Establishment Of A Lectin Microarray Method For The Rapid Analysis Of Glycoprotein And Its Application

Purpose: Glycosylation is one of the most significant posttranslational modifications of proteins, which plays a indispensable role in a wide variety of biological processes including bacterial infection, cell differentiation, tumor metastasis and cell cancerization. Hence, the study on glycosylation draws the attention of researchers widely, but the process develops slowly due to lacking a method can investigate protein-carbohydrate interactions in a rapid, exact and high-throughput manner. The purpose of this experiment was integrating the principle of lectin to glycan binding affinity and high-throughput microarray technology to established lectin microarray. The serum of liver cancer patien, patients with Kashin-Beck disease, membrane protein of liver cell and their controls were initially analyzed by our homemade lectin microarrays. Comparing the glycopattern between different samples and its controls, differences in glycoprotein were found, which were possibly used for early diagnosis and treatment.Method: The lectins were immobilized on the epoxysilane-caoted slides, the different samples and its controls were labled with Cy3 fluorescent dye, and the principle of lectin to glycan binding affinity was used to establish lectin microarray, which analyzed glycoprotein in serum of liver cancer patien, patients with Kashin-Beck disease, membrane protein of liver cell and their controls.Result: The consequence of experiment indicated that phosphate buffer containing 1%BSA was the optimal blocking buffer,the optimal incubation time and temperature were 3 hours and room temperature, the optimal probing buffer was phosphate buffer containing 1%BSA and 0.05% Tween-20. Additionally, we validated the specificity of lectin microarray through the mannose competition assay. The serum of liver cancer patien, patients with Kashin-Beck disease, membrane protein of liver cell and their controls were initially analyzed by lectin microarrays. The results were as follow:1 .The branches of N-glycans increased in serum of liver cancer patiens. Fucose structure increased and Fucoseα-1,6GlcNAc(core fucose) existed in serum of liver cancer patiens. What's more, the quantity of GalNAcβ-1,4-GlcNAc was higher in serum of liver cancer patiens. The mannose structure existed as form as non-substitutedα-1,6Man or terminalα-1,3 mannose, the quantity of Gal andαGalNAc were higher in healthy people. And Fucoseα-1,3-GlcNAc (core fucose) existed in serum of healthy people.2.The quantity of fucose was a little bit higher in the membrane glycoprotein of Hepatocellular carcinoma and existed as form as fucoseα-1,3GlcNAc(core fucose). However fucoseα1-2Galβ1- 4Glc(NAc) existed in the membrane glycoprotein of normal liver cells. The sialic acid, gal/GalNAc were higher in the membrane glycoprotein of Hepatocellular carcinoma and the sialic acid existed as form as sia2-3Galβ1-4Glc(NAc).The mannose existed as structure as terminal branched mannose in membrane glycoprotein of Hepatocellular carcinoma, and non-substitutedα-1,6Man existed in the membrane glycoprotein of normal liver cells. Further, GlcNAc existed in the membrane glycoprotein of Hepatocellular carcinoma. O-glycan structure Galβ1-3GalNAcα-Ser/Thr(Tn) existed in the membrane glycoprotein of normal liver cells and the branches of N-glycan increased as well.3.The O-glycan Galβ1-3GalNAcα-Ser/Thr(T) or GalNAcα- Ser/Thr(T) decreased and GlcNAc gone down as well in the serum of patients with Kashin-Beck disease. The quantity of Fucose was higher in serum of patients with Kashin-Beck disease and existed as form as fucoseα-1,6GlcNAc(core fucose).The mannose structure increased and existed as form as terminal branchedα-1,3mannose. Sialic acid increased significantly and existed as form as Sia2-6Galβ1- 4Glc(NAc) in the serum of patients with Kashin-Beck disease. And finally the branches of N-glycan and Gal/GalNAc increased in the serum of patients with Kashin-Beck disease as well.