Determination of terminal glycan and total monosaccharide profiles of reelin glycoprotein in SH-SY5Y neuroblastoma cell line by lectin blotting and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry system.


Demir R., Şahar U. , Deveci R.

Biochimica et biophysica acta. Proteins and proteomics, vol.1869, pp.140559, 2021 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 1869
  • Publication Date: 2021
  • Doi Number: 10.1016/j.bbapap.2020.140559
  • Title of Journal : Biochimica et biophysica acta. Proteins and proteomics
  • Page Numbers: pp.140559
  • Keywords: Glycosylation, LC-MS, Lectin blot, Monosaccharide, Reelin, SH-SY5Y, CONTROL NEURONAL MIGRATION, SIALIC ACIDS, PARACENTROTUS-LIVIDUS, PROTEIN GLYCOSYLATION, NERVOUS-SYSTEM, VLDL RECEPTOR, SEA-URCHIN, RECOGNITION, DISABLED-1, EXPRESSION

Abstract

Reelin (400 kDa) is an extracellular matrix glycoprotein that is a key regulator of the many significant biological processes including the brain formation, cell aggregation, and dendrite formation. The glycosylation contributes to the nature of the protein through folding, localization and trafficking, solubility, antigenicity, biological activity, and half-life. Although reelin is to be known as a glycoprotein, the knowledge of its glycosylation is very limited. In this study, we aimed to characterize the terminal glycan profile of reelin by lectin blotting and monosaccharide analysis of glycan chains by capillary liquid chromatography electrospray ionization ion trap tandem mass spectrometry (CapLC-ESI-MS/MS) in SH-SY5Y neuroblastoma cell line. According to our results, reelin was detected in different protein fragments (310, 250, and 85 kDa) in addition to full-length form (400 kDa) in the cell line. The reelin glycoprotein was found to carry the p-N-Acetylglucosamine, a-Mannose, 13-Galactose, and a-2,3 and a2,6 linked sialic acids by lectin blotting. Nevertheless, these terminal monosaccharides were found in different intensity according to reelin fragments. Besides, we purified a reelin fragment (250 kDa), and we analyzed it for their monosaccharide by CapLC-ESI-MS/MS. We found that reelin contained five types of monosaccharides, which were consisted of N-Acetylgalactosamine, N-Acetylglucosamine, Galactose, Glucose, Mannose and Sialic acid, from high to low abundance respectively. The present results provide a valuable guide for biochemical, genetic, and glycobiology based further experiments about reelin glycosylation in cancer perspective.