Paramagnetic lanthanide tagging for nmr conformational analyses of N-linked oligosaccharides

Article abstract of DOI:10.1002/chem.201100856

Sweet measurement: Paramagnetic tags are proposed as new tools for NMR analyses of carbohydrate conformations. A newly designed, lanthanide-chelating tag can be attached to the common disaccharide core shared among all N-linked oligosaccharides (see figur

Full text of DOI:10.1002/chem.201100856

DOI: 10.1002/chem.201100856
Paramagnetic Lanthanide Tagging for NMR Conformational Analyses of
N-Linked Oligosaccharides
Sayoko Yamamoto,[a, b] Takumi Yamaguchi,[b] Mꢀtꢁ Erdꢁlyi,[c, d]
Christian Griesinger,*[c] and Koichi Kato*[a, b]
It is estimated that more than half of all proteins in
nature are post-translationally modified with various oligo-
saccharides.[1] The oligosaccharides are critical for biomolec-
ular recognition events that mediates cell–cell communica-
tion and viral infections. N-linked oligosaccharides form a
major class of the glycoprotein glycans, which commonly
share the innermost N,N’-diacetylchitobiose structure cova-
lently attached to the amide group of the asparaginyl side
chains. Accumulating evidence[2] indicates that the N-linked
oligosaccharides not only regulate biological functions of ex-
tracellular or cell-surface proteins but also serve as tags of
proteins, determining their fates in cells, that is, folding,
translocation, and degradation. The biological codes carried
by the N-linked oligosaccharides are expressed as specific
conformations recognized or selected by the carbohydrate-
binding proteins collectively termed lectins.[2] Recent prog-
ress in glycomics has made it possible to profile N-linked
oligosaccharides and determine their sequences, linkages,
and positions on proteins.[3] However, conformational char-
acterization of the individual oligosaccharides remains a
challenging task because the flexible properties preclude X-
ray crystallographic approaches. Although NMR spectrosco-
py has great potential to provide information on structure
and dynamics of oligosaccharides,[4] the applicability of the
NOE-based approach, widely used for protein-structure de-
termination, is limited by the insufficiency of distance-re-
straint information as a consequence of the low proton den-
sity in oligosaccharides and the exceedingly low number of
proton–proton NOEs that restrain interglycosidic linkages.
Hence, it is highly desirable to develop NOE-independent
approaches for determining the oligosaccharide conforma-
tions and dynamics.
Paramagnetic effects, such as pseudocontact shifts (PCSs)
induced by lanthanide ions with an anisotropic magnetic-
susceptibility tensor (Dc tensor), offer long-distance infor-
mation on conformations and dynamics of biological macro-
molecules.[5] Indeed, over the last few years, several lines of
NMR studies have been reported by using PCSs as confor-
mational restraints of proteins.[6] Herein we present an ap-
plication of paramagnetic effects to characterize the carbo-
hydrate conformations by using new lanthanide tags at-
tached to the reducing end of an N-linked oligosaccharide.
To develop a general method, we focused on the common
core structure shared among all N-linked oligosaccharides,
that is, N,N’-diacetylchitobiose. We focused on the induced
PCSs of the CH groups of this disaccharide and extracted
unique information on the glycosidic-linkage conformation.
The modified disaccharide 1 was synthesized as shown in
Scheme 1. An ethylenediaminetetraacetic acid (EDTA) de-
rivative was designed to serve as the paramagnetic tag by
chelation to a lanthanide ion. A rigid phenylene spacer was
inserted to suppress unfavorable relaxation enhancement of
the carbohydrate resonances originating from the protons
spatially proximal to the coordinated paramagnetic metal
ion. The rigidity of the tag as well as the stability of the lan-
thanide complex are crucial factors for unambiguous inter-
pretation of the PCS data. By selective amination and subse-
quent acylation reactions, N,N’-diacetylchitobiose was at-
tached to this EDTA derivative through an amide linkage
that mimics the “N-linked” oligosaccharides.
[a] S. Yamamoto, Prof. K. Kato
Graduate School of Pharmaceutical Sciences
Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku
Nagoya 467-8603 (Japan)
Fax : (+81)52-836-3447
E-mail: kkato@phar.nagoya-cu.ac.jp
[b] S. Yamamoto, Dr. T. Yamaguchi, Prof. K. Kato
Institute for Molecular Science
and Okazaki Institute for Integrative Bioscience
5–1 Higashiyama, Myodaiji
Okazaki, 444-8787 (Japan)
[c] Dr. M. Erdꢀlyi, Prof. C. Griesinger
Department for NMR-Based Structural Biology
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax : (+81)52-836-3447
1H NMR spectral changes of an aqueous solution of 1
(3 mm) were observed upon titration with paramagnetic
Tm3+. The addition of up to one molar equivalent of the ion
generated a new set of peaks originating from 1 with con-
comitant disappearance of the original peaks. No further
chemical-shift changes were induced with excess amounts of
the lanthanide ion. These observations indicate that the lan-
thanide ion is bound at the specific site of 1 in a slow ex-
change regime; this gives rise to a stable 1:1 complex. By
E-mail: cigr@nmr.mpibpc.mpg.de
[d] Dr. M. Erdꢀlyi
Department of Chemistry and the Swedish NMR Centre
University of Gothenburg
Kemigꢁrden 4, 412 96 Gçteborg (Sweden)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100856.
9280
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Chem. Eur. J. 2011, 17, 9280 – 9282

COMMUNICATION
Information). These results in-
dicate that the common inner-
most part of the N-linked oligo-
saccharides exhibits a rigid con-
formation, which is little affect-
ed by the attachment of a tag.
The conformational rigidity of
the glycosidic linkage of this
disaccharide agrees with results
from molecular-dynamics simu-
lations during 10 ns (data not
shown). The behavior of N,N’-
diacetylchitobiose is thus differ-
ent from that of lactose investi-
Scheme 1. Introduction of the lanthanide-chelating unit to N,N’-diacetylchitobiose (HATU=2-(7-aza-1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, TFA=trifluoroacetic acid).
gated with the same ap-
proach.[9]
1H–13C HSQC experiments, the PCS values were measured
1
as the differences of H and 13C chemical shifts compared
with the compound chelated to the diamagnetic La3+ ion
(Figure 1). The observed PCS values were largest (1.1 ppm)
Figure 2. Analysis of the PCS data: a) Correlation between the experi-
mentally observed and back-calculated PCS values. b) The 3D model of
the carbohydrate moiety of 1. The observed PCS values (ppm) of each
proton are inserted. Determination of the Dc tensor and back-calculation
were performed with a modified version of MSpin.[11]
In conclusion, we have demonstrated the utility of the lan-
thanide tagging method, which provides valuable informa-
tion on carbohydrate conformations in solution. Especially,
the success of introducing a tag at the reducing-terminal,
rigid disaccharide will open up a new avenue for NMR char-
acterization of conformations, dynamics, and interactions
with lectins of a variety of the N-linked oligosaccharides, in-
cluding high-mannose-type oligosaccharides involved in the
glycoprotein-fate determination in cells. To deal with these
larger, N-linked oligosaccharides, the lanthanide ions with
larger Dc components, such as Dy3+ and Tb3+ would need
to be used as sources of long-distance information. It is also
expected that PCSs induced by lanthanide tagging contrib-
ute to peak separation in the highly degenerate spectra of
the larger oligosaccharides. The applicability of our ap-
proach will be strengthened by combining it with stable iso-
tope labeling of the oligosaccharides.[10] This line of studies
is underway in our laboratories.
Figure 1. 1H–13C HSQC spectra of 1 complexed with Tm3+ (red) and
La3+ (blue). The chemical-shift perturbations of the anomeric CH groups
are indicated by arrows.
for C1, the anomeric carbon located at the reducing termi-
nus, and smaller for the more distal atoms (Table S1 in the
Supporting Information).
For quantitative validation of our approach, the experi-
mentally obtained PCS values were compared with those
calculated from the 3D model of 1, which was built based
on a reported conformation of N,N’-diacetylchitobiose (with
the torsional angles defined by O5-C1-O4’-C4’ and C1-O4’-
C4’-C5’ of À56 and À1068, respectively).[7] The components
of the Dc tensor were determined for this model by employ-
ing the experimental PCSs. The value of these components,
Dcax and Dcrh, were estimated to be À7.66ꢃ10À32 and
À2.29ꢃ10À32 m3. As shown in Figure 2, the back-calculated
PCS values are in excellent agreement with the experimen-
tal data (Q=0.03).[8] Likewise, excellent agreement, with Q
values of 0.02, 0.05, and 0.07, was observed by using other
lanthanide ions, Ho3+, Er3+, and Yb3+ (see the Supporting
Experimental Section
All NMR spectra were recorded on JEOL JNM ECA-600 spectrometer
equipped with a 5 mm FG/HCN probe. For PCS observation, 1H–13C
Chem. Eur. J. 2011, 17, 9280 – 9282
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C. Griesinger, K. Kato et al.
HSQC spectra were recorded at 300 K with 512 (t1) and 1024 (t2) com-
plex points. NMR spectra were processed and analyzed with the pro-
grams NMRPipe[12] and Sparky.[13]
625; b) T. E. Klepach, I. Carmichael, A. S. Serianni, J. Am. Chem.
Soc. 2005, 127, 9781–9793; c) V. Slynko, M. Schubert, S. Numao, M.
Kowarik, M. Aebi, F. H. Allain, J. Am. Chem. Soc. 2009, 131, 1274–
1281; d) G. Widmalm in Comprehensive Glycoscience, Vol. 2 (Ed.:
J. P. Kamerling), Elsevier, Amsterdam, 2007, pp. 101–132.
[5] a) I. Bertini, C. Luchinat, G. Parigi, Prog. Nucl. Magn. Reson. Spec-
trosc. 2002, 40, 249–273; b) G. Otting, J. Biomol. NMR 2008, 42, 1–
9; c) P. H. J. Keizers, M. Ubbink, Prog. Nucl. Magn. Reson. Spec-
trosc. 2011, 58, 88–96; d) P. Haberz, F. Rodriguez-CastaÇeda, J.
Junker, S. Becker, A. Leonov, C. Griesinger, Org. Lett. 2006, 8,
1275–1278.
[6] a) T. Ikegami, L. Verdier, P. Sakhaii, S. Grimme, B. Pescatore, K.
Saxena, K. M. Fiebig, C. Griesinger, J. Biomol. NMR 2004, 29, 339–
349; b) F. Rodriguez-CastaÇeda, P. Haberz, A. Leonov, C. Griesing-
er, Magn. Reson. Chem. 2006, 44, S10–16; c) I. Bertini, S. Ciurli, A.
Dikiy, C. O. Fernandez, C. Luchinat, N. Safarov, S. Shumilin, A. J.
Vila, J. Am. Chem. Soc. 2001, 123, 2405–2413; d) I. Bertini, A.
Bhaumik, G. De Paepe, R. G. Griffin, M. Lelli, J. R. Lewandowski,
C. Luchinat, J. Am. Chem. Soc. 2010, 132, 1032–1040; e) X. C. Su,
B. Man, S. Beeren, H. Liang, S. Simonsen, C. Schmitz, T. Huber,
B. A. Messerle, G. Otting, J. Am. Chem. Soc. 2008, 130, 10486–
10487.
Acknowledgements
This work was supported by KAKENHI (20107004, 21370050, and
21850029) and Nanotechnology Network Project of MEXT and the Max
Planck Society. We thank Dr. Tomoki Kameda (AIST) for the MD calcu-
lation. We also thank Dr. Yoshiki Yamaguchi, Dr. Hirokazu Yagi, Dr.
Yukiko Kamiya, Erina Ohno, Yuki Horikawa, and Masahiro Yamamoto
(NCU) for their contributions at the early stage of this study. We also
thank Dr. Edward dꢄAuvergne (MPI) for useful discussion. M.E is grate-
ful for the research grant (no. 2004-3073) of the Swedish Research Coun-
cil.
Keywords: conformation analysis
· lanthanides · NMR
spectroscopy · oligosaccharides · paramagnetism
[7] M. Aida, Y. Sugawara, S. Oikawa, K. Umemoto, Int. J. Biol. Macro-
mol. 1995, 17, 227–235.
[8] The Q value is defined in the Supporting Information.
[9] M. Erdꢀlyi, E. dꢄAuvergne, A. Navarro-Vꢅzquez, A. Leonov, C.
Griesinger, Chem. Eur.J. 2011, DOI: chem.201100854.
[10] a) K. Kato, Y. Yamaguchi, Y. Arata, Prog. Nucl. Magn. Reson. Spec-
trosc. 2010, 56, 346–359; b) Y. Yamaguchi, K. Kato, Methods Enzy-
mol. 2010, 478, 305–322.
[11] V. M. Sꢅnchez-Pedregal, R. Santamarꢆa-Fernꢅndez, A. Navarro-
Vꢅzquez, Org. Lett. 2009, 11, 1471–1474.
[12] F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, A. Bax, J.
Biomol. NMR 1995, 6, 277–293.
[1] R. Apweiler, H. Hermjakob, N. Sharon, Biochim. Biophys. Acta.
1999, 1473, 4–8.
[2] a) K. Kato, Y. Kamiya, Glycobiology 2007, 17, 1031–1044; b) M.
Aebi, R. Bernasconi, S. Clerc, M. Molinari, Trends Biochem. Sci.
2010, 35, 74–82; c) Y. Kamiya, D. Kamiya, R. Urade, T. Suzuki, K.
Kato in Glycobiology Research Trends (Eds.: G. Powell, O.
McCabe), NOVA Science, New York, 2009, pp. 27–40.
[3] a) K. Kato, N. Takahashi in Experimental Glycoscience: Glycochem-
istry (Eds.: T. Kawasaki, S. Hase), Springer, Tokyo, 2008, pp. 413–
416; b) A. Dell, S. Chalabi, P. G. Hitchen, J. Jang-Lee, V. Ledger,
S. J. North, P.-C. Pang, S. Parry, M. Sutton-Smith, B. Tissot, H. R.
Morris, M. Panico, S. M. Haslam in Comprehensive Glycoscience,
Vol. 2 (Ed.: J. P. Kamerling), Elsevier, Amsterdam, 2007, pp. 69–
100.
[13] SPARKY 3, T. D. Goddard, D. G. Kneller, University of California,
San Francisco.
[4] a) K. Kato, H. Sasakawa, Y. Kamiya, M. Utsumi, M. Nakano, N. Ta-
kahashi, Y. Yamaguchi, Biochim. Biophys. Acta 2008, 1780, 619–
Received: March 20, 2011
Published online: July 19, 2011
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Chem. Eur. J. 2011, 17, 9280 – 9282