Vol.5 No.3 2012

74/94

Research paper : Development of basic tools for glycoscience and their application to cancer diagnosis (H. Narimatsu)−209−Synthesiology - English edition Vol.5 No.3 (2012) 5 Search and practical application of disease biomarkers by accumulated base technologies for glycoscience5.1 Strategy of the disease glycobiomarker searchThe search for disease biomarkers by application of proteomics technology is largely pursued. In proteomics, the concept of biomarkers is based on the quantitative difference of proteins. However, our principle is totally different. In our glycoproteomics approach for development of biomarkers, we are targeting on finding qualitatively changed glycoproteins based on the fact that the glycan structure of the disease-derived glycoproteins is altered from that derived from normal cells. Such glycoproteins can be called post-translationally modified isomers. The amount of in vivo glycobiomarkers (isomers) is considered to be very slight. Especially in the search for the early stage cancer markers, the amount is sparse as earlier the stage is. Therefore, it is impossible to find such a molecule in a serum. We fully utilized the preliminarily developed technologies and established a developmental scheme for cancer markers as shown in Figure 6.1.The RNA is extracted from both cancerous and normal tissues, and expression patterns of their glcyogenes are comprehensively analyzed by the real-time PCR. As a result, the glycan structures receiving modification by canceration are determined.2.Glycan profiles in the total glycoproteins obtained from cancerous tissue and cultured cancer cells are comparatively analyzed by lectin microarray. Characteristic lectin is selected as the probe. 3.The candidate marker glycoproteins are comprehensively identified with the selected lectin by the LC/MS/IGOT method. The number of candidate glycoprotein is narrowed to about a few hundred at this point. 4.To detect a marker in serum, glycoproteins that are abundantly present in serum even in a normal state are advantageous. We utilized bioinformatics to do the following: (i) Estimation of the serum concentration of candidate glycoproteins, and select candidates with sufficient amounts. (ii) Confirmation of the origin of the candidate is the target tissue. If the candidate has multiple origins, it is avoided due to dilution of the target molecule. (iii) Selection of the molecules with many N- or O-glycan binding sites, as more binding sites induce higher affinity to the probes. We prioritized the candidate molecules based on these parameters. 5.The candidate molecules are analyzed by western blotting with purchased antibodies according to the priority, and the serum concentrations are estimated.6.We further narrow down the promising candidates, and immunoprecipitate for rough purification. These concentrated molecules are again analyzed by lectin microarray, and the lectin indicated the most different profiles between cancerous patients and healthy controls (lectin A) is selected.7.In general, most of commercial antibodies are weak in affinity. In such cases, suitable antibody is also originally developed for the protein moiety of the candidate glycoprotein. 8.With the developed antibody possessing high affinity Confirmation of structural change by MSn Production of probe and antibodyLectin microarray Comparative analysis of glycan profile Identification of carrier proteinLectin-captured IGOT-LC/MS ValidationSandwich ELISA Bioinfomatics examination of tissue expression and expression levelComparative analysis of glycan profileAntibody-assisted lectin profiling (ALP)Immunoprecipitation of serum with antibody against carrier protein Antibody against carrier proteinAntibody against glycanCancer cell line Cancer cell Identification of cancer-related glycoproteinBiomarkerProbe lectin adoptionmRNA ProteinCancer-specific glycan structureAdoption of tissue-specific and cancer-related glycoproteinReal-time PCRExpression analysis of glycogenes Prediction of the cancer-related glycan transformationFig. 6 Strategy for development of disease glycobiomarkers

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