Advertisement

Journal of The American Society for Mass Spectrometry

, Volume 29, Issue 9, pp 1881–1891 | Cite as

Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry

  • Yannick Baschung
  • Loredana Lupu
  • Adrian Moise
  • Michael Glocker
  • Stephan Rawer
  • Alexander Lazarev
  • Michael Przybylski
Research Article

Abstract

Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains.

Graphical Abstract

Keywords

Mass spectrometry Human galectin-3 Glycine max lectin Blood group oligosaccharides CRD Recognition sites Proteolytic excision, proteolytic extraction Ligand epitope peptides SAW-biosensor analysis 

Abbreviations

CRD

Carbohydrate recognition domain

Aoa

Aminooxyacetic acid

DVS

Divinyl sulfone

GalNAc

Acétylgalactosamine

PBS

Phosphate-buffered saline

DTT

Dithiothreitol

SAM

Self-assembled monolayer

SAW

Surface acoustic waves

Notes

Acknowledgements

We thank Drs. Stefan Maeser and Elisa Peroni for the valuable discussions and critical reading of the manuscript.

Funding Information

This work has been partially supported by the European Union through the Marie-Curies IRSES grant “Integrating high performance mass spectrometry with applications in life science” (MSLife). Partial support is also acknowledged from the Bundesministerium für Wirtschaft (BMWi; SPR-MS).

Supplementary material

13361_2018_1998_MOESM1_ESM.docx (27 kb)
ESM 1 (DOCX 26 kb)
13361_2018_1998_MOESM2_ESM.pptx (85 kb)
ESM 2 (PPTX 85 kb)
13361_2018_1998_MOESM3_ESM.pptx (99 kb)
ESM 3 (PPTX 98 kb)

References

  1. 1.
    Lobsanov, J.D., Gitt, M.A., Leffler, H., Barondes, S.H., Rini, J.M.: X-ray crystal structure of the human dimeric S-lac lectin, L-14-II, in complex with lactose at 2.9 A resolution. J. Biol. Chem. 268, 27034–27038 (1993)Google Scholar
  2. 2.
    Moise, A., Andre, S., Eggers, F., Krzeminski, M., Przybylski, M., Gabius, H.J.: Toward bioinspired galectin mimetics: identification of ligand-contacting peptides by proteolytic-excision mass spectrometry. J. Am. Chem. Soc. 133, 14844–14847 (2011)CrossRefGoogle Scholar
  3. 3.
    Jimenez-Castells, C., Defaus, S., Moise, A., Przbylski, M., Andreu, D., Gutierrez-Gallego, R.: Surface-based and mass spectrometric approaches to deciphering sugar-protein interactions in a galactose-specific agglutinin. Anal. Chem. 84, 6515–6520 (2012)CrossRefGoogle Scholar
  4. 4.
    Stefanescu, R., Born, R., Moise, A., Ernst, B., Przybylski, M.: Epitope structure of the carbohydrate recognition domain of Asialoglycoprotein receptor to a monoclonal antibody revealed by high-resolution proteolytic excision mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 148–157 (2011)CrossRefGoogle Scholar
  5. 5.
    Stefanescu, R., Iacob, R.E., Damoc, E.N., Marquardt, A., Amstalden, E., Manea, M., Perdivara, I., Maftei, M., Paraschiv, G., Przybylski, M.: Mass spectrometric approaches for elucidation of antigenantibody recognition structures in molecular immunology. Eur. J. Mass. Spectrom. 13, 69–75 (2007)CrossRefGoogle Scholar
  6. 6.
    Iurascu, M.I., Marroquin-Belaunzanar, O., Petrausch, U., Renner, C., Przybylski, M.: An HLA-B27 homodimer specific antibody recognozes a discontinuous mixed-disulfide epitope identified by affinity-mass spectrometry. J. Am. Soc. Mass Spectrom. 27, 1105–1112 (2016)CrossRefGoogle Scholar
  7. 7.
    Juszczyk, P., Paraschiv, G., Szymanska, A., Kolodziejczyk, A.S., Rodziewicz-Motowidlo, S., Grzonka, Z., Przybylski, M.: Binding epitopes and interaction structure of the neuroprotective protease inhibitor cystatin C with beta-amyloid revealed by proteolytic excision mass spectrometry and molecular docking simulation. J. Med. Chem. 52, 2420–2428 (2009)CrossRefGoogle Scholar
  8. 8.
    Krzeminski, M., Singh, T., Andre, S., Lensch, M., Wu, A.M., Bonvin, A.M., Gabius, H.J.: Human galectin-3 (Mac-2 antigen): defining molecular switches of affinity to natural glycoproteins, structural and dynamic aspects of glycan binding by flexible ligand docking and putative regulatory sequences in the proximal promoter region. Biochim. Biophys. Acta. 1810, 150–161 (2011)CrossRefGoogle Scholar
  9. 9.
    Merrifield, R.B.: Solid-phase peptide synthesis. 3. An improved synthesis of bradykinin. Biochemistry. 3, 1385–1390 (1964)CrossRefGoogle Scholar
  10. 10.
    Carpino, L.A., Han, G.Y.: 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J. Am. Chem. Soc. 92, 5748–5749 (1970)CrossRefGoogle Scholar
  11. 11.
    Gronewold, T.M.: Surface acoustic wave sensors in the bioanalytical field: recent trends and challenges. Anal. Chim. Acta. 603, 119–128 (2007)CrossRefGoogle Scholar
  12. 12.
    Drǎguşanu, M., Petre, B.A., Przybylski, M.: Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry. J. Pept. Sci. 17, 184–191 (2011)CrossRefGoogle Scholar
  13. 13.
    Ma, Y., Wang, T.: Deactivation of soybean agglutinin by enzymatic and other physical treatments. J. Agric. Food Chem. 58, 11413–11419 (2010)CrossRefGoogle Scholar
  14. 14.
    López-Ferrer, D., Petritis, K., Hixson, K.K., Heibeck, T.H., Moore, R.J., Belov, M.I., Camp, D.G., Smith, R.D.: Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly. J. Proteome Res. 7(8), 3276–3281 (2008)CrossRefGoogle Scholar
  15. 15.
    Balny C. Biochimica et Biophysica Acta-Proteins and Proteomics 1764 (2006) 632–639Google Scholar
  16. 16.
    Sharon, N., Lis, H.: Detection, occurrence and isolation. In: Sharon, N., Lis, H. (eds.) Lectins, 2nd edn, pp. 33–62. Kluwer Academic Publishers, Dordrecht, Boston, MA (2003a)Google Scholar
  17. 17.
    Sharon, N., Lis, H.: Specificity and affinity in lectins. In: Sharon, N., Lis, H. (eds.) , 2nd edn, pp. 63–104. Kluwer Academic Publishers, Dordrecht, The Netherlands; Boston, MA (2003)Google Scholar
  18. 18.
    Rao, V.S.R., King, L., Pradman, K.: Three dimensional structure of the soybean agglutinin-Gal/GalNAc complexes by homology modeling. J. Biomol. Struct. Dyn. 15(5), 853–860 (1998)CrossRefGoogle Scholar
  19. 19.
    Seetharaman, J., Kanigsberg, A., Slaaby, R., Leffler, H., Barondes, S.H., Rini, J.M.: X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution. J. Biol. Chem. 273, 13047–13052 (1998)CrossRefGoogle Scholar
  20. 20.
    Saraboji, K., Håkansson, M., Genheden, S., Diehl, C., Qvist, J., Weininger, U., Nilsson, U.J., Leffler, H., Ryde, U., Akke, M., Logan, D.T.: The carbohydrate-binding site in galectin-3 is preorganized to recognize a sugarlike framework of oxygens: ultra-high-resolution structures and water dynamics. Biochemistry. 51, 296–306 (2011)CrossRefGoogle Scholar
  21. 21.
    Diehl, C., Engström, O., Delaine, T., Håkansson, M., Genheden, S., Modig, K., Leffler, H., Ryde, U., Nilsson, U.J., Akke, M.: Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3. J. Am. Chem. Soc. 132, 14577–14589 (2010)CrossRefGoogle Scholar
  22. 22.
    Henrick, K., Bawumia, S., Barboni, E.A., Mehul, B., Hughes, R.C.: Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3. Glycobiology. 8, 45–57 (1998)CrossRefGoogle Scholar
  23. 23.
    Murakami, T., Yoshioka, K., Sato, Y., Tanaka, M., Niwa, O., Yabuki, S.: Synthesis and galectin-binding activities of mercaptododecyl glycosides containing a terminal beta-galactosyl group. Bioorg. Med. Chem. Lett. 21, 1265–1269 (2011)CrossRefGoogle Scholar
  24. 24.
    Stowell, S.R., Arthur, C.M., Mehta, P., Slanina, K.A., Blixt, O., Leffler, H., Smith, D.F., Cummings, R.D.: Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens. J. Biol. Chem. 283, 10109–10123 (2008)CrossRefGoogle Scholar
  25. 25.
    Bachhawat-Sikder, K., Thomas, C.J., Surolia, A.: Thermodynamic analysis of the binding of galactose and poly-N-acetyllactosamine derivatives to human galectin-3. FEBS Lett. 500, 75–79 (2001)CrossRefGoogle Scholar
  26. 26.
    Liu, J., Gray, W.D., Davis, M.E., Luo, Y.: Peptide- and saccharide-conjugated dendrimers for targeted drug delivery: a concise review. Interface Focus. 2, 307–324 (2012)CrossRefGoogle Scholar
  27. 27.
    Xiao, S., Abu-Esba, L., Turkyilmaz, S., White, A.G., Smith, B.D.: Multivalent dendritic molecules as broad spectrum bacteria agglutination agents. Theranostics. 3, 658–666 (2013)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Steinbeis Centre for Biopolymer Analysis and Biomedical Mass SpectrometryRüsselsheim am MainGermany
  2. 2.Department of ImmunologyUniversity of RostockRostockGermany
  3. 3.Department of Chemistry and Steinbeis Center for Biopolymer Analysis and Biomedical Mass SpectrometryUniversity of KonstanzKonstanzGermany
  4. 4.Thermofisher ScientificDarmstadtGermany
  5. 5.Pressure BioSciences, Inc.South EastonUSA

Personalised recommendations