Molecular Regulation And Biological Function Analysis Of Bisecting GlcNAc Structures In Breast Cancer Cells And Clinical Samples

Glycosylation is one of the most essential post-translational modifications in proteins. Glycosylation plays important roles in many biological processes, including protein folding, degradation and cell-to-cell interaction. ?1,4-N-Acetylglucosaminyltransferase ?(MGAT3 or GnT?) catalyzes the addition of GlcNAc to the core ?-mannose residue of N-glycans with ?1,4-linkage, and resulting ?1,4-linked GlcNAc is named as bisecting GlcNAc structure. Bisecting GlcNAc structures is associated with many biological processes, such as cellular signaling, cell adhesion and cell migration. Bisecting GlcNAc structures and MGAT3 are closely associated with tumor migration and invastion. However, the global analysis of the alteration of bisecting GlcNAc structures and its biological functions during breast cancer progression still need clarified, which may provide theoretical basis to the development of breast cancer biomarkers and cancer therapy.In this study, the aberrant bisecting GlcNAc structures and expression of MGAT3 were analyzed in breast cancer and epithelial-to-mesenchymal transition(EMT) progression. The target proteins with bisecting GlcNAc structures were identified, and the biological functions of glycoprotein with bisecting GlcNAc structure were studied. The main results are as follows:(1) Glycomic study of breast epithelial cell undergoing EMT. We established the TGF?-induced EMT model using normal mouse mammary gland epithelial(NMuMG) cell line. The aberrant N-glycosylation and glycogenes in EMT process was studied by the combination of mass spectrometry, gene microarray and lectin microarray, and comfirmed by lectin staining and qRT-PCR. In TGF?-induced EMT, levels of high mannose type N-glycan were enhanced, bisecting GlcNAc structure and fucosylation were suppressed. The expression of seven N-glycan-related genes were significantly changed. The expression of ALG9 and MGAT3 were decreased.(2) Variation of bisecting GlcNAc structures and MGAT3 expression in breast cancer. N-glycans from human and mouse breast cell lines and human breast cancer tissues were subjected to mass spectrometry, and results indicated that bisecting GlcNAc structures were suppressed during breast cancer progression. Lectin staining of human breast cancer tissue microarray was performed, and the suppression of bisecting GlcNAc glycosylation was detected in 24 of 30 pairs of breat cancer tissues. The expression of MGAT3 was decreased at both mRNA and protein levels, which was determined by ONCOMINE database, Western blot and immunofluorescence. The down-regulation of MGAT3 appears to contribute to the suppression of bisecting GlcNAc glycosylation. The promoter of MGAT3 was hypermethylated, which was determined by online database(MethHC). Expression of MGAT3 was up-regulated by treating cells with inhibitor of DNA methylation(decitabine), with the down-regulation of DNA methyltransferase(DNMT) 1. The results revealed that DNA methylation plays regulatory roles in expression of MGAT3. The relapse free survival of breast cancer patients with high expression of MGAT3 was higher than which with low expression of MGAT3 analyzed by online survival analysis tool.(3) Identification and functional analysis of target proteins with bisecting GlcNAc structure using PHA-E enrichment. Glycoproteins with bisecting GlcNAc structures were identified in human and mouse breast cell lines, and bioinformatically analyzed. Target proteins including EGFR and integrin were identified and the pathway including ERK and AKT et al were involved.(4) Establishment of a lectin-based isolation/ enrichment strategy for N-glycan analysis. High-abundant N-glycans were separated from low-abundant N-glycans by lectin enrichment, and the two parts of N-glycans were desalted and detected by mass spectrometry with enhanced coverage and sensitivity. This strategy was applied to standard glycoprotein(ovalbumin), complex biological samples including cells and serum. Our strategy allows comprehensive detection of low-abundant and high-abundant N-glycan structures using a similar amount of proteins as in other methods, but with enhanced coverage and sensitivity.