A method for fluorescent HPLC analysis of CS/DS glycosaminoglycans

Lauder, R (2004) A method for fluorescent HPLC analysis of CS/DS glycosaminoglycans. International Journal of Experimental Pathology, 85 (4). A70-A70. ISSN 1365-2613

Abstract

Introduction Chondroitin and dermatan sulfate (CS/DS) show considerable species, tissue, age and pathology-related structural heterogeneity. In addition within chains their sulfation patterns are not random. To elucidate their structure/function relationships, methods for complete characterization are required. A method for keratan sulfate (KS) fingerprinting (Whitham et al. 1999) has been extended for the linkage, repeat and chain cap regions of CS/DS, including the acquisition of CS/DS ratios. Methods Chains were depolymerized by 1 U/100 mg of chondroitin ABC endolyase or ACII lyase at 50 mg/ml in 0.1 m ammonium acetate, pH 8 and for 15 h at 37 °C. Alternatively, chains were de-N-acetylated by hydrazinolysis at 98 °C for 24 h at 10 mg/1 ml in anhydrous hydrazine with 100 mg/ml hydrazine sulfate. Then, they were depolymerized by 3.9 m sodium nitrite/0.28 m acetic acid at 0 °C for 4 h. Unreduced chains were released from their protein core in 0.5 m LiOH at 4 °C for 12 h. Materials were fluorescently labelled with 2-AA as previously described (Whitham et al. 1999) and characterized by HPAEC using a Dionex AS4-SC column at 50 °C and 2 ml/min with constant 15% 1 m NaOH. A 5-min isocratic period of 85% H2O/0% 2 m NaCl was followed by a linear gradient of 0–30% 2 m NaCl over 60 min. The oligosaccharides were monitored using a λex of 315 nm and a λem of 400 nm. Results and discussion This method resolves repeat region di-, tri- and tetrasaccharides, capping oligosaccharides and linkage regions and can be used to profile known and unknown oligosaccharides. Unsulfated oligosaccharides elute between 2 and 10 min, monosulfated between 7 and 30 min, disulfated between 25 and 40 min and trisulfated between 49 and 54 min. Allied with data on size, oligosaccharide identification is facilitated. Hydrazinolysis/nitrous acid depolymerization of CS/DS chains results in disaccharides from CS with 4- or 6-sulfation and from DS with 4-sulfation which retain IdoA and GlcA structures and which can be distinguished chromatographically. The methodology was used to examine CS/DS from shark, whale, bovine and human tracheal, articular and meniscal cartilage and cornea. Tracheal cartilages show predominantly 4-sulfation with porcine sources being more highly 4-sulfated (ca. 75%) than bovine (ca. 65%). Articular cartilage comprises mainly 6-sulfated GalNAc (ca. 95% in the adult), while adult meniscal cartilage shows only ca. 85%. Tracheal and articular cartilage aggrecan showed no IdoA; however, it represented ca. 20% of the uronic acids of bovine meniscal aggrecan, showing the presence of DS. Corneal CS/DS has a very low level of 6-sulfation (<ca. 5%) but shows an equal abundance of unsulfated and 4-sulfated residues and contains high levels, ca. 50%, of IdoA residues. Shark cartilage shows ca. 75% 6-sulfation with significant levels of uronic acid 2-sulfation found only between a 4-sulfated residue and a 6-sulfated residue, reflecting sulfotransferase specificity. Shark cartilage contains modest (ca. 1–5%) levels of DS that may be contaminants of preliminary isolation. This method extends a previous method to now allow the complete examination of KS, CS and DS chains by a single rapid chromatographic method.