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

Highlights

• Reelin was detected as distinct fragments (310, 250, and 85 kDa) as well as full-length form (400 kDa) in SH-SY5Y cells

• The reelin was found to carry the β-GlcNAc, α-Man, β-Gal, and Neu5Ac by lectin blotting.

• The purified reelin fragment (250 kDa) was found to contain GalNAc, GlcNAc, Gal, Glc, Man and Neu5Ac by CapLC-ESI-MS/MS.

• This is the first study for the characterization of reelin glycoprotein with CapLC-ESI-MS/MS system.

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 β-N-Acetylglucosamine, α-Mannose, β-Galactose, and α-2,3 and α2,6 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.

Introduction

Protein glycosylation is the most widespread co- and post-translational modification, which is resulting in the addition of carbohydrates to proteins. During the cellular interaction events, it actually affects proteins by means of folding, localization and trafficking, protein solubility, antigenicity, biological activity, and half-life [1,2]. There are two main types of protein glycosylation, which are termed as N- and O- linked glycosylation. The first one, called N-glycosylation, is identified by attachment of precursor oligosaccharide, GlcNAc2-Man9-Glc3, from the dolichol-linked pyrophosphate donor to the amino side chain of the asparagine (Asn) amino acid within a consensus sequence of Asn-Xaa-Ser/Thr (Xaa is any amino acid except Pro) on proteins. The second one, called O-glycosylation, is identified by attachment of monosaccharide/glycan units to the hydroxyl side chain of serine(Ser) or threonine(Thr) amino acids of proteins [3]. Following the transfer of glycan to the polypeptide, N- and O- linked glycans have been processed to construct the structure of mature glycan by a variety of glycosidase and glycosyltransferase enzymes [4,5]. The process significantly influences biochemical, physicochemical, and structural characteristics of glycoproteins [6].

Reelin (400 kDa) is an extracellular glycoprotein composed of approximately 3461 amino acids and secreted mainly from the Cajal-Retzius cells in the marginal zone of the developing cortex and a subpopulation of GABAergic interneuron in the mature brain [7]. It acts as a key regulator of several biological processes in laminar formation including neuronal migration, cell aggregation, and dendrite formation [8]. From the N- to the C-termini, reelin is composed of a signal peptide, F-Spondin-like domain, unique region called ‘H' subdivided into three subdomains termed X, Y, Z and eight tandem reelin repeats (RR1 to 8) followed by basic C-terminal domain respectively. Each of these repeats includes epidermal growth factor (EGF) like motif at its center and links up two sub-repeats [9,10].

During the reelin signaling pathway, it activates the tyrosine phosphorylation-dependent signal transduction [11,12]. When the reelin binds to its receptors, very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) [[13], [14], [15]], it induces phosphorylation of the adapter protein disabled-1 (Dab1) through the Src family tyrosine kinases (SFKs) Fyn and Src [16,17]. Phosphorylated Dab1 in turn recruits several signaling molecules, including the Crk family adaptor proteins, Crk and CrkL, [18]. Alternatively, it is identified that reelin signaling activates the integrin α5β1 [19] and N-Cadherin [20].

Although reelin is to be known as a glycoprotein, there is limited knowledge about its glycosylation. Reelin actually possesses a mass of N-linked and a little O-linked glycan [21]. Recent data has shown that reelin glycosylation could be important in non-neurodegenerative diseases and some types of cancers [22]. In some neurological diseases including Alzheimer's disease, reelin glycosylation is differentiated, when it is compared to healthy (control) groups [23]. Our preliminary bioinformatics data have shown that reelin carries a variety of possible N- and O-glycosylation positions (Unpublished). However, there is no detailed information about glycosylation capacity, which is obtained analytical techniques such as liquid chromatography-mass spectrometry. In this study, we aimed to characterize the terminal glycan profile of reelin by lectin blotting and monosaccharide analysis of glycan chains by CapLC-ESI-MS/MS.