A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

Abstract

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology, 4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to ∼0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di-and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sβ(1-3)GalNAc6S structure.

Increased incidence of unsulphated and 4-sulphated residues in the chondroitin sulphate linkage region observed by high-pH anion-exchange chromatography

“…significantly in advance of all monosulphated disaccharides [38], with only a slight retardation when compared with an unsulphated disaccharide. Oligosaccharides Octa-II, Octa-III, Hexa-II and Hexa-III are eluted very closely to each other, and also very closely to ∆UAβ(1-3)GalNAc6S-ol (∆di-6S), whereas Octa-IV, Octa-V and Hexa-IV are eluted very closely together at the start of a region in which a series of disulphated tetrasaccharides are eluted, but significantly in advance of a disulphated disaccharide.…”

“…saccharides (see Figure 4) [38]. The Carbopac PA1 column was initially calibrated with reduced repeat region di-and tetrasaccharides that were derived from mixed-whale and -shark CS by chondroitin ABC endolyase digestion, as described elsewhere [38].…”

“…The Carbopac PA1 column was initially calibrated with reduced repeat region di-and tetrasaccharides that were derived from mixed-whale and -shark CS by chondroitin ABC endolyase digestion, as described elsewhere [38]. The structure and purity of these standard oligosaccharides were confirmed by HPAEC analysis and both 1D and 2D "Hand "$C-NMR spectroscopy.…”

Glycoform Quantification of Chondroitin/Dermatan Sulfate Using a Liquid Chromatography−Tandem Mass Spectrometry Platform

“…In adult articular cartilage CS 6-sulfation is known to dominate over non-sulfated and 4-sulfated GalNAc residues (38). The level of IdoA sulfation is also known to be low (39). Previous research has shown that the GAG composition of aggrecan in normal human adult articular cartilage was predominantly Δdi6S while Δdi4S was low in all normal samples (40).…”

The Streptococcus equi prophage-encoded protein SEQ2045 is a hyaluronan-specific hyaluronate lyase that is produced during equine infection

“…Inhibition studies with ascorbic acid were performed using the standard reaction conditions, except that ascorbic acid was added to final concentrations of 2-20 mM. High-performance anion-exchange chromatography was performed according to the method of Lauder et al (2000).…”