecules and the ensuing spectral changes have to be taken into account. The essence of the new steric situation is in the occurrence of sugar rings vicinally substituted by two other ones, as opposed to those substituted at the more remote relative 1,3 or 1,4 positions (see Scheme 1, and the formulae in Table I). As a consequence of this vicinal (i.e., relative 1,2) substitution pattern, the mutual influence between sugar rings is no longer restricted to pairs of neighboring units but rather is exerted between the first and the third member of each given triad as well. Indirectly, the interaction between the middle sugar residue and each of the outer ones can also be changed because the r$ and %f angles between them

405

0003-9361/81/090405-07$02.00/O Copyright All righta

0 1981 by Academic Press. Inc. of reproduction in any form reeerved.

406

DABROWSKI ET AL.

o-R

C

R = Coramidr SCHEME1. The Leb-blood-group-active glycosphingolipid

may readjust while the outer rings escape steric strain. The relevant NMR spectral implications will be dealt with in the Discussion. It should be added that a similar type of crowding due to a vicinal substitution is encountered in all oligosaccharides containing 2-deoxy-2-acetamido hexose units l-linked to sugar aglycons, particularly (but not necessarily so) if these amino sugar units themselves are glycosylated at sites 3. Several examples pointing to this problem were discussed in our previous paper (3) but, unfortunately, the H-3 and H-4 resonances of Nacetylglucosamines could not be detected at that time. Now, these signals were found by NOR3 and shown to be informative with respect to the structures under study. The precursors of I, III, and IV (substance V), and II (substance VI) were also investigated. The combined data enable one to distinguish native glycosphingolipids containing type 1 and type 2 structures of blood group determinants. Recently, the Karlsson group described a solution to this problem by comparing the NMR spectra of totally methylated glycosphingolipids with those of the methylated-reduced ones (6, 7). The solution found in the present work is more direct and seems to be simpler. MATERIAL AND METHODS The isolation and purification of the substances IVI (for formulae, see Table I and Scheme 1) was de3 Abbreviations used: NOE, nuclear Overhauser enhancement, or effect; SDDS, spin decoupling difference spectroscopy; GlcNAc, N-acetylglucosamine.

IV.

scribed in the following papers: I, V, and VI in (l), III in (8), II in (9), and IV in (10). Of the four Lem (I) samples [Le”n-I, -11, -111,and -IV, this enumeration from Ref. (1) is not to be confused with the present one] the first, which was practically identical with the second, was investigated in detail; the third and the fourth ones were identical between them, but differed from the former pair by the Glc (H-l) resonance shifted downfield (4.21 vs 4.16 ppm; see Table I). This difference points to some interaction of the head glucose unit with differing ceramide parts. The three samples (10) of the Leb-blood-group-active glycosphingolipid (IV) [Leb-I, -11, and -111;enumeration from Ref. (lo)] exhibited NMR spectra only slightly differing in the sugar proton region [A 6 for Glc (H1) was 0.02 ppm]. The spectrum of the ceramide tetrasaccharide (VI) from Ref. (1) was practically identical with that of neo-lactotetraosyl ceramide investigated previously (3); a sample of the latter was used here to perform NOE experiments. The ‘H NMR spectra were obtained on a Bruker HX-360 spectrometer using 16 K data points for 3.3kHz spectral width. The excitation pulse angle was 90”. Other details were reported elsewhere (3). NOE was measured in the manner described by Wagner and Wiithrich (11). RESULTS

The assigned signals of the sugar ring protons of substances I-VI are presented in Table I. The signals of all anomeric protons, as well as those of H-2 of glucose, H5 of fucose, H-4 of galactose, and partly H-3 of N-acetylglucosamine residues occurred outside of the bulk of overlapping resonances but all others were found in the bulk with the aid of SDDS (3,12) and NOE (4,5) measurements. SDDS, and conventional decoupling performed for unhidden resonances, revealed connectivities between signals of coupled protons whereas

PROTON NUCLEAR

MAGNETIC

RESONANCE TABLE

OF LEWIS-ACTIVE

GLYCOLYPIDS

407

I

SUGAR PROTON CHEMICAL SHIFTS (ppm from MedSi) AND sJl,p COUPLING CONSTANTS (Hz) OF GLYCOSPHINGOLIPIDS IN Me&304

I 6(H-1) J 6(H-2) 6(H-3) 6(H-4) 6(H-5)

D Fucc~l 4.99 3.0 3.51 3.51 3.51C 4.07

II 6(H-1) J 6(H-2) 6(H-3) 6(H-4) b(H-5)

D Fueal 5.03 3.0 3.55 3.55 3.42 4.96

-

-

-

-

B 3GlcNAcfil 4.66 8.5 3.49 3.81”*b 3.21 3.18d

* 3Galj31 4.28 7.3 3.43 3.47* 3.84*

B 4GlcNAqTl 4.64 7.5 3.45 3.53” 3.49’ 3.23d

+ 3Gal61 4.26 7.1 3.42 3.45* 3.85*

B + 3GlcNAc 4.77 7.5 3.62 3.87f 3.56“

A (4+laFuc)j31 4.78 4.0 3.56’ 3.59

-

3GalSl 4.28 7.0 3.45 3.45 3.87

- -P4Glc61

1Cer

4.16 7.7 3.04 3.33

- + 4Glcj31

1Cer

4.16 8.0 3.04 3.33

- -, 4Glcj31

1Cer

4.16 7.5 3.04 3.33

4.58 D Fucal 4.91 4.0 3.56 3.66

-

C 2Galj31 4.53 7.1 3.46 3.42” 3.70

B + 3GlcNAc 4.64 7.7 3.57 3.95* 3.49

C GaltTl 4.15 -7 3.34 3.32 3.62 C Gal61 4.23 6.5 3.32 3.32” 3.64

VI d(H-1) J d(H-2) d(H-3) a( H-4) d(H-5) by by by by by by by

A (I+laFuc)@l 4.77 4.0 3.49 3.61

+

3GalSl 4.28 8.0 3.44 3.43 3.85

- + 4Glc61 -

1Cer

4.17 8.0 3.04 3.33

4.60

4.19

&l) J 8(H-2) 6(H-3) d(H-4)

DObtained * Obtained ’ Obtained ’ Obtained e Observed *Obtained g Obtained

C 2GalSl 4.33 7.4 3.54 3.55& 3.66 C Gala1 4.31 7.5 3.28 3.32 3.66

III d(H-1) J d(H-2) d(H-3) d(H-4) 6(H-5) IV d(H-1) J d(H-2) b(H-3) d(H-4) 6(H-5)

C 2Galj31 4.45 7.3 3.51 3.52 3.66

B + 3GlcNAfll 4.80 8.4 3.44 3.63 3.24 B + 4GlcNAc61 4.70 7.7 3.44 3.56” 3.47” 3.31d

* 3Gal@l 4.28 7.1 3.46 3.48 3.86 * 3Galj31 4.28 7.7 3.44 3.46 3.85

SDDS, and confirmed by NOE (H-l of the same ring irradiated). SDDS, and observed by NOE (H-l of the next ring irradiated). NOE (H-6, and H-5 of the same residue irradiated). NOE (H-l of the same ring irradiated). NOE (H-l of the next ring irradiated). SDDS, and observed by NOE (H-l of Gal C irradiated). SDDS, and observed by NOE (H-l of Fuc A irradiated).

- + 4Glc@l -

1Cer

4.16 7.5 3.06 3.33

- + lGlc@l 4.17 7.7 3.05 3.33

1Cer

408

DABROWSKI

the NOES observed upon preirradiation of anomeric protons had a dual function of (a) identifying the resonances of protons of the same ring located close in space and (b) revealing the proximity to the protons of the aglyconic sugar residues in order to establish the sequence, the site of glycosidic linkage, and possibly the conformation of this fragment of the oligosaccharide chain. The typical identification scheme, based on both the actual and the previously ascertained (3,4,13) data, will be illustrated by the assignments of the LedH glycolipid (I) resonances. There are four /I (J1,2= 7.38.5 Hz) and one (Y(J1,z= 3.0) anomeric proton signals. Two of them (64.16 and 4.28), together with the signals of H-2, 3, and 4 of established connectivities, are practically identical with those of the lactose headgroup of many glycosphingolipids (3, 4) and were so assigned. The low field /3(H-l) signal (64.60) indicates an amino sugar (confirmed by the acetyl methyl singlet at 1.82 ppm). The question of whether this is a glucosamine or galactosamine residue is unequivocally answered in favor of the former by its 63.49 (H-2), because ca. 4 ppm would be required for the latter (3) due to the 1,3-diaxial orientation of its C-2-H and C-4-O bonds. The partial sequence GlcNAc - lactose and the site of glycosidic linkage follows from the NOE of the already identified H-3 of galactose, obtained upon preirradiation at 4.60 ppm. In this same measurement, a NOE was observed for H-3 of the N-acetylglucosamine, thus confirming the assignment made with the aid of SDDS via H-2. In turn, a NOE of the same H-3 resonance was obtained when irradiating H-l of the remaining /3-hexose at 4.45 ppm. Leaving for a while the identity of this hexose aside, we identify first the last sugar as an CYfucose on grounds of (a) the low-field position and the J1,2coupling constant of its anomeric proton signal, and (b) the methyl doublet at 1.04 ppm coupled to the broadened H-5 quartet at 4.07 ppm. Since the protons located at sites of glycosidic linkage and at the two contiguous positions are known to be deshielded (3, 14), we consider the chemical shifts of H-l, H-

ET AL.

2, and H-3 of the hexose C as conforming with the values obtained for galactose units (3), but modified by glycosylation at site 2. The practically unchanged chemical shift of H-4 of C (3.66 ppm vs 3.62 for V, and 3.64 for VI) lends additional support to this assignment. The assignments for the type 2 H-I glycosphingolipid (II) are fully analogical, except for the obvious difference concerning the NOE of H-4 of GlcNAc B observed on irradiating H-l of Gal C. Of other assignments, the unusual highfield position of the H-l resonance of Gal C of substance V (as compared with the data of Ref. (3)) deserves mention. This deviation can possibly be explained by crowding caused by the bulky acetamido group at the neighboring position 2 of the aglyconic GlcNAc B. Another deviation from the expected values will be dealt with in the Discussion. DISCUSSION

All compounds studied here contain the trisaccharide ceramide fragment GlcNAcj31- 3Gal/?l-+ 4GluBl+ 1Cer which was already fully characterized, both as such and as a part of more complex glycosphingolipids (3,4). In accord with those earlier findings, the chemical shifts of protons of the lactose headgroup are little affected by structural changes occurring at the Nacetylglucosamine residue or farther along the oligosaccharide chain (Table I). Considering the effects of these structural changes on the proton chemical shifts of N-acetylglucosamine, the most important comparison is between LedH(I) and HI(I1) which have disaccharide terminals of the type 1 and 2, respectively (Table I). It was shown for noncrowded situations that substitution at a saccharide ring by another sugar residue induces large downfield shifts of the signals of protons at the site of glycosidic linkage or vicinal to it (3,14). Since the relative magnitude of these shifts is unpredictable, this would mean that 3- vs I-glycosylated N-acetylamine units can possibly be distinguished without prior knowledge by comparing their H-2 and/or H-5, but not H-3 and/or H-4

PROTON

NUCLEAR

MAGNETIC

RESONANCE

resonances. Unfortunately, there is little comparative data on H-5 and the H-2 ones differ only slightly (I vs II) or not at all (V vs VI). Obviously, the diagnostically useful regularity previously observed (3, 4) for @H-2) of P-galactose residues is invalidated here due to crowding in the environment of H-2. However, since the substitution site is directly determinable by NOE (Table I, footnotes b, e, f, and g), the problem can be approached from the reverse side, i.e., one can return to the question whether the H-3 and H-4 resonances do contain the relevant information after all. This seems to be the case, particularly if H-3 is concerned: for I-, III-, and IVcontaining terminals of type 1 the H-3 protons are very strongly deshielded (3.813.95 ppm) compared to the same protons of II with its type 2 terminal (3.53 ppm); for the precursors V and VI this difference is smaller, but even so, the H-3 resonance of V (63.63), like those of I, II, and IV, occurs downfield of the bulk of overlapping signals. As for H-4 resonances, they are shifted downfield for 4-glycosylated Nacetylglucosamine units (3.49 and 3.47 ppm for II and VI, respectively) as compared to the 3-glycosylated one (3.21 ppm for I). Additional 4-glycosylation of the type 1 N-acetylglucosamine unit by cr-fucase A has a deshielding effect on H-4 of the former (3.56 and 3.50 ppm for III and IV, respectively). Certainly, more examples are desirable to confirm these regularities. Another example of a proton which is sensitive to crowding is H-l of N-acetylglucosamine B, in that it is shielded upon glycosylation of the next galactose C at site 2. Understandably, this effect is larger for type 1 (-0.20 ppm for I vs V, and -0.13 for IV vs III) than for type 2 terminals (-0.66 ppm for II vs VI), but these differences are useless for discrimination of those two substitution types, as the shifted signals fall into the same spectral region. It should be pointed out that, among the glycosphingolipids studied by us (3, 4), this is the first example of sensitivity of chemical shifts to components of structure other than those of the next rings. Such a sensitivity is best described in terms of deviations from the

OF LEWIS-ACTIVE

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409

additivity of substituent effects on chemical shifts. Let us consider a trisaccharide ABC, either as such, or as a fragment of a larger molecule ABCX containing some unchangeable part X. If additivity is obeyed, the chemical shift of a given proton (i) of the middle residue B of the trisaccharide is composed of the value obtained for the monosaccharide B and the changes induced by its substitution when forming the disaccharides AB and BC:

or sp

- (6;; + lip) + SIi = 0 .

Ul

Any deviation from additivity will make this sum not equal zero. Such deviations can occur for steric reasons related to changed shielding by reoriented anisotropic groups, and/or because of nonadditive electronegativity through-bond effects. However, since the electronegativities of the OH and OR groups differ only slightly, the effect of replacing two OH groups of B by OR ones when forming ABC from B, A, and C must be almost perfectly additive, even if this replacement occurs at vicinal positions. Hence, any deviation experimentally observed will indicate that steric factors are operating, most probably by distorting the dihedral angles 4 and \k between sugar rings. The situation is completely different if the outer sugar residues of this triad are concerned, in that the conformation-dependent part of the shielding of A by C (and vice versa) cannot be detected by any additive combination. This does not mean, however, that the role of the conformational factor must remain concealed under all circumstances. For example, if attaching of A to B caused shifts of signals of protons of C distant from the anisotropic grumps of A, these shifts would strongly suggest a conformational change in all the system. An extension of the above scheme suited for a tetrasaccharide ABCD treats the chemical shifts of protons of any of the inner residues B or C of the tetrasaccha-

410

DABROWSKI

ET AL.

TABLE

II

DIFFERENTIAL CHEMICAL SHIFTS (ppm) CALCULATED ACCORDING TO ADDITIVE SCHEMES’ Difference

H-l

1 6’” - (6”’ + 6’) + 6” -0.08 2 6’ - 6” 3 #I - p

H-2

0.01

4 6’” - p

-0.13

-0.05

5 6’” - 6’

-0.08

-0.01

H-3

H-4

&Gal C -0.10

0.00

j3-GlcNAc B 0.08 -0.07 cu-Fuc D 0.03

b

H-5

H-l

H-2

*

0.07 -0.10 -0.20 0.05 -0.06 0.01

*

-0.01

0.12

H-3

H-4

@-GlcNAc B -0.10 -0.04 0.18 -0.03 -0.03 0.02

-0.01

a-FUC A 0.02

0.08 -0.05

&Gal C -0.10

b

0.04

H-5

* * -0.08 0.02 *

a See text. * Not measured.

ride as composed of the values obtained for the disaccharide BC and the shift differences resulting from its substitution by A and D:

+ (snyD- sF>, or 8fyCD-
[2]

The expression for S$FcDis fully analogical. Again, the outer residues A and D must be treated differently, as described above for the trisaccharide. Table II shows several differential chemical shifts. Item 1 shows the deviations from additivity calculated for the tetrasaccharide terminal of IV by using Eq. [2]. The absolute values of these deviations exceed up to five. times the estimated experimental error. Since all shielding effects resulting from the glycosylation of the disaccharide by a third sugar residue have been taken into account by those calculations, the obtained deviations are believed to evidence a change in conformation of the oligosaccharide chain occurring on glycosylation of either ABC by D or BCD by A to form ABCD. The conformational changes are also believed to be reflected by other substitution-induced shifts observed for protons of the next but one, and the three

residues removed sugar rings (items 2-5). Although the conformation-dependent part cannot be separated here from the total shielding effect (vide supra), large A 6 values for protons located at a large distance from the introduced sugar residue obviously indicate that relative orientation of structure elements has changed. Thus, Fuc A introduced into I to form IV has a strong effect on 6 (H-3) of Fuc D and 6 (H-3) of Gal C which are very far removed (item 5). The same applies to 6 (H-l) and 6 (H-4) of GlcNAc B if I is compared with V (item 2) oi IV with III (item 4); remarkably, the fucose unit A resonances remain unaffected when Fuc D is introduced into III to form IV, but it must be taken into account that, while shielding changes are conclusive, their lack is not, because effects of opposite signs may cancel out. Our interpretation of the NMR data is at variance with the conclusions drawn by Lemieux et al. (5) who hold that the 9 and \k angles between the sugar residues are practically constant in all synthetic oligosaccharides identical with the terminals of the native Lewis-blood-group-active glycosphingolipids investigated by us. Since those authors did not take into account all elements of the full additive scheme, the calculated differential shifts are not directly comparable. In fact, the values calculated (5) for the combinations leading to the tetrasaccharide Leb termi-

PROTON NUCLEAR

MAGNETIC

RESONANCE

nal correspond to simple differences of the @CD - @FBcand 8&scD- @CDtype. Nevertheless, recalculation using Eq. [2] reveals marked deviations from additivity for several protons, the largest amounting to 0.24 ppm; hence, the arguments just discussed should be applicable in this case as well. Moreover, even the incomplete schemes yielded differences (5) which, to our opinion, can more plausible be explained by chain deformations, as they refer to protons too remote from the entering sugar residue to be directly shielded by it; so, e.g., A 6 (H-5) of GlcNAc (-0.08) or Gal (-0.15) of Leb (which correspond to our #v-6111and 6iv-6r, respectively). Regarding the particularly transparent case of a trisaccharide, it can hardly be agreed that “the spectrum for the Le” trisaccharide [BGall - 3bGlcNAc (4 + laFuc)] .is closely approximated by the addition of the appropriate chemical shifts for the disaccharides [@Gall + 3@GlcNAc] and [crFucl + 4@GlcNAc]” (5). In fact, the large deviations from additivity for H-3 (0.30 ppm) and H-4 (0.17 ppm)4 of the middle N-acetylglucosamine residue cannot be explained by shielding effects of neighboring groups, as this would mean to have counted those effects twice. Summing up, it should be emphasized that the resonances of all sugar ring protons are likely to contain structural information hence every effort should be undertaken to identify them. The application of nuclear Overhauser and spin decoupling difference spectroscopy for this purpose was a decisive point, as it made it possible to find most of these resonances buried under the unresolved bulk. Other methods are expected to follow in the near future.5 4 0.22 and 0.11 ppm, respectively,

if calculated

using

Eq.111. ’ For a short review, see J. Dabrowski, P. Hanfland, and H. Egge, in Methods in Enzymology (Ginsburg, V., ed.), Academic Press, New York, in press.

OF LEWIS-ACTIVE

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Another aspect of the NOE spectroscopy, viz., the detection of short through-space distances between protons, is a further important step toward the determination of both the primary (sequence, linkage analysis) and secondary structure of glycosphingolipids and other oligosaccharide derivatives by NMR alone, without resorting to data obtained by other methods. ACKNOWLEDGMENT Thanks are due to Mrs. Sylvia assistance.

Kuhn for skillful

REFERENCES 1. HANFLAND, P., AND GRAHAM, H. A. (1981) Arch. Biochem. Biophys. 210,383-395. 2. EGGE, H., AND HANFLAND, P. (1981) Arch. Biochem. Biophys. 210,396404. 3. DABROWSKI, J., HANFLAND, P., AND EGGE, H. (1980) Biochemistry 19, 5652. 4. HANFLAND, P., EGGE, H., DABROWSKI, U., KUHN, S., ROELCKE, D., AND DABROWSKI, J., (1981) Biochemistry, in press. 5. LEMIEUX, R. U., BOCK, K., DELBAERE, L. T. J., KOTO, S., AND RAO, V. S. (1980) Canad. J. Chem. 58,631. 6. FALK, K.-E., KARLSSON, K.-A., AND SAMUELSSON, B. E. (1979) Arch. B&hem. Biophys. 192,164, 177, 191. 7. KARLSSON, K.-A., AND LARSON, G. (1979) J. Biol.

Chem. 254,931l. 8. HANFLAND, P., KLADETZKY, R.-G., AND EGLI, H. (1978) Chem. Phys. Lipids 22.141. 9. HANFLAND, P. (1975) Chem. Phys. Lipids 15,105. 10. HANFLAND, P. (1978) Eur. J. Biochem. 87,161. 11. WAGNER, G., AND W~HRICH, K. (1979) J. Magn.

Rest. 33, 675. 12. GIBBONS, W. A., BEYER, C. F., DAWK, J., SPEICHER, R. F., AND WYSSBROD, H. R. (1975) Biochemistry 14,420. 13. DABROWSKI, J., HANFLAND, P., AND EGGE, H. (1980) Chem. Phys. Lipids 26,187. 14. DEBRUYN, A., ANTEUNIS, M., DEGUSSEM, R., AND DU~ON, G. G. S. (1976) Carbohydr. Res. 47, 148, and references therein.