Technology

The principle of glycan structure profiling is based on the principle of lectin to glycan binding affinity. Lectin's are glycan binding proteins and certain lectins will only bind with particular glycans. A Moritex's LecChip™ 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 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 profiling pattern generated from a Moritex LecChip™ scanned on a Moritex 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 Moritex technology allows for generation of lectin-glycan affinity data from unwashed samples thereby taking advantage of the higher signal strength.

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 is formed on the surface of the LecChip™ when light is input into the edges of the slide glass 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 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. GlycoAnalyser™ (Ver 1.0) is a glycan structure inference software package based on the principle of fingerprint authentication which references the AIST database. GlycoAnalyser™ is a GlycoStation™ Reader 1200 system option.

Applications

The potential applications for glycan profiling are numerous and wide ranging. Moritex currently sees three areas of short term application of the technology:

1. Biomaker Screening (focusing on glycan structure changes)
2. Therapeutic Protein Glycan Structure Analysis
3. Stem Cell Characterization

References

• Data-mining system for differential profiling of cell glycoproteins:
  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 of detailed information: 834KB

• Glycan profiling targeting formalin-embedded tissue sections:
  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).
• Special Feature on Drug Discovery:
  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 of detailed information: 2.3MB

• Living Cell Surface Glycome Profling:
  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).
• Antibody-Overlay Lectin Microarray Assey:
  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.
• Application to Crude Samples:
  Application of Lectin Microarray to Crude Samples: Differential Glycan Profiling of Lec Mutants: Youji Ebe, Atsushi Kuno, Noboru Uchiyama, Shiori Koseki-Kuno, Masao, Takashi Sato, Hisashi Narimatsu and Jun Hirabayashi, J Biochem, 139(3), p.323 (2006)
• Frontal Affinity Chromatograph:
  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)
• Principle and Performance of Glycan Profiling Technology:
  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)