Application of GlycoStation™ Glycan Profiling Analysis Technology

The potential applications of glycan profiling analysis are numerous and wide ranging. GlycoTechnica 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™(GlycoTechnica’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.

Outline of Application of GlycoStation™ and LecChip™

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 Profling Analysis of EPOs

EPO(Erythropoietin) is a drug for amenia. It is well know that drug efficacy of biotechnology-based drugs such as EPO and IgG is greatly affected by the glycosylation. According to a paper published in Folio Pharmacol., Jpn, 131, pp192-199, the second generation EPO, of which glycosylation is modulated with introducing two more N-glycans, has higher homatopoietic effct comparing with the first generation one. The figure below shows compariosn of glycan profiles between the fisrt generation and the second generation EPOs (red color shows the first genetation and blue color shows the second generation). The difference shown by PHA-L suggests that tetra-antennary N-glycans are more expressed in the second generation, the strong signal of MAL_I indicates that those N-glycans are heavily terminated by &alpha2,3 Sia, the differences shown by AOL and AAL suggest that those N-glycans are more fucosylated in the second generation, and also you can find that there are some differences in polylactosamine binder LEL and lactosamin binder STL. Thus, we believe that lectin microarray is very powerful in comparing differences of glycosylation among biotechnology-based drugs and useful in accerelating drug R&D. Of course, it is also possible to do glycan profiling of secreted glycoproteins into culture supernatant.

New Biomarker Development: 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.

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 were applied 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.

Characterization of Stem Cells (hES, iPS, MSC etc.) and Regenerative Medicine

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.

Intestinal Bacteria and Probiotics

It is well know that intestinal bacteria are deeply involved in human immune activity, health, and longlife. It is also known that carbohydrate moiety in the cell wall of intestinal bacteria is playing a pivotal role as a possible immune modulator. Recently, it was demonstrated that lectin microarray is able to profile bacterial cell surface glycome quite simply and quickly by combining a labeling method using SYTOX Orange. This was a joint R&D result between Yakult Central Institute for Microbiological Research and AIST. This method is now attracting so much attention as a powerful method promoting probiotics related research and development.