ABSTRACT
The chondroitin sulphate (CS) linkage regions have been isolated from human articular cartilage aggrecan (from 10- to 72-year-olds) by chondroitin ABC endolyase digestion and size-exclusion chromatography. Linkage region hexasaccharides have been characterized and their abundance estimated by high-pH anion-exchange chromatography. The basic structure for the CS linkage region oligosaccharides identified from human aggrecan is as follows: DeltaUA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)Gal[0S/6S](beta1-3)Gal(beta1-4)Xyl, where DeltaUA represents 4,5-unsaturated hexuronic acid, 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively, and 0S represents zero sulphation. There are significant age-related changes in the abundance of the various N-acetylgalactosamine (GalNAc) sulphation forms identified, occurring up to approx. 20 years old. During the period from 10 to 20 years old the level of GalNAc 6-sulphation at the linkage region increases from approx. 43% to approx. 75%, while there is a corresponding reduction in unsulphated (approx. 30% to approx. 20%) and 4-sulphated (approx. 25% to approx. 6%) GalNAc residues. There is also an increase in the incidence of linkage region galactose 6-sulphation (approx. 2% to approx. 10%) which was only observed in linkage regions with GalNAc 6-sulphation. Beyond 20 years old there are few changes in the relative abundance of these GalNAc sulphation variants; however, there is a slight increase in the abundance of 6-sulphation between approx. 20 years old and approx. 40 years old and a slight decrease in its abundance beyond approx. 40 years old. Our data show that in the majority of chains from tissues of all ages the GalNAc residue closest to the linkage region is 6-sulphated, but the level of GalNAc 6-sulphation within the linkage region is lower than the average level observed within the repeat region.
Subject(s)
Aging , Cartilage, Articular/physiology , Chondroitin Sulfates/chemistry , Extracellular Matrix Proteins , Proteoglycans/chemistry , Adolescent , Adult , Aged , Aggrecans , Carbohydrate Sequence , Child , Chromatography, Ion Exchange , Humans , Lectins, C-Type , Magnetic Resonance Spectroscopy , Middle Aged , Molecular Sequence DataABSTRACT
Chondroitin sulfates were fragmented using the enzymes chondroitin sulfate ABC endolyase and chondroitin ACII lyase; both disaccharide and tetrasaccharide fragments were isolated after reduction to the corresponding 2-deoxy-2-N-acetylamino-D-galactitol (GalNAc-ol) form. These have the structures: Delta UA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S-ol, Delta UA2S(beta 1--3)GalNAc6S-ol, Delta UA(beta 1--3)GalNAc4S(beta 1--4)L-IdoA(alpha 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc4S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc6S-ol, Delta UA2S(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol and Delta UA2S(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc6S-ol, where Delta UA represents a 4,5-unsaturated hexuronic acid (4-deoxy-alpha-Lthreo-hex-4-enepyranosyluronic acid) and 6S/4S/2S represent O-ester sulfate groups at C6/C4/C2 sites. Complete (1)H-NMR and (13)C-NMR data are derived for these species, which may help to alleviate some of the significant difficulties resulting from signal complexity that are currently hindering the characterization and assignment of major and minor structural components within chondroitin sulfate and dermatan sulfate polymers.
Subject(s)
Chondroitin Sulfates/chemistry , Dermatan Sulfate/chemistry , Oligosaccharides/analysis , Oligosaccharides/chemistry , Animals , Carbon , Cartilage , Cattle , Chondroitin ABC Lyase/metabolism , Chondroitin Lyases/metabolism , Chondroitin Sulfates/metabolism , Dermatan Sulfate/metabolism , Disaccharides/analysis , Disaccharides/chemistry , Disaccharides/metabolism , Hydrogen , Magnetic Resonance Spectroscopy , Oligosaccharides/metabolism , ProtonsABSTRACT
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 approximately 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 GlcA2Sbeta(1-3)GalNAc6S structure.
Subject(s)
Chondroitin Sulfates/analysis , Chromatography, Ion Exchange/methods , Dermatan Sulfate/analysis , Hyaluronic Acid/analysis , Oligosaccharides/analysis , Animals , Anions , Carbohydrate Sequence , Cartilage/chemistry , Cattle , Chondroitin Lyases/metabolism , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Sharks , WhalesABSTRACT
We report the isolation, characterization and quantification of five octasaccharides, four hexasaccharides and two tetrasaccharides, derived from the chondroitin sulphate (CS) linkage region of 6-8-year-old bovine articular cartilage aggrecan, following digestion with chondroitin ABC endolyase. Using a novel high-pH anion-exchange chromatography (HPAEC) method, in conjunction with one- and two-dimensional (1)H-NMR spectroscopy, we have identified the following basic structure for the CS linkage region of aggrecan: DeltaUA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)Gal[0S/6S](beta1-3)Gal(beta1-4)Xyl, where DeltaUA represents 4,5-unsaturated hexuronic acid, and 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively. The octa-, hexa- and tetra-saccharide linkage region fragments were used to develop a HPAEC fingerprinting method, with detection at A(232 nm), and a linear response to approx. 0.1 nmol of substance. The sulphation patterns of CS linkage regions, of up to octasaccharide in size, from articular and tracheal cartilage aggrecan were examined. The results show that in articular cartilage, for the majority (53%) of octasaccharides the 2-deoxy-2-N-acetyl amino-D-galactose (GalNAc) residues closest to the linkage region are both 6-sulphated; however, in a significant portion (34%), one or more of these GalNAc residues are unsulphated, and in 8% both are unsulphated. Approximately 10-18% of the chains have a 4-sulphated GalNAc in the first disaccharide, and 12% have a sulphated linkage region Gal residue. No evidence was found for uronic acid sulphation. These data show that there is a significant increase in the incidence of unsulphated and 4-sulphated GalNAc residues adjacent to the linkage region compared with the rest of the chain. Bovine tracheal cartilage linkage regions displayed very similar sulphation profiles to those from articular cartilage, despite the presence of a higher level of GalNAc 4-sulphation within the repeat region of the main CS chain.
Subject(s)
Chondroitin Sulfates/chemistry , Chondroitin Sulfates/metabolism , Extracellular Matrix Proteins , Proteoglycans/chemistry , Proteoglycans/metabolism , Aggrecans , Animals , Anions , Carbohydrate Sequence , Cattle , Chondroitin ABC Lyase/metabolism , Chondroitin Sulfate Proteoglycans/chemistry , Chondroitin Sulfate Proteoglycans/metabolism , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Lectins, C-Type , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptide MappingABSTRACT
Keratan sulfate (KS) proteoglycans are of importance for the maintenance of corneal transparency as evidenced in the condition macular corneal dystrophy type I (MCD I), a disorder involving the absence of KS sulfation, in which the cornea becomes opaque. In this transmission electron microscope study quantitative immuno- and histochemical methods have been used to examine a normal and MCD I cornea. The monoclonal antibody, 5-D-4, has been used to localize sulfated KS and the lectin Erythrina cristagalli agglutinin (ECA) to localize poly N -acetyllactosamine (unsulfated KS). In normal cornea high levels of sulfated KS were detected in the stroma, Bowman's layer, and Descemet's membrane and low levels in the keratocytes, epithelium and endothelium. Furthermore, in normal cornea, negligible levels of labeling were found for N -acetyllactosamine (unsulfated KS). In the MCD I cornea sulfated KS was not detected anywhere, but a specific distribution of N -acetyllactosamine (unsulfated KS) was evident: deposits found in the stroma, keratocytes, and endothelium labeled heavily as did the disrupted posterior region of Descemet's membrane. However, the actual cytoplasm of cells and the undisrupted regions of stroma revealed low levels of labeling. In conclusion, little or no unsulfated KS is present in normal cornea, but in MCD I cornea the abnormal unsulfated KS was localized in deposits and did not associate with the collagen fibrils of the corneal stroma. This study has also shown that ECA is an effective probe for unsulfated KS.
Subject(s)
Cornea/pathology , Corneal Dystrophies, Hereditary/pathology , Keratan Sulfate/analysis , Adult , Cornea/metabolism , Cornea/ultrastructure , Corneal Dystrophies, Hereditary/metabolism , Humans , MaleABSTRACT
The use of high-field two-dimensional 1H-correlation data is described for the detailed comparison of intact keratan sulfate polymer chains derived from human articular cartilage sources as a function of age. For fetal material the nonreducing chain termini are shown to be sparsely capped by sialyl groups which, if present, are exclusively (alpha2-3)-linked to an unsulfated galactose residue. The asialo capping segment has the structure: Gal-GlcNAc6S-Gal-GlcNAc6S-. Examination of keratan sulfate from 10-year-old cartilage shows that capping by sialyl groups is complete, with (alpha2-3)-linkages predominant; for both this and the 38-year-old cartilage the three capping structures: NeuAc(alpha2-3)-Gal-GlcNAc6S-Gal-GlcNAc6S-, NeuAc(alpha2-3)-Gal-GlcNAc6S-Gal6S-GlcNAc6S-, and NeuAc(alpha2-3)-Gal6S-GlcNAc6S-Gal6S-GlcNAc6S- are clearly recognizable. The level of (alpha2-6)-linked chain capping sialyl groups is significant for 38-year-old cartilage keratan sulfate. Structural information concerning the linkage region to protein and the distribution of galactose environments is readily obtained from the spectra. Signal complexities severely limit the usefulness of two-dimensional correlation spectroscopy at 600 MHz for the examination of N-acetylglucosamine residues within the poly(N-acetyllactosamine) repeat sequence and signals representing fucose placements remain undifferentiated. This nondestructive approach complements current degradative methods for the structural examination of keratan sulfates.
Subject(s)
Cartilage, Articular/chemistry , Keratan Sulfate/chemistry , Adult , Carbohydrate Conformation , Carbohydrate Sequence , Child , Fetus/chemistry , Humans , Keratan Sulfate/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Sequence DataABSTRACT
Proteoglycans are of fundamental importance to the normal functioning of the cornea. They consist of a core protein to which one or more glycosaminoglycan chains are attached. Cell surface proteoglycans are known to mediate many aspects of cell behaviour including cell adhesion, control of extracellular matrix deposition, cell proliferation, cell migration, leukocyte adhesion and modulation of growth factor activity. This paper describes the first investigation into the distribution and function of the three main classes of proteoglycans on human corneal endothelium. Immuno-gold labelling techniques were used at the light, scanning and transmission electron microscope level to localise heparan sulphate, chondroitin sulphate and keratan sulphate proteoglycans on human corneal endothelium. Human corneas were freeze-wounded and kept in organ culture for 3 days in order to study the distribution of proteoglycans on migrating corneal endothelium. An Optimas image analysis system was used to quantify the change in proteoglycan labelling during cell migration. Labelling for chondroitin sulphate and heparan sulphate was at very low levels on normal corneal endothelium while keratan sulphate labelling was at high levels. The wound healing experiments showed that migrating cells had increased labelling for heparan sulphate and chondroitin sulphate with greatly decreased labelling for keratan sulphate. Statistical analysis showed these changes were highly significant (P<0.001). Transmission electron microscopy revealed that chondroitin sulphate and keratan sulphate were present throughout Descemet's membrane while heparan sulphate was concentrated at the interface of Descemet's membrane and the migrating corneal endothelial cells. The pattern of occurrence of chondroitin sulphate, heparan sulphate and keratan sulphate on the human endothelium in normal and wounded cornea suggests that these proteoglycans are linked to the process of cell migration.
Subject(s)
Epithelium, Corneal/metabolism , Proteoglycans/analysis , Cell Movement , Chondroitin Sulfates/analysis , Epithelium, Corneal/injuries , Epithelium, Corneal/physiology , Heparitin Sulfate/analysis , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Keratan Sulfate/analysis , Microscopy, Electron, Scanning , Wound HealingABSTRACT
A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311-317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram ( approximately 100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed.
Subject(s)
Brain Neoplasms/chemistry , Chromatography, Liquid/methods , Keratan Sulfate/chemistry , Oligosaccharides/chemistry , Ovarian Neoplasms/chemistry , Acetylglucosaminidase/metabolism , Animals , Carbohydrate Sequence , Cattle , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Female , Fluorometry , Humans , Molecular Sequence Data , Oligosaccharides/metabolism , Swine , ortho-AminobenzoatesABSTRACT
Alkaline borohydride-reduced keratan sulfate chains were isolated from human articular cartilage aggrecan from individuals of various ages (0-85 years old). The chains were structurally characterized using 1H NMR spectroscopy, gel permeation chromatography, and oligosaccharide profiling (after digestion with the enzymes keratanase and keratanase II). The results show that from birth to early adolescence (0-9 years) the levels of alpha(1-3)-fucosylation, alpha(2-3)-sialylation, and galactose sulfation increase. Also, the weight-average molecular weight of the chains increases. During maturation (9-18 years) the levels of fucosylation and galactose sulfation continue to increase and alpha(2-6)-sialylation of the chains occurs. In adult life (18-85 years) there is little change in the weight-average molecular weight of the chains, and the levels of fucosylation, sialylation, and sulfation remain fairly constant.
Subject(s)
Extracellular Matrix Proteins , Keratan Sulfate/chemistry , Proteoglycans/chemistry , Acetylglucosaminidase/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Aggrecans , Carbohydrate Conformation , Carbohydrate Sequence , Fucose/metabolism , Humans , Hydrolysis , Keratan Sulfate/metabolism , Lectins, C-Type , Magnetic Resonance Spectroscopy , Middle Aged , Molecular Sequence Data , N-Acetylneuraminic Acid/metabolism , Proteoglycans/metabolismABSTRACT
Bovine corneal keratan sulphate has been fragmented using the enzyme endo-beta)-galactosidase and 1H-NMR chemical shift data are reported for five newly isolated tetrasaccharides which derived from the repeat region. They have the structures: GlcNAc(beta1-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal-ol, GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal-ol, GlcNAc(beta1-3)Gal6S(beta1-4)GlcNAc6S(beta1-3)Gal-ol, GlcNAc6S(beta1-3)Gal6S(beta1-4)GlcNAc(beta1-3)Gal-ol, and GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal-ol. Structures for two trisaccharides formed by a peeling reaction are also given. These are GlcNAc(beta1-3)Gal6S(beta1-4)GlcNAc-ol and GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6S-ol where GlcNAc6S-ol represents N-acetylglucosaminitol 6-O-sulphate. Characterisation of such structures and their spectral assignments will be of considerable value for the studies of both selectin ligands and undersulphated keratan sulphates such as those occurring on cell surfaces and in brain tissue.
Subject(s)
Acetylglucosamine/chemistry , Cornea/chemistry , Glycoside Hydrolases , Keratan Sulfate/metabolism , Oligosaccharides/chemistry , beta-Galactosidase/metabolism , Acetylglucosamine/metabolism , Amino Sugars/chemistry , Animals , Cattle , Glycosaminoglycans/chemistry , Keratan Sulfate/chemistry , Magnetic Resonance Spectroscopy , Polysaccharides/chemistryABSTRACT
PURPOSE: We report an investigation into the distribution of proteoglycans (PGs) in normal, organ-cultured and dextran-treated human corneas. METHODS: Immunogold labeling was carried out at the electron microscope level to localize keratan sulphate (KS), chondroitin sulphate (CS), and heparan sulphate (HS) PGs. RESULTS: High levels of labeling for CS was found in the epithelium, endothelium, and keratocytes, with light labelling present in the basement membranes and the corneal stroma. Labeling for HS was present in the epithelium, endothelium, and keratocytes, with intense labeling present at the endothelium/Descemet's membrane interface and the epithelium/Bowman's layer interface. Large filaments were also observed in these regions in cuprolinic blue-stained specimens. Keratan sulphate was present at high levels in the stroma and the basement membranes with low levels present within the keratocytes, epithelium, and endothelium. The pattern of KS labeling along the collagen fibrils in the stroma sometimes showed evidence of periodicity. Organ-cultured corneas had extensive collagen-free "lakes," the interior of which immunolabeled positively for KS and showed staining with cuprolinic blue. The lakes were greatly reduced in the dextran-treated samples. CONCLUSION: This investigation determined the ultrastructural distribution of KS, CS, and HS PGs in human cornea and showed that organ culture is associated with a change in distribution of stromal PGs.
Subject(s)
Chondroitin Sulfates/ultrastructure , Cornea/ultrastructure , Heparitin Sulfate/ultrastructure , Keratan Sulfate/ultrastructure , Chondroitin Sulfates/metabolism , Cornea/metabolism , Dextrans/pharmacology , Heparitin Sulfate/metabolism , Humans , Keratan Sulfate/metabolism , Microscopy, Immunoelectron , Organ Culture TechniquesABSTRACT
Bovine articular cartilage fibromodulin has been isolated from animals aged 3 months to 8 years, and the attached keratan sulphate (KS) chains digested with keratanase II. The oligosaccharides generated have been reduced, examined by high-pH anion-exchange chromatography and their structures identified by comparison with standards. It has been shown that in fibromodulin from young articular cartilage, the KS chains do not possess either non-reducing terminal (alpha2-6)-linked N-acetylneuraminic acid or fucose (alpha1-3)-linked to sulphated N-acetylglucosamine residues. However, an age-related increase has been observed in the abundance of both (alpha2-6)-linked N-acetylneuraminic acid and (alpha1-3)-linked fucose, neither of which is found in KS isolated from non-articular cartilage, irrespective of the age of the source. Interestingly, the KS chain length remains constant as a function of age, which possibly relates to a role in collagen fibril assembly. In addition, no significant age-related changes were identified in levels of galactose sulphation.
Subject(s)
Aging/metabolism , Carrier Proteins/metabolism , Cartilage, Articular/metabolism , Extracellular Matrix Proteins , Keratan Sulfate/metabolism , Proteoglycans , Acetylglucosaminidase/metabolism , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cattle , Chromatography, Ion Exchange , Fibromodulin , Hydrogen-Ion Concentration , Molecular Sequence Data , Oligosaccharides/chemistryABSTRACT
Skeletal keratan sulphate has been fragmented using the enzyme keratanase II, and 13C chemical-shift data are reported for five reduced sialylated pentasaccharides that derived from the non-reducing chain terminal region. They have the structures: NeuAc(alpha2-6)Gal(beta1-4)GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6 S-ol, NeuAc(alpha2-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6 S-ol, NeuAc(alpha2-6)Gal(beta1-4)GlcNAc6S(beta1-3)Gal6S(beta1-4)++ +GlcNAc6S-ol, NeuAc(alpha2-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal(6S)(beta1-4)Glc NAc6S-ol, and NeuAc(alpha2-3)Gal(6S)(beta1-4)GlcNAc6S(beta1-3)Gal(6S)(beta1-4)++ +GlcNAc6S-ol, where GlcNAc6S-ol represents N-acetyl-glucosaminitol 6-O-sulphate and NeuAc represents N-acetylneuraminic acid. The use of these 13C-NMR spectroscopy data for the recognition of specific chain-capping structures within native keratan sulphates is discussed. In addition, examination of the data derived from the NeuAc(alpha2-6) capping structures strongly suggests that sulphation three residues away from the neuraminic acid cap has a profound effect upon the conformation of the capping region.
Subject(s)
Acetylglucosaminidase/metabolism , Cartilage, Articular/chemistry , Keratan Sulfate/chemistry , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Carbon Isotopes , Cattle , Keratan Sulfate/isolation & purification , Keratan Sulfate/metabolism , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Oligosaccharides/chemistryABSTRACT
Five octasaccharides derived from the protein carbohydrate linkage region of chondroitin sulphate (CS) have been isolated from the large aggregating proteoglycan (aggrecan) extracted from the bovine articular cartilage of 6-year-old to 8-year-old animals. Following the purification of aggrecan the attached CS chains were digested with CS ABC endolyase and subsequently released from the protein core by beta-elimination. The individual oligosaccharides were purified by strong anion-exchange chromatography and their structures determined by very high-field one-dimensional and two-dimensional 1H-NMR spectroscopy. They were found to be octasaccharides, comprised of tetrasaccharide repeat-region extensions to the core tetrasaccharide linkage region structure. They have the following structures: deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4)+ ++GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc6S(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol and deltaUA(beta1-3)GalNAc4S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol. They differ only in the nature of the sulphation of the GalNAc residues of the tetrasaccharide-repeat-region extension, which forms the first two disaccharides of the repeat region. No sulphation of any of the uronic acid residues has been identified and in one oligosaccharide neither of the GalNAc residues were sulphated. The majority of the linkage regions contained GalNAc residues which were fully 6-sulphated. However, in a significant amount, only one of the residues was 6-sulphated while the other was either unsulphated or 4-sulphated. There was no evidence either for sulphation of the linkage region galactose residues or for phosphorylation of the xylose residue, through which the chain is attached to the core protein.
Subject(s)
Cartilage, Articular/chemistry , Chondroitin Sulfates/chemistry , Extracellular Matrix Proteins , Oligosaccharides/chemistry , Proteoglycans/chemistry , Aggrecans , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cattle , Lectins, C-Type , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Sequence AnalysisABSTRACT
The keratan sulfate-containing proteoglycans were isolated from fourteen pooled human corneas (thirteen from 61- to 86-year-olds, plus one from a 12-year-old). These proteoglycans were subjected to digestion with the enzyme keratanase II, and the released oligosaccharides, which included nonreducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and identified on two separate ion-exchange columns. Both of the latter had been calibrated with samples, most of which had been derived from bovine corneal keratan sulfate (Tai, G.-H., Huckerby, T. N., and Nieduszynski, I. A. (1996) J. Biol. Chem. 271, 23535-23546) and all of which had been fully characterized by NMR spectroscopic analysis. The capping structures identified in human corneal keratan sulfates occurred in the relative proportions: NeuAcalpha(2-6)- >NeuAcalpha(2-3)- >GalNAc(S)beta(1-3)-. The other groups of capping structures which had been identified in bovine corneal keratan sulfate, i.e. NeuGcalpha(2-3)-, NeuGcalpha(2-6)-, GlcNAc(S)beta(1-3)- were absent, although the possibility of the presence of some Galalpha(1-3)- structures could not be excluded. In addition, the human sample showed significantly higher levels of alpha(1-3)-fucosylated repeat region structures than did the bovine sample, and it is not clear whether this reflects a species or age dependence as the bovine corneas were from young animals, whereas the human corneas were predominantly from an older group. The charge densities and keratan sulfate chain sizes of the human and bovine keratan sulfate-containing proteoglycans were seen to be similar.
Subject(s)
Cornea/metabolism , Keratan Sulfate/metabolism , Acetylglucosaminidase/metabolism , Aged , Aged, 80 and over , Animals , Carbohydrate Sequence , Cattle , Child , Chromatography, Ion Exchange , Humans , Middle Aged , Molecular Sequence Data , Molecular WeightABSTRACT
The small keratan sulphate proteoglycan, fibromodulin, has been isolated from pooled human articular cartilage. The main chain repeat region and the chain caps from the attached N-linked keratan sulphate chains have been fragmented by keratanase II digestion, and the oligosaccharides generated have been reduced and isolated. Their structures and abundance have been determined by high pH anion-exchange chromatography. These regions of the keratan sulphate from human articular cartilage fibromodulin have been found to have the following general structure: [structure: see text]. Significantly, both alpha(2-6)- and alpha(2-3)-linked N-acetyl-neuraminic acid have been found in the capping oligosaccharides. Fucose, which is alpha(1-3)-linked as a branch to N-acetylglucosamine, has also been found along the length of the repeat region and in the capping region. The chains, which have been found to be very highly sulphated, are short; the length of the repeat region and chain caps is ca. nine disaccharides. These data demonstrate that the structure of the N-linked keratan sulphate chains of human articular cartilage fibromodulin is similar, in general, to articular cartilage derived O-linked keratan sulphate chains. Further, the general structure of the keratan sulphate chains attached to human articular cartilage fibromodulin has been found to be generally similar to that of both bovine and equine articular cartilage fibromodulin.
Subject(s)
Carrier Proteins/chemistry , Cartilage, Articular/chemistry , Extracellular Matrix Proteins , Keratan Sulfate/chemistry , Proteoglycans , Acetylglucosaminidase , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cattle , Chromatography, Gel , Chromatography, Ion Exchange , Femur , Femur Head , Fibromodulin , Horses , Humans , Molecular Sequence Data , N-Acetylneuraminic Acid/analysis , Oligosaccharides/chemistry , Species SpecificityABSTRACT
PURPOSE: A study was made of the distribution of keratan sulphate in the human anterior chamber. METHODS: The monoclonal antibody, 5-D-4, was used in immuno-electron microscopy to visualise keratan sulphate distribution in the anterior chamber of 16 normal eyes, 7 Fuchs' dystrophy corneas, and a macular dystrophy cornea. RESULTS: Keratan sulphate was detected in normal human aqueous humour and also on the surface of trabecular cells in the uveal meshwork. Normal corneal stroma showed an increase in keratan sulphate labelling from anterior to posterior, with marked labelling in the posterior region of Descemet's membrane. The apical surface of the corneal endothelium labelled positively, but showed considerable variation in the level of labelling from cell to cell. The macular dystrophy cornea had the classic histopathological features of a type I case, including a highly abnormal Descemet's membrane. No keratan sulphate was detected in the macular dystrophy patient's corneal stroma or serum. The Fuchs' endothelial dystrophy corneas showed a normal distribution of keratan sulphate labelling in the stroma. The Fuchs' endothelial cells labelled for keratan sulphate but were highly abnormal in appearance, often exhibiting long filopodia and appearing to be actively migrating. CONCLUSIONS: This work has shown that keratan sulphate has a much wider distribution than was previously believed. The detection of keratan sulphate on the trabecular and endothelial cell surfaces also suggests a possible role for this molecule in cell adhesion and/or migration.
Subject(s)
Cornea/metabolism , Keratan Sulfate/metabolism , Trabecular Meshwork/metabolism , Antibodies, Monoclonal , Aqueous Humor/metabolism , Corneal Dystrophies, Hereditary/metabolism , Corneal Dystrophies, Hereditary/pathology , Fuchs' Endothelial Dystrophy/metabolism , Fuchs' Endothelial Dystrophy/pathology , Humans , Microscopy, Electron, Scanning , Microscopy, Immunoelectron , Reference ValuesABSTRACT
Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N-linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both alpha(2-6)-linked N-acetylneuraminic acid and alpha(1-3)-linked fucose. It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non-reducing end of the chain. The galactose residue adjacent to the non-reducing cap is sulphated in only 1-3% of chains, compared with a sulphation level of over 40% closer to the reducing end. This highlights the difference between the chain termini and the repeat region in terms of structure and points to the potential for functional importance. The repeat region and capping fragments of the N-linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have the following general structure: NeuAc-(alpha 2-3/6)Gal[6SO3-](beta 1-4)GlcNAc6SO3-(beta 1-3)Gal[6SO3-] (beta 1-4)¿[Fuc(alpha 1-3)]0-1GlcNAc6SO3-(beta 1-3)Gal-[6SO3-](beta 1-4)¿ 6-7GlcNAc6SO3-.
Subject(s)
Carrier Proteins/chemistry , Cartilage, Articular/chemistry , Extracellular Matrix Proteins , Keratan Sulfate/chemistry , Oligosaccharides/chemistry , Proteoglycans , Acetylglucosaminidase , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Carrier Proteins/isolation & purification , Cattle , Chromatography, Ion Exchange , Fibromodulin , Horses , Keratan Sulfate/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Oligosaccharides/isolation & purificationABSTRACT
Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals) and digested with keratanase II, an endo- beta-N-acetylglucosaminidase. The resulting oligosaccharides were borohydride-reduced and fractionated on a strong anion-exchange column. 1H-NMR spectroscopic analysis of the products revealed one containing both alpha(2-3)-linked sialic acid and alpha(1-3)-linked fucose which was shown to have the structure (I) shown. This structure is a sulphated variant of the VIM-2 epitope (CD65), a putative ligand of E-selectin. No oligosaccharide containing the sialyl-Le(+) structure [NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-] was identified in this study. [equation: see text]
Subject(s)
Cartilage, Articular/chemistry , Epitopes/chemistry , Keratan Sulfate/chemistry , Oligosaccharides/chemistry , Acetylglucosaminidase/metabolism , Animals , Carbohydrate Sequence , Cattle , Chromatography, Ion Exchange , Fucose , Magnetic Resonance Spectroscopy , Molecular Sequence Data , N-Acetylneuraminic AcidABSTRACT
Keratan sulfate-containing proteoglycans were isolated from bovine cornea (15-month-old to 3-year-old animals) and digested with the enzyme, keratanase II. The released oligosaccharides, which included non-reducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and fractionated on a Spherisorb column. These oligosaccharides were examined by 600-MHz 1H NMR spectroscopy using one- and two-dimensional methods and, in addition to some oligosaccharide alditols previously recovered from skeletal keratan sulfate, the following new capping structures were identified: NeuAcalpha2-6Galbeta1-4GlcNAc(S)-ol, NeuAcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-6Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-o l, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc( S)-ol, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)- ol, GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol, and GalNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol. These structures represent seven families of capping residues, whose relative molar proportions are given in parentheses: NeuAcalpha(2-3)- (12%), NeuAcalpha(2-6)- (41%), NeuGcalpha(2-3)- and NeuGcalpha(2-6)- families (12%), Galalpha(1-3)- (26%), GalNAc(S)beta(1-3)- (5%), and GlcNAc(S)beta(1-3)- (4%). It is not clear, at present, where each of these structures occurs on the bi-antennary N-linked corneal keratan sulfate chains, which themselves occur within three keratan sulfate proteoglycan species. However, examination of the relative proportions of the capping to the repeat structures and knowledge of the average molecular size suggests that the sum of these non-reducing termini represents the caps of two antennae.