Establishment And Application Of Glycosphingolipid Glycan Profile Analysis Based On Lectin Microarray

PurposeGlycosphingolipids are bioactive components distributed on the surface of plasma membrane with multiple biological functions such as cell-cell recognition, signal transduction, cell migration, and angiogenesis. In the recent years, researches on GSLs have become a hotspot in the aspect of cancer research due to the connection between GSL alteration and tumor growth. Currently, people have known much more about the structure and chemosynthesis of GSL than they knew about the alterations and biological functions during diseases. The main techniques to analyze GSLs are MS and LC-MS. Research focused on MS is costly and it requires a high-level sample pretreatment. Furthermore, the fact that MS analysis cannot provide a full-scaled illustration of glycan makes GSL research a hard undertaking.Compared to MS, lectin microarray is an ideal technique to help people investigate glycopatterns in an easy, low-consuming, and high throughput way. With high potential of being discovered as tumor biomarkers as well as therapeutic targets, GSL glycopatterns are crucial to be studied in a more high-efficiency way.MethodFirst the hydrophilization of GMla and the formation of -NH2werecatalyzed by the sphingolipid ceramide deacylase, as a result, GMla could be successfully dissolved into the aqueous phase and dyed with Cy3. Hence the experimental conditions of lectin microarray were optimized. Finally, both neutral and acid GSL from cell lines (HepG2 and HL-7702) and serum (patients with hepatocellular carcinoma and healthy volunteers) were applied to lectin microarray with proper conditions to highlight the significantly altered lectins and the corresponding glycans.ConclusionThe optimized conditions for GSL lectin microarray are as follows. Use Sephadex G-25 column to perform the separation and purification of the Cy3-Lyso-GM1a.0.05?g/?L of GSL glycan was the best dose to perform lectin microarray. The optimal blocking buffer was 50mmol/L MEA-50mmol/L sodium borate. Phosphate buffer containing0.06% Triton X-100,1% BSA and 1.5mol/L glycine were the optimal incubation buffer. The optimal incubation time was 3h.The total GSL should be first divided into neutral and acid part before the detection of lectin microarray. GOD oxidization showed no obvious influence on the result of serum GSL glycopatterning.Researches on GSL glycopatterns from HepG2 and HL-7702 cell lines indicated that when compared to HL-7702, GSLs from HepG2 cell showed a higher level of T/Tn antigen, (GlcNAc?1-4)n, Gal?1-4GlcNAc, terminal GalNAc?/?1-3/6Gal, and a lower level of a-Gal, ?-GalNAc, Sia?2-3, Gal?-1,3Gal, Gal?-1,6Glc, high mannose, GlcNAc, (GalNAc)n, bisecting GlcNAc, biantennary complex-type N-glycan.Researches on GSL glycopatterns from serum of HCC patients and healthy volunteers indicated that when compared to normal control, GSLs from HCC serum showed a higher level of T/Tn antigen, high mannose N-glycan, Fucal-6GlcNAc(core fucose), Fucal-3(Gal?1-4)GlcNAc, Fuc?l-2Gal-, and a lower level of?-Gal, ?-Gal, Gal?-1,4GlcNAc, Gal?1-3 GlcNAc, GalNAc?1-3Gal, (GalNAc)n, Sia?2-3, (GalNAc)n, terminal GalNAc and Gal. The high similarity between fucosylated GSL in this research and other reports manifested that fucosylated GSL might be a diagnostic biomarker or a therapeutic target aganist HCC.T/Tn antigen, a-Gal, (GalNAc)n, Siaa2-3 are glycans which showed mutual variation trends in both cell and serum groups. Fucosylated GSL increased in HCC serum samples while did not altered in HepG2 cell. Siaa2-6 showed no evidence of change in all 3 cell groups, however, Siaa2-3 decreased in both HepG2 cell and HCC serum samples, which indicated that the down-regulation of Sia?2-3 might be a diagnostic biomarker and a hotspot in cancer-related GSLs.