Glycosaminoglycans from earthworms (Eisenia andrei)

Abstract

The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC-ESI-MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.

Fig. 1

Picture of earthworm

Fig. 2

Profile of DEAE-Sepharose ion-exchange chromatography. No. 0–240 is the 0.0 M NaCl fraction, No. 241–480 is the 0.5 M NaCl fraction, No. 481–720 is the 1.0 M NaCl fraction and No. 721–960 is the 2.0 M NaCl fraction

Fig. 3

Agarose gel electrophoresis. Lane 1, chondroitin sulfate; lane 2, hyaluronan; lane 3, DS; lane 4, heparin; lane 5, HS; lane 6, blank; lane 7, 1.0 M NaCl fraction from DEAE Sepharose chromatography; lane 8, 2.0 M NaCl fraction from DEAE Sepharose chromatography. 25 μg of each sample was loaded and stained with Azure A

Fig. 4

Disaccharide compositional analysis of CS by LC-ESI-MS. a CS/DS disaccharide standards, b EIC of CS/DS disaccharide analysis, c UV (232 nm) detection of CS/DS disaccharide analysis, d MS spectrum of 0S, ΔUA-GalNAc, e MS spectrum of 4S, ΔUA-GalNAc4S, f MS spectrum of 6S, ΔUA-GalNAc6S, and g MS spectrum of 2S4S, ΔUA2S-GalNAc4S

Fig. 5

Disaccharide compositional analysis of HS by LC-ESI-MS. a EIC of heparin/HS disaccharides, b MS spectrum of NS, ΔUA-GlcNS, c MS spectrum of NS6S, ΔUA-GlcNS6S, and d MS spectrum of NS2S6S, ΔUA2S-GlcNS6S