GP Biosciences Ltd.

Application of GlycoStation™ Glycan Profiling Analysis Technology

The potential applications of glycan profiling analysis are numerous and wide ranging. GP Biosciences currently sees that the following three areas are the highest priprity as short term applications of the technology:

A various types of samples can be applied onto LecChip™(GP Bio's Lectin Microarrays). GlycoStation™ (Evanescent-field fluorescence excitation profiler) is a very powerful and versatile system designed for glycan profiling analysis to have a quickest, easiest and high sensitive analysis way.

The most important differentiating factor of GlycoStation is the sensitivity. As you know, LC-MS/MS is very powerful in determining carrier proteins and N-glycan structures, however, it is not strong enough to screen glyco-biomarkers from a small amount of serum sample because of the low sensitivity. Although GlycoStation can not identify glycan structures digitally, it can give you meaningful results from only a few milli-liter of serum sample for not only N-glycans but also O-glycans. That's a good example to explain you the merit of GlycoStation™. Also, we should emphaseize that lectin microarray is an easiest way to identify isomers, such as α2,3-Sia and α2,6-Sia, which is quite tough with a conventional LC-MS/MS system, though.

Glycan Profling Analysis of Crude Samples

GlycoStation™ system is not just for purified samples, but also very effective for differential analysis of crude samples such as cell lysate, serum, and so forth. One of the good examples is a comparison between CHO and Lec1 mutant cells with the membrane faction. The difference in N-glycan related binders is relatively strong, whereas that of O-glycan related binders is relatively weak. The following is just some of the interesting information easily discovered through differential profiling. Signals from branched complex-type N-glycan binders (PHA(L), PHA(E), ACG), α2,3-Sialic acid binder(MAL I), lactose binder (RCA120) drastically decrease in Lec1 (shown in the graph of the lower left side). On the contrary, signals from high-mannose type N-glycan binders (GNA, HHL, PWM, Calsepa, PSA, LCA) increase in Lec1 (shown in the graph of lower right side). These observations are quite reasonable, taking into consideration of the lack of glycosyltransferase GlcNAc-T1 in Lec1 mutant cells.

Glycan Profiling Analysis of Crude Samples

Antibody Overlay Lectin Microarray Method

This is a quickest shortcut to investigate glycan structures of a target protein. That is because the fluorescence coming from the labeld antibody reflects interactions of glycans of the target protein with immobilized lectins directly. It is possible to study glycan structures of a specific protein from crude samples. For instance, Kato-et al. has actually investigated glycan structures of podoplanin which is a mucin-type sialoglycoprotein and acts as a platelet-aggregating factor. They used a highly ractive anti-podoplanin antibody: NZ-1 for this purpose. This method is a kind of sandwich assay. After the incubation of podoplanin sample on LecChip, biotinated NZ-1 was applied, and the fluorescence was measured by using a Cy3 labeled streptavidin. Please refer to a reference listed below.

Glycan Analysis using an Antibody Overlay Lectin Microarray Methods

Cell Surface Glycome Profling Analysis of Living Cells

It is well known that the cell surface glycome changes from species to species, with differentiation stages, and development of malignant variations. Our standard protocol is based on Cy3 labeling onto the protein part (actually to the amino-group) of glycoproteins. However, recently, Tateno et al. has succeeded in investigating cell surface glycome of living cells using GlycoStation. They labeled living cells metabolically by a Cell-Tracker Orange CMRA regent and applied those directly onto LecChip. One of the biggest merits of this method is that it becomes possible to investigate not only glycoproteins but also glycolipids on cell surfaces. They applied this method to CHO and Lec mutant cells (Lec1, Lec2 and Lec.8), and demonstrated that the result coincides very well with the expectation inferred from the lack of specific glycosyltransferase.
They also applied the same method to K562 cells to see the difference of cell surface glycome before and after the differentiation, and demonstrated how powerful this technology is in terms of characterizing stem cells.

Glycan Profiling Analysis of Paraffin-embedded Tissue Arrays

Matsuda et al. have investigated glycan profiling of formalin-fixed parafin-embedded tissue samples. They succeeded in showing that good quality of glycan profiling is possible with a very small amount of tissue scratch section (1.5mm in diameter) which contains roughly only 500cells. So, this must be an ultra-sensitive method for glycan profiling. We believe that this protocol is useful to screen glycobiomerkers, because there are so many paraffin-embedded tissue samples stocked waiting for awaiking from deep sleep. Glycolipids are eliminated during the deparaffinizing process.

Glycan Profiling Analysis of Paraffin-embedded Tissue Arrays

Clear-cut discrimination of F9(mEC) from mES: Characterization of Stem Cells (hES, iPS, MSC etc.)

It was shown that mES(mouse ES), F9(mouse embryonic carcinoma cell), and differentiated cells by retinoic acid were clearly discriminated each other by appliying hierarchical clustering and principal component analyses to those glycan profiling patterns using LecChips. This result was presented at ISSCR 2009 by Dr. Toyoda and Dr. Umezawa, NCCHD, et al.

Stem Cell Characterization

References

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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
  8. 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.
  9. New GlycoBiomarker for Cholangiocarcinoma:
    Wisteria floribunda Agglutinin-Positive Mucin 1 is a Sensitive Biliary Marker for Human 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.
  10. 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.
  11. 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.
  12. 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
  13. 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.
  14. 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.
  15. 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).
  16. 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).
  17. 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.
  18. 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).
  19. 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).
  20. 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
  21. A Review Paper:
    Concept, Strategy and Realization of Lectin-based Glycan Profiling, J. Hirabayashi, J. Biochem. 144, pp.139-147 (2008).
  22. 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).
  23. 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
  24. 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).
  25. 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.
  26. 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).
  27. 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).
  28. 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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