Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

Abstract

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: -[3GlcAbeta1-4(GalNAcalpha1-3)Fucalpha1](n)-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique (0,2)A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position-3 of the disaccharide repeat.

Transportation of squid ink polysaccharide SIP through intestinal epithelial cells and its utilization in the gastrointestinal tract

“…SIP was prepared as previously reported (Chen et al, 2008). Briefly, after squid ink of O. bartrami was acidified to pH 4.0-5.0 with 0.1 M HCl and the solution stood for 24 h at 4°C to precipitate melanin, melanin was removed by centrifugation at 5000 × g for 1 h. Then melanin-free ink was digested with 2 volumes of 1% (w/v) papain in Tris-HCl buffer (50 mM, pH 6.8) containing 5 mM Cys and 5 mM ethylenediaminetetraacetic acid (EDTA) at 60°C for 24 h. Digestion was repeated twice to ensure the cleavage of the protein/peptide moiety.…”

“…NMR analysis was performed as previously reported (Chen et al, 2008). Briefly, SIP (20 mg) and FTSC-SIP (20 mg) were coevaporated with D2O (99.8%) twice by lyophilization before final dissolution in 500 µL high-quality D2O (99.96%), containing 0.1 mL lacetone.…”

“…As one ingredient in squid, squid ink is nowadays becoming more and more popularly used in food in Asian countries, mostly in forms of noodles and dumplings; it is necessary to elucidate the functional components of the squid ink. Our previous studies have identified a polysaccharide SIP from squid ink of O. bartrami, with a chemical structure -[3GlcAβ1-4(GalNAcα1-3)Fucα1]n-, 55 kD (Chen et al, 2008). It was demonstrated that the SIP has a wide range of functions, such as promoting intestinal secretion of secretory Immunoglobulin A (SIgA) (Zuo et al, 2014); protecting intestinal epithelial lineages, Paneth cells and goblet cells (Zuo, Cao, Xue, & Tang, 2015;Zuo, He, Cao, Xue, & Tang, 2015); up-regulating epithelial tight junctions (Zuo et al, 2015); and even modulating gut microbiome (Lu et al, 2016).…”

Interventional effects of squid ink polysaccharides on cyclophosphamide-associated testicular damage in mice

“…SIP is a type of glycosaminoglycon with a unique structure Fig. 1) (11,12) and it has been discovered that the bioactive polysaccharides could increase antioxidant capabilities of some tissues including the liver, heart, lungs, and kidneys of model animals exposed to CP (13), which implies that SIP protects normal tissues of model animals from damage induced by the chemotherapeutic drug. Especially, our latest preliminary fi ndings revealed intervention of SIP on CP-induced spermatogenetic damage in mice, the investigated parameters including sperm density, survival rate, abnormality rate, as well as superoxide dismutase activity in testis indicated that SIP could protect the testes from chemotherapeutic damage caused by CP (14,15) and that the marine polysaccharides could be potentially developed as a cytoprotector.…”

Preparation of water-soluble melanin from squid ink using ultrasound-assisted degradation and its anti-oxidant activity

“…The squid ink has been reported to have anti-radiation activity, antitumor activity, immunomodulatory activity, procoagulant function and so on (Takaya 2000). Several bioactive molecules, including a glycosaminoglycan like polysaccharides (Chen et al 2008), a tyrosinase (Naraoka et al 2003) and an angiotensin-converting enzyme inhibitor (Kim et al 2003) have been identified from squid ink. Among them, the melanin has attracted most research interests.…”