Glyan Analysis Technology

GlycoStation™, LecChip™, Introduction of Glycan Profiling Analysis Technology

glycan analysis technology

The principle of glycan structure profiling analysis is based on the principle of lectin to glycan binding affinity. Lectins are glycan binding proteins and certain lectins will only bind with particular glycans. A GP Bio's LecChip™ (Lectin Microarray) contains 45 different lectins immobilized on a slide glass in a X-Y array. Cy3 labeled glycoproteins are applied to the LecChip surface and their glycans then bind to the lectin with which it has affinity. The LecChip is then loaded into the GlycoStation™ Reader 1200 where an evanescent-field is generated above the LecChip array. The Cy3 marker attached to glycans that have successfully bound with lectins will fluoresce. The total LecChip™ fluorescence pattern generated by the glycan to lectin binding is then captured by the scanner (profiler) and analyzed by the on-board software which then provides the most likely glycan structure.

For a detailed explanation, please refer to "Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profling," A. Kuno et al., Nature Methods 2, p. 851 (2005)

Shown below is an actual glycan profiling pattern generated from a GPB LecChip™ scanned on a GPB GlycoStation™ Reader 1200

an actual glycan profiling pattern generated from a LecChip™ scanned on a GlycoStation™ Reader 1200

The evanescent-field fluorescent excitation method used to excite the Cy3 marker on the glycans is an essential enabling technology as it allows for detection of very weak molecular interactions. Lectin-glycan interactions are well known to be relatively weak compared to antigen-antibody and biotin-avidin interactions (see Table 1). Given this fact, if a washing process is applied to the LecChip™ to remove non-lectin binding redundant glycoproteins, much of the affinity information will be lost. Fortunately, the GPB technology allows for generation of lectin-glycan affinity data from unwashed samples thereby taking advantage of the higher signal strength.

Interaction between lectin and glycan is so weak

Table 1: Range of Kd Values For Typical Molecular Interactions
  Kd (M)
Lectin-glycan 10-3~10-7
Antigen-antibody 10-6~10-9
Biotin/avidin 10-12

The evanescent field that is formed has a depth equal to the wavelength of the light and the field strength decreases exponentially with the distance away from the slide surface.

The evanescent-field is formed on the surface of the LecChip™ when light enters into the slide glass from the sidewall and propagates through the glass. This is known as the principle of internal reflection mode. The evanescent field that is formed has a depth equal to the wavelength of the light and the field strength decreases exponentially with the distance away from the slide surface. Therefore, the further away from the slide glass, the weaker the evanescent-field becomes. Therefore Cy3 tagged glycans floating above the slide glass in a liquid phase exhibit a relatively low level of excitation as they are in the weakest portion of the evanescent-field. Whereas Cy3 tagged glycans that interact with the lectins located on the LecChip™, are contained in the stronger portion of the evanescent-field and are effectively excited. This technology, jointly developed by Moritex and the National Institute of Advanced Industrial Science and Technology (AIST), allows monitoring of very weak molecular interactions directly from a liquid phase without washing of the sample.

A significant piece of the overall technology used in this glycan profiling is the comprehensive database developed by AIST which covers more than 10,000 lectin-glycan interactions and developed utilizing Frontal Affinity Chromatography (FAC). The AIST database is an indispensable tool for understanding and calculating any given sample's most probable glycan structure taken from its complex lectin-glycan affinity pattern. The database has already been open to the public.

You can take a look of outline of GlycoStation operation on video.

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Q&A

We have summarized the most frequently asked questions obtained from this web site, and answers for the questions, for your information.

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References

  1. Lectin Microarrays
    Lectin Microarrays: concept, principle and applications, J. Hirabayashi, M. Yamada, A. Kuno and H. Tateno, Chemical Society Reviews, 2013, doi:10.1039/C3CS35419A.
  2. Liver Fibrosis and Glyco-biomarker
    A serum "sweet-doughnut" protein facilitaes fibrosis evaluation and therapy assessment in patients with viral hepatitis, A. Kuno, Y. Ikehara, Y. Tanaka, K. Ito, A Matsuda, S. Sekiya, S. Hige, M. Sakamoto, M. Kaga, M. Mizokami and H. Narimatsu, Scientific Reports 3, Article number: 1065, doi:10.1038/srep01065.
  3. Endometrial cancers
    Glycan profiling of endometrial cancers using lectin microarray, Y. Nishijima, M. Toyoda, M. Yamazaki-Inoue, T. Sugiyama, M. Miyazawa, T. Muramatsu, K. Nakamura, H. Narimatsu, A. Umezawa, and M. Mikami, Genes Cells, 2012 Oct. 17(10):826-36. doi: 10.1111/gtc. 12003.
  4. Metastasis-associated changes in glycosylation
    Lectin array-based strategies for identifying metastasis-associated changes in glycosylation, S. Fry, B. Afrough, A. Leathem, and M. Dwek, Methods Mol Biol., 2012;878:276-72
  5. Barrett's esophagus
    Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus, Bird-Lieberman EL, Neves AA, Lao-Sirieix P, O'Donovan M, Novelli M, Lovat LB, Eng WS, Mahal LK, Brindle KM, Fitzgerald RC., Nature Med. 2012 Jan 15. doi: 10.1038/nm.2616.
  6. Application to iPS
    Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis, YC. Wang, M. Nakagawa, I Garitaonandia, I. Slavin, G. Altun, RM Lacharite, KL Nozor, HT Tran, CL Lynch, TR Leonardo, Y. Lue, SE Peterson, LC Laurent, S. Yamanaka, and JF Loring, Cell Res. 2011 Nov;21(11):1551-63. doi: 10.1038/cr.2011.148. Epub 2011 Sep 6.
  7. A triplex lectin-santibody sandwich immunoassay for liver fibrosis:
    LecT-Hepa: A triplex lectin-antibody sandwich immunoassay for estimating the progression dynamics of liver fibrosis assisted by a bedside clinical chemistry analyzer and an automated pretreatment machine, A. Kuno, Y. Ikehara, Y. Tanaka, K.Saito, K. Ito, C. Tsuruno, S. Nagai, Y. Takahama, M. Mizokami, J. Hirabayashi, and H. Narimatsu, Clinica Chimica Acta 412 (2011) 1767-1772.
  8. Lectin Microarray Profiling of PSGL-1/mIgG:
    Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan subsitution as targeting molecules for mannose-specific receptors of the immune system, A. Gustafsson, M. Sjoblom, L. Strindelius, T. Johansson, T. Fleckenstein, N. Chatzissavidou, L. Lindberg, J. Angstrom, U. Rova, and J. Holgersson, Glycobiology, 2011 Aug. 21(8), 1071-1086.
  9. Glycan Profiling of Intestinal Bacteria:
    Lectin microarray reveals binding profiles of Lactobacillus casei strains in a comprehensive analysis of bacterial cell wall polysaccharides, E. Yasuda, H. Tateno, J. Hirabayashi, I. Iino, and T.Sako, Appl. Eviron Microbiol., 2011 Jul. 77(13), 4539-4546.
  10. Lectin Microarray Profiling of Breast Cancer:
    Lectin Microarray Profiling of Metastatic Breast Cancers, S. A. Fry, B. Afrough, H. J. Lomax-Browne, J. F. Timms, L. S. Velentzis, A. J.C. Leathem, Glycobiology (2011) doi: 10.1093/glycob/cwr045, First published online: April 19, 2011.
  11. A review paper: A lectin-based practical approach to complex glycans:
    Lectin-based structural glycomics: A practical approach to complex glycans, J. Hirabayashi, A. Kuno, H. Tateno, Electrophoresis, 2011 May;32(10):1118-28. doi: 10.1002/elps.201000650.
  12. A possible biomarker for iNPH
    A unique N-glycan on human transferrin in CSF: a possible biomarker for iNPH, S. Futakawa, K. Nara, M. Miyajima, A. Kuno, H. Ito, H. Kaji, K. Shirotani, T. Honda, Y. Tohyama, K. Hoshi, Y. Hanzawa, S. Kitazume, R. Imamaki, K. Furukawa, K. Tasaki, H. Arai, T. Yuasa, M. Abe, H. Arai, H. Narimatsu, and Y. Hashimoto, Neurobiol Aging. 2011 Apr 1. [Epub ahead of print]
  13. Lectin Microarray analysis of MSC, ES, and iPS:
    Lectin microarray analysis of pluripotent and multipotent stem cells, M. Toyoda, M. Yamazaki-Inoue, Y. Itakura, A. Kuno, To. Ogawa, M. Yamada, H. Akutsu, Y. takahashi, S. Kanzai, H. Narimatsu, J. Hirabayashi, and A. Umezawa, Genes to Cells, Vol.16, Issue 1, 1-11, January (2011), DOI: 10.1111/j.1365-2443.2010.01459.x
  14. Stem Cell Characterization by the Cell Surface Glycome:
    Potential Linkages Between the Inner and Outer Cellular States of Human Induced Pluripotent Stem Cells, S. Saito, Y. Onuma, Y. Ito, H. Tateno, M. Toyoda, H. Akutsu, K. Nishino, E. Chikazawa, Y. Fukawatase, Y. Miyagawa, H. Okita, N. Kiyokawa, Y. Shimma, A. Umezawa, J. Hirabayashi, K. Horimoto, and M. Asashima, ISB2010, Suzhou, China, September 9-11, 2010, pp.381-388.
  15. New GlycoBiomarker for Cholangiocarcinoma:
    A. Matsuda, A. Kuno, H. Matsuzaki, T. Irimura, Y. Ikehara, Y. Zen, Y. Nakamura, M. Yamamoto, N. Ohkuhchi, J. Shoda, J. Hirabayasi, and H. Narimatsu, Hepatology. 2010 Jul;52(1):174-82.
  16. Xenotransplantatio of GalT knockout pig:
    S. Miyagawa, S. Takeishi, A. Yamamoto, K. Ikeda, H. Matsunari, M. Yamada, M. Okabe, E. Miyoshi, M. Fukuzawa, and H. nagashima, Xenotransplantation, 2010 Jan. Vol 17(1), pp.61-70.
  17. Relationship between activities of IL23R-Fc protein and its glycan structure:
    Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure, K. F. Suen, M. S. Turner, F. Gao, B. Liu, A. Althage, A. Slavin, W. Ou, E. Zuo, M. Eckart, T. Ogawa, M. Yamada, T. Tuntland, J. L. harris, and J. W. Trauger, Protein Expr. Purif. (2010), doi:10.1016/j.pep.2009.12.015.
  18. A Strategy for Discovery of Cancer Glyco-biomarkers:
    A Strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics, H. Narimatsu, H. Sawaki, A. Kuno, H. Kaji, H. Ito, and Y.Ikehara, FEBS Journal (2009), doi:10.1111/j.1742-4658.2009.07430.x
  19. Chemoenzymatic Synthesis of N-glycan Clusters and those Affinity to Lectin Microarrays:
    Chemoenzymatic Synthesis and Lectin Microarray Characterization of a Class of N-Glycan Clusters, W. Huang, D. Wang, M. Yamada, and Lai-Xi Wang, J. Am. Chem. Soc., Nov.16, 2009.
  20. GPIase Activity of tACE:
    Testicular Angiotensin-Converting Enzyme with Different Glycan Modification: Characterization on Glycosylphosphatidylinositol-Anchored Protein Releasing and Dipeptidase Activities, G. Kondoh, Ho. Watanabe, Y. tashima, Y. Maeda, and T. Kinoshita, J. Biochem. 2009 145(1)115-121, doi:10.1093/jb/mvn148.
  21. A Potential Marker for Hepatic progenitor cells:
    High levels of E4-PHA-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells, N. Sasaki, K. Moriwaki, N.Uozomi, K. Noda, N. Taniguchi, A. Kameyama, H. Narimatsu, S. Takeishi, M. Yamada, N. Koyama, and E. Miyoshi, Glyconj J., DOI 10.1007/s10719-009-9240-2, May (2009).
  22. Strategy for Glycoproteomics:
    Strategy for Glycoproteomics: Identification of Glyco-Alteration Using Multiple Glycan Profiling Tools (dagger), H. Ito, A. Kuno, H. Sawaki, M. Sogabe, H. Ozaki, Y. Tanaka, M. Mizokami, JI. Shoda, T. Angata, T. Sato, J. Hirabayashi, Y. Ikehara, and H. Narimatsu., J Proteome Res., Mar 6;8(3), pp.1358-1367 (2009).
  23. Advanced Antibody-overlay Lectin Microarray Method:
    Focused differential glycan analysis with the platform antibody-assisted lectin profiling (ALP) for glycan-related biomarker verification. A. Kuno, Y. Kato, A. Matsuda, MK. Kaneko, H. Ito, K. Amano, Y. Chiba, H. Narimatsu, and J. Hirabayashi, Mol Cell Proteomics, Jan;8(1), p.99., 2009.
  24. Microarray Methods and Protocols: Chapter 9:
    Lectin Microarrays, Masao Yamada, "Microarray Methods and Protocols" edited by Robert S. Matson, CRC Press, Taylor &Francious Group, p.141 (2009).
  25. Optimization of High-sensitive Detection:
    Optimization of evanescent-field fluorescense-assisted lectin microarray for high-sensitive detection of monovalent oligozaccharides and glycoproteins, Noboru Uchiyama, Atsushi Kuno, Hiroaki Tateno, Yoshiko Kubo, Mamoru Mizuno, Midori Noguchi, and Jun Hirabayashi, Proteomics Vol.8, p.3042 (2008).
  26. Development of Data-mining System:
    Development of a data-mining system for differential profiling of cell glycoproteins based on lectin microarray, Atsushi Kuno, Yoko Itakura, Masashi Toyoda, Yoriko Takahashi, Masao Yamada, Akihiro Umezawa, and Jun Hirabayashi, Journal of Proteomics & Bioinformatics(JPB), Vol.1, p.68 (2008.5).     Download: 834KBlink to PDF
  27. A Review Paper:
    Concept, Strategy and Realization of Lectin-based Glycan Profiling, J. Hirabayashi, J. Biochem. 144, pp.139-147 (2008).
  28. Glycan Profiling of Paraffin-embedded Tissue Arrays:
    Development of an all-in-one technology for glycan profiling targeting formalin-embedded tissue sections, Atsushi Matsuda, Atsushi Kuno, Hiroyasu Ishida, Toru Kawamoto, Jun-ichi Shoda and Jun Hirabayashi, Biochemical and Biophysical Research Communications,370, p.259 (2008).
  29. The Era of Glycan Profiling has come:
    Glycomics's Infinite Potential and Applications to Healthcare, M. Yamada, GOR, Vol.9, No.1, p.16 (2007).     Download: 2.3MBlink to PDF
  30. Profiling of Living Cell Surface Glycome:
    A novel strategy for mammalian cell surface glycome profiling using lectin microarray, H. Tateno, T. Sato, H. Narimatsu, and J. Hirabayashi, Glycobiology, Vol.17, No.10, p.1138 (2007).
  31. Antibody-overlay Lectin Microarray Method:
    Inhibition of tumor cell-induced platelet aggregation using a novel anti-podaplanin antibody reacting with its platelet-aggregation-stimulating domain, Y. Kato, M. K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y. Chiba, Y. Hasegawa, J. Hirabayashi, H. Narimatsu, K. Mishima, and M. Osawa, doi:10.1016/j.bbrc.2006.08.171.
  32. Application to Crude Samples:
    Application of Lectin Microarray to Crude Samples: Differential Glycan Profiling of Lec MutantsF Youji Ebe, Atsushi Kuno, Noboru Uchiyama, Shiori Koseki-Kuno, Masao, Takashi Sato, Hisashi Narimatsu and Jun Hirabayashi, J Biochem, 139(3), p.323 (2006).
  33. Frontal Affinity Chromatography:
    High-throughput analysis of lectin-oligosaccharide interactions by automated frontal affinity chromatography, Methods, S. Nakamura-Tsuruta, N. Uchiyama, and J. Hirabayashi, Enzymol., Vol.415, p.311 (2006).
  34. Principle of Glycan Profiling Technology and the Performance:
    Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling: Atsushi Kuno, Noboru Uchiyama, Shiori Koseki-Kuno1, Youji Ebe, Seigo Takashima, Masao Yamada & Jun Hirabayashi, Nature Methods Vol.2, No.11, p.851 (2005).

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