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- Results and Discussion
- Experimental Procedures
Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: ΔUA(β1–3)GalNAc4S(β1–4)l-IdoA(α1–3)GalNAc4S(β1–4)l-IdoA(α1–3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(β1–4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: ΔUA(β1–3)GalNAc4S(β1–4)GlcA-ol and ΔUA(β1–3)GalNAc6S(β1–4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S-ol and GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.
There is considerable interest in the detailed molecular structure [1,2] and function  of glycosaminoglycans (GAGs) including chondroitin and dermatan sulfate (CS/DS) [4–6]. These structurally diverse polymers are abundant components of extracellular matrices and cell surfaces in humans and other mammals. Data are emerging that show roles for CS/DS in a variety of fundamental biological processes including neurite outgrowth , disease development  and growth factor binding . CS has also been found in invertebrates [10–12] including Drosophila melanogaster and Caenorhabditis elegans, where it has been shown to have fundamental roles in development .
CS/DS chains comprise a linkage region, a chain cap and a repeat region [4,5]. The repeat region of CS is a repeating disaccharide of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) [-4)GlcA(β1–3)GalNAc(β1-]n, which may be O-sulfated on the C4 and/or C6 of GalNAc and C2 of GlcA. GlcA residues of CS may be epimerized to iduronic acid (IdoA) forming the repeating disaccharide [-4)IdoA(α1–3)GalNAc(β1-]n of DS. Thus, CS and DS may be found as pure polymers or a mixed copolymer in which the DS residues and GalNAc sulfation isoforms may be located together in large blocks or distributed throughout the chain. These will have very different effects on molecular interactions and biological function.
Both the concentrations and locations of sulfate ester substituents vary with GAG source [4,16]. For example, in adult human articular cartilage, extensive 6-O-sulfation of GalNAc is observed (≈ 95%) . In shark cartilage, lower levels of 6-O-sulfation are found (≈ 70%), with 4-O-sulfation making up the balance along with ≈ 25% 2-sulfation of the uronic acid residues , and in tracheal cartilage lower levels of 6-O-sulfation are found (≈ 20–40%), with the balance being mainly GalNAc 4-O-sulfation. In D. melanogaster, 4-O-sulfation, but not 6-O-sulfation, is observed whereas in C. elegans the chondroitin is unsulfated [13,14].
Within a tissue, the sulfation profile and levels of epimerization of GlcA to IdoA change with age. For example, the level of GalNAc 6-O-sulfation reported above for human articular cartilage applies only to the adult; at birth this level is close to zero but rises significantly during the first 20 years of life [5,17].
Not only does CS/DS structure change with tissue source and age, but within a single chain there is variability. The chain cap of CS is a GalNAc or GlcA residue; a 4,6-disulfated GalNAc residue, rare in the repeat region of human articular cartilage CS, represents over 50% of the chain caps for a normal adult, but only ≈ 30% at the termini of CS chains from osteoarthritic cartilage [18,19]. Whereas the CS chain caps may be highly sulfated, the linkage regions, via which these pendant polymers are attached to a protein core, have been shown to exhibit low levels of sulfation relative to residues within the repeat region [5,6,20], with preferential localization of unsulfated and 4-O-sulfated GalNAc residues at linkage regions [5,6,20].
The overall structure of CS chains is thus highly complex, showing significant variation in composition across materials from diverse tissue sources, from tissues of differing ages and also within a single chain. Enhanced availability of data to facilitate the characterization of these structures is therefore of value.
GAGs are not primary gene products and therefore their analysis cannot rely on genomic approaches; structural analysis requires their isolation followed by a complex characterization process. In our previous work we have used the paradigm of isolation and depolymerization of GAG chains to generate oligosaccharides, the structures of which are determined using NMR spectroscopy [6,16]. These oligosaccharides are then integrated into a chromatographic fingerprinting method which can be used for the analysis of biological samples .
Chondroitin lyase enzymes are eliminases which cleave the -3)GalNAc(β1–4)GlcA(β1-/IdoAα(1- bond in CS/DS in the case of chondroitin ABC lyase (EC 126.96.36.199), whereas chondroitin AC lyases act on CS alone. Chondroitin AC and ABC lyases generate disaccharides and tetrasaccharides  and have been widely used for the analysis of CS/DS composition. These studies have yielded crucial data allowing an understanding of species, tissue, age and pathology related differences and the estimation of changes in CS/DS abundance and composition. However, the reduction of the polymer to its individual disaccharide units removes any possible sequence data that would allow the reconstruction of biologically important functional motifs. In addition, the action of chondroitin lyase enzymes generates a 4,5-unsaturated hexuronic acid (ΔUA) from the uronic acid of the cleaved bond. Thus, the distinction between IdoA and GlcA, epimerization at C5, is lost and it is impossible to distinguish between disaccharides derived from DS and those derived from CS.
We have previously reported 1H-NMR data for disaccharides and tetrasaccharides from CS/DS , and Sugahara et al.  have examined, by 1H NMR and MS, a series of chondroitin ABC lyase-resistant fragments derived from CS or DS. Several of these were trisaccharides, including ΔUA(β1–3)GalNAc4,6diS(β1–4)GlcA, terminated by an unreduced GlcA ring, which could have been derived from polymer chain-reducing termini by a peeling reaction, or, through the action of a tissue endo-β-d-glucuronidase. A series of reduced and unreduced oligosaccharides obtained from DS were previously characterized by 1H and 13C NMR , including the trisaccharide GalNAc4S(β1–4)l-IdoA(α1–3)GalNAc4S-ol.
More recently, the preparation and structural characterization of unreduced DS oligosaccharides of up to dodecasaccharide in size has been discussed ; a combination of 1D and 2D NMR together with electrospray MS was employed.
The employment of nondestructive analytical methods in the characterization of GAGs is becoming more important now that full structural information for large domains is required as part of the examination of function for these species. Data from other GAG fragments has already proved valuable in the study of intact parent polymers; the architecture of keratan sulfate chains has been explored in this manner [24,25]. There have already been attempts to examine intact CS chains isolated from various sources. Considerable difficulties were met when specific assignments of structural components were sought. It is thus important that polymer characterizations should be facilitated through the availability of comprehensive parameters describing the structures of a wide range of oligosaccharide structures. The complete assignments of 1H and 13C NMR spectra from a series of disaccharides and tetrasaccharides derived from CS/DS chains have already been given . In this report we present 1H-NMR and some 13C-NMR data for trisaccharides and hexasaccharides from CS/DS. These have the structures shown below:
GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S-ol: CS#604 GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S-ol: CS#606 ΔUA(β1–3)GalNAc4S(β1–4)GlcA-ol: CS040# ΔUA(β1–3)GalNAc6S(β1–4)GlcA-ol: CS060# GalNAc6S(β1–4)GlcA-ol: CS#60# ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S-ol: CS060606 ΔUA(β1–3)GalNAc4S(β1–4)l-IdoA(α1–3)GalNAc4S(β1–4)l-IdoA(α1–3)GalNAc4S-ol: DS040404