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Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis   

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Original research article

A brief version of this protocol appeared in:
Biochemical Journal
Sep 2016

Abstract

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), heparin (HP), heparan sulfate (HS) and keratan sulfate (KS) are linear, sulfated repeating disaccharide sequences containing hexosamine and uronic acid (or galactose in the case of KS). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. Here, we report the isolation protocol for KS-branched CS from M. chinensis.

Keywords: Mactra chinensis, Glycosaminoglycan, Chondroitin sulfate, Keratan sulfate, Galactose

Background

GAGs are found in tissues as the glycan moieties of proteoglycan (PG) glycoconjugates. CS is a GAG type composed of linear, sulfated repeating disaccharide sequences of N-acetyl-D-galactosamine (GalNAc) and glucuronic acid (GlcA). Another GAG type, DS, is biosynthesized through the action of glucuronyl C5-epimerase on CS, converting its GlcA to the CS epimer, iduronic acid (IdoA). The other GAG types HS and HEP are consisted of sulfated repeating disaccharide sequence of N-acetyl-D-glucosamine (GlcNAc) and GlcA/IdoA. KS is composed of sulfated repeating disaccharide sequences of GlcNAc and galactose. Among them, structural variations of CS, such as sulfation patterns and fucosylation, depend on the species and tissue of origin. For example, the A-unit with the structure [-4)GlcA(β1-3)GalNAc4S(β1-] (where S designates a sulfonate residue) is a predominant disaccharide found in mammalian or chicken tracheal cartilage CS, while the C-unit with the structure [-4)GlcA(β1-3)GalNAc6S(β1-] is a major disaccharide found in shark cartilage or salmon nasal cartilage CS. In contrast, a significant amount of the D-unit disaccharide [-4)GlcA2S(β1-3)GalNAc6S(β1-] is characteristically found in CS isolated from shark cartilage, while the E-unit disaccharide [-4)GlcA(β1-3)GalNAc4S,6S(β1-] is characteristic in squid cartilage. In sea cucumber, a sulfated fucose branches at the 3-OH position of GlcA. The disaccharide composition of CS governs its biological activities, including cell proliferation, migration, differentiation, cell-cell crosstalk, adhesion and wound repair through the interaction with growth factors, receptors, and other CS-binding proteins. Evidence suggests that CS structure is tightly correlated with function. For example, consecutive and disulfated disaccharide units including B, D and E-units in CS are critical for the interaction between CS and binding proteins (Hikino et al., 2003). A branched fucose at the 3-OH position of GlcA is also required for the anticoagulant activity of fucosylated CS (Mourão et al., 1996).

In general, isolation of GAGs is carried out as follows. 1) acetone defatting, 2) proteolysis, 3) collection of the GAGs, 4) fractionation of GAGs by anion-exchange chromatography and 5) desalting (Maccari et al., 2015). In our protocol, actinase E from Streptomyces griseus (step 2) and cetylpyridinium chloride precipitation (step 3) were used for the isolation of GAGs.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Higashi, K. and Toida, T. (2017). Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis. Bio-protocol 7(15): e2441. DOI: 10.21769/BioProtoc.2441.
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