Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Abstract

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low-and high-grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low-grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma-specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC-MS/MS. The identified proteins from the two cell types were quantified using label-free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin-based immunosorbent assay.

Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma

“…High-grade glioma cells showed overexpression of several sialyl-and fucosyltransferases involved in the biosynthesis of Lewis glycans and the high expression of SLe x could be attributed to the expression of FUT7/11 and ST3GAL3. Similar results were recently reported by Park and colleagues, they found that the high-grade glioma cell line T98G expressed higher levels of α1,2 fucose and α2,3 sialic acid in comparison with the low-grade glioma cell line Hs683 [36]. In the same line, a microarray analysis of glyco-gene expression in human glioblastomas carried out by Kroes reported the identification of glycosyltransferases gene expression profiles able to classify cancer types employing expression data taken from cancer patient samples in TCGA and, remarkably, ST3GAL3 gene was found to be highly overexpressed in GBM [38].…”

Clinical Significance of Extracellular Vesicles in Plasma from Glioblastoma Patients

“…The main differentially expressed protein domains in GBM plasma extracellular vesicles include members of complement and coagulation cascade and regulators of iron metabolism. The role of these proteins has been already described in GBM biology, thus making these targets extremely interesting for extracellular vesicle-based biomarker development (46)(47)(48)(49)(50)(51)(52)(53). We demonstrate that the clinical correlation of plasma extracellular vesicles is with the presence of the tumor.…”

Site‐ and structure‐specific characterization of the human urinary N‐glycoproteome with site‐determining and structure‐diagnostic product ions

“…Therefore, relative quantitative analysis was also performed for S1M, S1N, S1E, S2M, and S3M. A label‐free quantitative method approach using spectral counting and normalized spectral abundance factor (NSAF) 50 was used to calculate the relative abundance of each identified intact N‐glycopeptide: The total number of GPSMs of each glycopeptide (SpGPSMs, defined by the peptide sequence, N‐glycosite and N‐glycan, composition) was divided by the sum of all the GPSMs. The relative quantitative results of all the datasets are listed in Sheets 1–5 of Table S3 (supporting information) .…”