Studing Glycoproteins Of Cancer Cells Using Glycoproteomic Techniques

In this paper, we separated glycoproteins using glycoproteomic techniques andidentifed glycoproteins from human tumor cells. The influent of glycoproteins ontumor cell behavior was also investigated. This research reveals several glycoproteinscould be acted as potential biomarker and they provided options in tumor prognosisand targets in therapy.To identify potential cancer related glycoproteins in breast cancer cells, weenriched N-linked glycoproteins by lentil lectin from the human breast cancer cellline Hs578T and the normal breast cell line Hs578BST for proteomics comparison.Glycoproteins were separated and compared by two-dimensional electrophoresisanalysis. Twenty-two glycoproteins were identified which expressed remarkablydifferently, among which9were involved in the progress of collagen synthesis. Prolyl4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breastcancer was further investigated. Immunohistochemistry revealed that P4HA2wasup-regulated in breast tumor cells compared with its adjacent normal tissues.Moreover, over-expression and RNA interference of P4HA2showed that P4HA2expression suppressed cell proliferation and migration in Hs578T in vitro.Click chemistry refers to chemical reactions that can occur inside of livingsystem without altering native biochemical processes. Applications of this conceptextended the studies on a group of biomolecules including glycans, proteins andlipids. An alkyne reagent containing one disulfide bond was synthesized for theenrichment of glycoproteins metabolized with peracetylatedN-azidoacetylmannosamine (Ac4ManNAz), which was applied on three differentcancer cell lines and all isolated proteins were analyzed by High Performance LiquidChromatography/Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS).Expression patterns of glycoproteins were acquired and confirmed by real-time PCRand western blot. Immune tissue microarray analysis was performed to confirm theexpression of Neuronal cell adhesion molecule (NrCAM) in different types of cancertissues involved in this study. The strategy of purifying cell surface glycoproteins introduced in this paper was shown to be reliable and a total of56cell surfaceglycoproteins were identified. NrCAM, a member of immunoglobulinlike celladhesion molecule family, was found uniquely expressed in lung adenocarcinomaA549. NrCAM expression was only detected in non-small cell lung carcinomas(NSCLC) by immunohistochemistry, which coincided with MS result. Furthermore,significant increment of NrCAM expression was identified in NSCLCadenocarcinoma tissues compared to adjacent noncancerous tissues however thisincrement was not present in NSCLC squamous carcinoma cells. In this study,bio-orthogonal chemistry was employed to identify new cancer related glycoproteins.A strategy for isolating cell-surface glycoproteins by introducing an azido-decoratedprecursor of sialic acid (ManNAz) into cells was described. The specificup-regulation of NrCAM suggests that it might take part in some kind of function inNSCLC adenocarcinoma cells and is a novel potential target and marker in cancertreatment and detection.As the strategy for isolating cell-surface sialylated glycoproteins was establishedand applied on three different cancer cell lines: A549lung adenocarcinoma, HeLacervical carcinoma, and SW1990pancreatic adenocarcinoma. gp96was identified inglycoprotein fraction of all the three cell lines. Western and realtime PCR were usedto investigate the expression of gp96in whole cell level. The content of gp96inpurified glycoprotein fraction was also be detected. As results, gp96was identified inpurified glycoprotein fraction of all the three cell lines and its content was significantlow in SW1990than Hela and A549. However, no significant difference of gp96expression was detected at whole cell level in the three cell lines. ELISA was used tomeasure the level of biotinylation and sialylation of gp96in different cells.Biotinylation of gp96in SW1990was30-40%less than A549and Hela. Sialylationof gp96and of gp96in SW1990was30%less than A549and Hela. Conclusion Thisstudy revealed a diversity of sialylation on gp96in different cancer cell lines, andsuggested its different immunological and pathological properties in pancreaticadenocarcinoma cells.