The Quantitative DSA Affinity Glycoproteomics Of Hepatocellular Carcinoma Patients And Functional Research Of L-Selectin

Protein glycosylation is one of the most important post-translational modifications, it mainly involves in signal transduction, regulation of protein translation, protein degradation, immune response, cell apoptosis,development, and the process of many diseases. Most of the tumor biomarkers are glycoprotein. More and more studies paid their attention to the hepatocelluar carcinoma (HCC) glycoproteomics research based on abnormal glycosylation and glyco-markers. In this study, datura stramonium agglutinin (DSA) was selected to enrich serum N-linked glycoproteins for its broader specificity with tri-or tetra-antennary complex type. Serum DSA affinity glycoproteins were identified and quantified by isobaric tags for relative and absolute quantitation (iTRAQ) based two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) analysis. Subsequently, the differential proteins of HCC were screened out. Furthermore, the function of L-Selectin in HCC cell adhesion was further explored and it might provide novel insight for metastasis and prognosis of HCC.Part One The quantitative glycoproteomics of serum DSA affinity glycoproteins of HCC patientsThe goal of this part was to quantitatively analyze the serum DSA affinity glycoproteins alterations of HCC. First, the pooled serum from HCC group and control group were treated by ProteoExtract Albumin/IgG Removal Kit to remove albumin and IgG. Then, DSA was chosen to enrich serum glycoproteins for its broader specificity with tri-or tetra-antennary complex type. The chromatography yield of DSA affinity glycoproteins was 5% with the comparison of loaded total serum protein. A total of 76 distinct serum glycoproteins were identified and quantified by iTRAQ based 2DLC-MS/MS analysis and compared. Subsequently,24 differential glycoproteins were screen out and filtered with manually selected filter exclusion parameters (P<0.05, proteins were considered up-or down-regulated as their ratios were>1.2 or <0.8). Among them,11 proteins were up-regulated and 13 proteins were down-regulated in HCC group. For these identified differential glycoproteins, gene ontology (GO) analysis showed that the subcellular distributions were enriched in extracellular region. And they were involved in regulation of response to external stimulus, defense response, and response to wounding. Ingenulty Pathways Analysis (IPA) indicated that most of the differential glycoproteins were mainly related to LXR/RXR activation, FX activation, coagulation system, acute phase response signaling. Western blot, lectin blot, and enzyme-linked immunosorbent assay (ELISA) were applied to further validate three candidate proteins (L-Selectin, Gal-3BP, and HRG). Our results presented that the candidates have similar trends with the iTRAQ results, which implied the credibility of proteomics analysis.Part Two Promotive effect on HCC cell adhesion of glycocylated L-SelectinAccording to bioinformatics analysis, L-Selectin was selected for further study. The objective of this part was investigated the effect of L-Selectin on HCC cell adhesion. L-Selectin recombinations were added to SMMC7721 and HCCLM3 cells in vitro. Our results showed that eukaryotic recombination L-Selectin could promote the adhesion activity of HCC cells, while the procaryotic recombination L-Selectin, without promotion. The results indicated that glycosylation may play a vital role in L-Selectin promoting cell adhesion of HCC.Conclusions1. Serum DSA affinity glycoproteins from HCC patients were analyzed and 24 potential differential serum glycoproteins associated with HCC were screened out and finally identified.2. Glycocylated L-Selectin could promote the adhesion activity of HCC cells.