Structural elucidation of N-glycans and bioactivity of sialoglycoprotein from crucian carp eggs structure and bioactivity of crucian egg SGP
Graphical abstract
Introduction
Protein glycosylation in eukaryotes is a universal post-translational modification with important roles in living activities and structure–function relationships (Ohtsubo & Marth, 2006). In vertebrates, N-acetyl-neuraminic (Neu5Ac; a sialic acid) is often present at the terminal of a carbohydrate chain in glycoproteins and is linked to galactose (Gal) or N-acetyl-d-galactosamine (GalNAc) by α-2,3 or α-2,6 glycosidic bonds (Varki, 2008). Given its unique location and structural characteristics (electronegativity), sialic acid is involved in the regulation of numerous physiological and pathological processes, such as cancer-cell migration, fertilization, and neonatal brain development (Ang et al., 2011; Martinez-Duncker et al., 2011; Schnaar et al., 2014). Some glycoproteins, such as sialylglycoproteins (SGP), contain a large amount of sialic acids. Most of them contain complex branched and sialylated oligosaccharide chains, such as double antennary, tri-antennary, and tetra-antennary structures. SGP exert a good anti-binding effect primarily by inhibiting the binding of bacteria and viruses to host cells. For example, oral administration of SGP can effectively inhibit Salmonella growth in the spleen of mice (Sugita-Konishi et al., 2002). In cell-culture tests, SGP also inhibits the binding of Salmonella enteritidis to Caco-2 cells but not to Escherichia coli K88. In terms of antiviral activity, the sialylated α-2,6-GalNAc residue of SGP can act as a ligand that binds to the hemagglutinin of human influenza A virus, thereby inhibiting its binding to human host cells (Makimura et al., 2006). When Helicobacter pylori binds to human host cells, the trisaccharide of a glycoprotein consisting of Neu5Ac-α2,3-Gal-β1,4-N-acetylglucosamine has an important role in molecular recognition (Johansson et al., 2005). Recently, Rhee et al. (2016) used casein glycopeptides rich in sialylated oligosaccharides to prepare an effective cure against H. pylori. Thus, SGP showed the potential as a good and safe preventive medicine or functional foods to prevent infection by pathogens or viruses.
Numerous SGP occur in the brain, blood, submandibular gland, mucins, colostrum, and reproductive cells. The typical SGP source is edible bird's nest (EBN) with a high SGP content of 40–50% (w/w) and the highest sialic acid content (5–15%, depending on habitat) (Zhang et al., 2012). Yagi et al. (2008) found that 70% of carbohydrate chains on the EBN SGP are in the form of tri-antenna or tetra-antenna oligosaccharide branches, and that the terminal sialic acid is linked to the galactosyl group by a α-2,3 glycosidic bond. EBN SGP primarily had molecular weights of 128, 106, and 43 kDa; the former two are rich in sialic acids but cannot bind to Sambucus nigra and Maackia amurensis lectins. The EBN SGP of 43 kDa consists of α-2,3 and α-2,6 linked sialic acid and is structurally similar to animal chitinase (You et al., 2015). EBN SGP can be directly used as health foods without the need for extraction, but the process is costly (1600–2600 US$/kg). Another source of SGP is hen eggs with a sialic acid content of ~24–29 mg/kg, and the N-glycans are found mostly in the form of double antenna oligosaccharide chains (Liu et al., 2017; Seko et al., 1997). However, different types of eggs have different sialic acid contents. For example, the sialic acid content in black bone chicken yolk can reach 205 mg/kg (Koketsu et al., 2003). Some fish eggs also contain a large amount of SGP. Eggs from crucian carp roes have sialic acid content reaching 5.9% (w/w). Sialylated glycoprotein can promote the proliferation, differentiation, and mineralization of MC3T3-E1 cells and prevent osteoporosis (Wang et al., 2016; Xia et al., 2016). SGP from Gadus morhua (Atlantic cod) eggs can be used as functional foods to enhance fracture healing (Zhan et al., 2017). Fish eggs do not contain N-glycolylneuraminic acid (a sialic acid) and are thus a good source of SGP.
In China, people consume ~30 million tonnes of freshwater fish each year (Fluet-Chouinard et al., 2018). Among these fishes, sexually mature crucian carps contain numerous fish eggs and are currently not well utilized. Except for a small amount as a foodstuff, most fish eggs are discarded. Crucian carps eggs contain a high SGP content (~20% of total protein). In the present study, SGP were extracted and purified from crucian carp eggs with high sialic acid content. The SGP oligosaccharide chain structure and biological activity were determined.
Section snippets
Materials
Commassie blue R-250, protein markers, and reagents for SDS-PAGE were purchased from Bio-Rad (Hercules, CA, USA). Q Sepharose Fast Flow (QFF) column (1.6 × 10 cm) was acquired from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Crucian carp eggs were collected from fresh carps after delivering a sharp blow to their head (Wuxi Ehu Agriculture Inc., Wuxi, Jiangsu, China) between March and July. The eggs were stored in −20 °C for a maximum of 4 wk.
Extraction of glycoprotein from crucian carp eggs
Crucian carp eggs (300 g) were added to
Isolation and purification of CcSGP
Fish eggs generally contain ovalbumin, ovoglobulin, and ovomucoid, most of which are modified with glycans. Herein, the lipid portion was removed to elucidate the glycan's structure of the glycoprotein from crucian carps. The recovery ratios of protein, total sugar, and sialic acid were 58.2, 36.0, and 72.0% (w/w), respectively. As shown in Fig. 1, the molecular weights of most of the crude proteins from crucian carp eggs were 100, 27, and 22 kDa, consistent with a previous report (Xia et al.,
Conclusions
CcSGP had molecular weights of 97, 27, and 22 kDa and sialic acid content of ~2.6% (w/w). MADLI-TOF analysis showed that CcSGP primarily contained 6 glycan chain structures, namely, H6HexN4, H4HexN4S1, H5HexN2S2, H5HexN4S1-Ac, H5HexN4S1, and H5HexN5S1. Most N-glycans had double-antenna structure with many GalNAc at the terminal of the glycan chain. CcSGP could delay the precipitation of calcium phosphate and the scavenging activity of DPPH free radicals. It also promoted the proliferation of
CRediT authorship contribution statement
Ning Wang: Investigation, Writing - original draft. Zeling Yang: Data curation. Jianrong Wu: Conceptualization, Writing, Supervision, Writing - review & editing. Yun Jiang: Project administration. Hongtao Zhang: Methodology. Xiaobei Zhan: Funding acquisition.
Declaration of competing interest
The authors declared that they have no conflicts of interest to this work.
Acknowledgments
This work was supported by the National Science and Technology Major Project (No. 2017YFD0400302), the Program of Introducing Talents of Discipline to Universities (111-2-06), the Fundamental Research Funds for the Central Universities (JUSRP51632A), and the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-17).
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