Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: Production of well-defined hexasaccharides

Abstract

Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.

Following the enzymatic digestion of chondroitin sulfate by a simple GPC analysis

“…Usually, a separation of the fraction with high Mw from the oligosaccharides is performed in the beginning. Oligosaccharides are then separated by anion exchange chromatography and MS is used to determine the Mw of each isolated component [9,16,17]. Herein, we propose the use of single measurement with GPC that gives information about Mw and concentration of all species (low and high Mw) present in the mixture.…”

“…Indeed, the protocols for characterization of CS oligosaccharides obtained via enzymatic digestion in vivo or in vitro usually include separation of the obtained oligosaccharides by gel permeation chromatography (GPC, also known as size-exclusion chromatography, SEC) or anion exchange HPLC and characterization of the individual oligosaccharides by nuclear magnetic resonance (NMR) and mass spectrometry (MS) to elucidate their structures [9]. One general disadvantage of these protocols is that they do not allow real time measurement of the molecular weight of the digested species.…”

Unique Properties of Human β-Defensin 6 (hBD6) and Glycosaminoglycan Complex

“…Fondaparinux was purchased from GlaxoSmithKline. The heparin-derived octasaccharide (degree of polymerization, dp8) was prepared by size exclusion chromatography (SEC) of a commercially available enoxaparin sample applied to a Bio-gel P10 column as described elsewhere (25).…”

Unravelling Glycobiology by NMR Spectroscopy

“…These initial assignments are crucial to further inform the chemical shift changes induced by the presence of increasing concentration of the ligants. The ligants used were two chondroitin sulfate hexasaccharides fully characterized by NMR [34]. They are named CS 6-6-4, and CS 4-4-4, which represent the following respective structures: ΔUA( 13)GalNAc6S( 14)GlcA( 13)GalNAc6S( 14)GlcA( 13)GalNAc4S-ol, and GlcA( 13)GalNAc4S( 14)GlcA( 13)GalNAc4S( 14)GlcA( 13)GalNAc4S-ol.…”

“…They are named CS 6-6-4, and CS 4-4-4, which represent the following respective structures: ΔUA( 13)GalNAc6S( 14)GlcA( 13)GalNAc6S( 14)GlcA( 13)GalNAc4S-ol, and GlcA( 13)GalNAc4S( 14)GlcA( 13)GalNAc4S( 14)GlcA( 13)GalNAc4S-ol. The abbreviations are ΔUA for Δ 4,5 unsaturated uronic acid; GalNAc for N-acetyl galactosamine; GlcA for glucuronic acid; "S" for sulfation group, digits before "S" represent the ring position; and -ol stands for reduced sugars (open rings at the reducing-end terminal units) [34]. As the concentration of the chondroitin sulfate ligants increases, either loss of signal intensity or chemical shift migration were observed on the 15 N-HSQC spectra of E66S CCL5 ( Figure 13).…”