Keratan Sulfate Fractions Research Articles

ETIOLOGY AND PATHOGENESIS OF IDIOPATHIC SCOLIOSIS

Objective. To study etiologic factors and pathogenetic mechanisms of scoliosis development. Material and Methods. We investigated vertebral body growth plates (GP) from convex and concave sides of the curve, intervertebral discs (IVD), and vertebral bone tissue – surgical material obtained from 100 patients at the age from 10 to 14 years with III–IV grade idiopathic scoliosis (IS). Structural components of the spine of 12–14 years old children obtained from the forensic medicine department were used as controls. The methods of morphohistochemistry, biochemistry, and ultrastructural analysis were used to study glycosaminoglycans (GAGs), oxidation-reduction enzymes, alkali and acid phosphatases, RNA, DNA, qualitative and quantitative composition of GAGs. The expression of proteoglycan genes of cartilage tissue and their protein products was investigated with molecular genetic assays. Results. Pathogenetic mechanism of spine deformity formation in idiopathic scoliosis was formulated. It was shown that idiopathic scoliosis development is predetermined by a disorder in regulation and synthesis of proteoglycans in vertebral GP. The decrease of chondroitin sulphate and increase of keratan sulphate components in proteoglycans indicate the change of proteoglycan spectrum in IS. The revealed keratan sulphate fraction is a result of increased expression of lumikan gene in condition of sharp decrease of aggrecan gene expression and its protein product quantity in chondroblasts of patients with III–IV grade IS. Conclusion. Alteration in aggrecan gene expression at the level of transcription and translation testifies for its involvement in scoliosis development.

Differential distribution of lumican and fibromodulin in tooth cementum.

The objectives of this study were to isolate and characterize the major proteoglycans of tooth cementum in relation to the tissue's mineralization. Cementum was collected from the root apex region of bovine molars and pulverized. It was first extracted with 6M guanidine-HCI, pH 7.4 (G-extract, mineral-unassociated) and then demineralized and extracted with 0.5M EDTA (E-extract, mineral-associated). Both extracts were applied to anion exchange and then molecular sieve chromatography to isolate proteoglycans. The fractions collected were assayed for chondroitin-(CS) and keratan sulfate (KS) containing proteoglycans using the monoclonal antibodies 2-B-6 and 5-D-4, respectively. It was found that the KS was the major glycosaminoglycan and was enriched in the G-extract fraction. The major KS fraction was then applied to 7.5% SDS-PAGE. The major broad band (69 kDa) was 5-D-4 positive in Western blot analysis and separated into two bands (46 kDa and 50 kDa) after treatment with keratanase II and endo-beta-galactosidase. These two proteins were transfered to PVDF membrane and analyzed for amino acid sequence. The results showed the major band (46 kDa) to be lumican and the minor (50 kDa) fibromodulin. In addition, based on the immunohistochemical study using a number of mono- and polyclonal antibodies including 5-D-4, anti-lumican core protein as well as anti-fibromodulin core protein antibodies, the KSPGs were found to be located almost exclusively in nonmineralized portions of cementum such as precementum and the pericementocyte area. These biochemical as well as immunohistochemical data suggest that the major KSPGs of cementum, lumican and fibromodulin, have a specific tissue distribution and may have regulatory roles in cementum mineralization.

Serum keratan sulfate. Quantitative and qualitative comparisons in inflammatory versus noninflammatory arthritides

The concentration of keratan sulfate (KS) epitope was measured in the serum of patients with osteoarthritis (OA) or rheumatoid arthritis (RA) by enzyme-linked immunosorbent assay and compared with that in the serum of patients with primary fibromyalgia syndrome (PFS) and of controls who had no joint disease. By Student's tau-test, the mean serum KS concentrations in OA and RA patients measured with monoclonal antibodies (MAb) 5-D-4 and 2-D-3 were significantly increased over those in the PFS and normal groups; similar findings were observed using a nonparametric test, except that levels in RA patients showed no difference from those in PFS patients and normal subjects. There was no significant correlation between joint scores or disease duration and KS levels in OA or RA patients. Gel filtration of sera revealed mainly large, polydisperse KS-bearing fragments which eluted in a broad profile. KS purified from sera by immunoaffinity chromatography consisted mainly of high-density proteoglycans. Electrophoresis of pooled high-density KS fractions in polyacrylamide-agarose gels followed by Western blotting with MAb 5-D-4 showed diffuse bands with relative mobilities corresponding to large proteoglycans. These findings are consistent with attachment of KS to protein core fragments of various sizes; KS in patient sera is comparable in size with that in normal sera. Elevations of serum KS levels occur in the presence of cartilage degradation, but do not quantitatively define the extent or duration of articular involvement.

The quantitative discrimination of corneal type I, but not skeletal type II, keratan sulfate in glycosaminoglycan mixtures by using a combination of dimethylmethylene blue and endo-β- d-galactosidase digestion

The quantitation of individual glycosaminoglycans in mixtures of polyanions using the dimethylmethylene blue (DMB) method described by R. W. Farndale, D. J. Buttle, and A. J. Barrett (1986, Biochim. Biophys. Acta 883, 173) is dependent on enzymatic hydrolysis by specific polysaccharidases. While using this method to examine the keratan sulfate (KS) of the intervertebral disc we found that digestion with commercially available keratanase decreased binding to DMB by less than 30%, whereas corneal KS was reduced by 85%. However, by preincubating the KS fractions with endo-β- d-galactosidase prior to keratanase treatment the corneal KS could be completely digested and disc KS digestion increased to 60%. It is suggested that the resistance of the disc KS to these digestive procedures arises from branching and/or sites of multisulfation on the polysaccharide chain. Agarose gel electrophoresis and compositional analyses of the keratan sulfates supported such an interpretation.

Structural changes in the large proteoglycan in differentiating chondrocytes from the chick embryo tibiotarsus.

35SO4(2-)- or [3H]GlcN-labeled heavy proteochondroitin sulfate was isolated from monolayer cultures of chondrocytes from the zones of dividing, elongated, and hypertrophying cells of chick embryo tibias, and the keratan sulfate (KS) component was characterized. The KS glycopeptides remaining after digestion of the proteoglycans with thermolysin and chondroitinases were isolated and depolymerized by hydrazinolysis and nitrous acid cleavage. The resulting KS disaccharides had nonreducing terminal D-galactose (Gal) residues and reducing terminal anhydro-D-mannose (AMan) residues. The KS fractions from all cultures had identical disaccharide compositions, with 18-20% Gal----AMan, 72-79% Gal----AMan(6-SO4), and 7-9% Gal(6-SO4)----AMan(6-SO4). The ratios of chondroitin sulfate (CS) to KS synthesized by cultures of dividing, elongated, and hypertrophied chondrocytes were 15, 27, and 30, respectively. Approximately 30% of the CS chains of the proteochondroitin sulfate in the cell matrix pools had nonreducing terminal GalNAc(4,6-diSO4) residues, but none of the CS chains in the proteochondroitin sulfate recovered from the culture medium pools were terminated with these residues.

On the structure of bovine articular cartilage high density proteoglycans. Isolation of the keratan sulfate and chondroitin sulfate side chains.

The structure of adult bovine articular cartilage high density proteoglycans (PG-I) was studied by degradation with Pronase, chondroitinase ABC, and alkaline borohydride treatments and fractionation and analysis of the products. The keratan sulfate (KS) peptides were rich in glutamic acid, proline, and serine and had a low glycine content. The chondroitin sulfate (CS) peptides had a high content of serine, glycine, and glutamic acid and a much lower proline content than the KS peptides. The data indicate that the KS and CS chains occur in more distinct regions of the protein core(s) than in bovine nasal cartilage PG. After alkaline borohydride treatment there was an almost quantitative conversion of xylose to xylitol and galactosaminitol was the only hexosaminitol detected in KS fractions. The results obtained indicated that the alkali-labile bonds linking the CS and KS chains are the same as those reported to occur in other cartilage PGs. The Mr of the KS chains calculated from the glucosamine and galactosaminitol contents gave values of 6,000-7,000, although gel chromatography and light scattering measurements indicated considerable heterogeneity. The KS and CS chains were quantitatively precipitated by cetylpyridinium chloride and the KS and a portion (15%) of the CS chains were found to be soluble in 1% cetylpyridinium chloride. The abnormal solubility properties of the CS chains in the presence of 1% cetylpyridinium chloride is thought to be due to their low sulfate content. The molecular weight of the remainder of the CS chains, based on the ratio of xylitol to galactosamine, varied from 6,500 to 16,000. The low Mr CS chains were rich in 6-sulfated disaccharides whereas the higher Mr chains had a higher content of 4-sulfated disaccharides. The ratio of galactose to xylitol also varied with Mr. These results indicate similarities in the structure of the adult bovine articular cartilage PG-Is to other cartilage high density PGs. The heterogeneities observed in the composition of the KS and CS chains, and their occurrence in relatively distinct regions of the protein core(s) indicate, however, that there is still much to be learned about the structure of these complex macromolecules.

Mannose in the keratan sulfate from bovine cornea.

After exhaustive digestion of bovine corneas with a protease, keratan sulfate fractions of different chain length were obtained by ethanol fractionation on a cellulose column, followed by ion-exchange column chromatography. Compositional analysis of these fractions showed that aspartic acid is the predominant amino acid and that the keratan sulfate in each fraction contains aspartic acid and mannose in the molar ratio of 1:3. Molecular weights of these fractions, estimated by gel chromatography, were close to the calculated values based on the molar ratios of the components to three moles of mannose. The results strongly suggested that the keratan sulfate of bovine cornea contains three mannose residues per chain, as an integral component of the linkage region to protein.

920 GAS LIQUID CHROMATOGRAPHY OF KERATAN SULFATE IN THE KNIEST SYNDROME

The Kniest syndrome is an autosomal dominant skeletal dysplasia characterized by dwarfism, dysmorphic facies, short extremities and joint swelling. Increased urinary excretion of keratan sulfate (KS) in this syndrome has recently been discovered. To identify further the KS in the Kniest syndrome, analysis of the purified KS was carried out using gas liquid chromato-graphy (GLC). Mucopolysaccharides from urine of 4 patients with Kniest syndrome were isolated and KS was purified using Dowex-1 ion exchange chromatography. Keratan sulfate fractions were meth-anolyzed at 80° for 18 h. Amino sugars were N-acetylated with trifluoroacetic anhydride, and following silylation the material was analyzed by GLC. The Kniest KS was compared to Morquio KS and also to standard KS isolated from corneas and rib cartilage. The results indicate that the KS from Kniest and Morquio syndrome are identical. Equimolar amounts of galactose and N-acetyl-glucosamine were found, fucose, mannose, N-acetylgalactosamine and sialic acid, which are found in KS, were also present. The chromatograms of standard skeletal KS correspond to KS from these 2 syndromes. Urinary mucopolysaccharides from normal individuals show different chromatograms as do mucopolysaccharides from urine of a patient with Sanfilippo syndrome. These results indicate that GLC can be used to characterize KS in urine of patients with the Kniest syndrome. GLC may be useful for screening of skeletal dysplasias for keratan sulfaturia.

Excretion patterns of glycosaminoglycans in the urine of normal children and adults.

The glycosaminoglycans excretion was studied in the urine of normal human subjects from newborns to 68 years of age. The glycosaminoglycans preparation was made by digesting the urine with proteolytic enzymes followed by removal of enzyme and undigested protein and finally freeze-drying of the supernatant. Cellulose acetate electrophoresis was carried out on these preparations. Electrophoresis of urinary concentrates results in three alcian blue positive fractions. The proportion with the mobility of chondroitin sulfates fraction was high in childhood and thereafter decreased gradually with age, and that of heparan sulfate and keratan sulfate fraction was low in childhood. Any significant change in the fraction corresponding with hyaluronic acid could not be observed throughout the life.

The effect of cations in equilibrium gradient centrifugation of mucopolysaccharides

When high-density, cartilage proteoglycan (and the lower molecular weight mucopolysaccharides derived from it by papain or trypsin digestion) is centrifuged to equilibrium in complex gradients of CsCl and guanidinium chloride, the peak density is markedly and reproducibly decreased. Hyaluronic acid, a less dense macromolecule, is also decreased in density by the presence of guanidinium ion in the gradient and the extent of the decrease is directly related to the concentration of the guanidinium ion in the starting solution. Other cations, such as Li + or Ca 2+, also produce marked, concentration-dependent, decreases in the density of hyaluronate. The effect is complicated by the fact that the equilibrium distribution of the added cation does not follow the CsCl gradient, but forms a reverse gradient. The density shift must involve an interaction of the added cation with the charged groups of the mucopolysaccharide polyanion since the added cations have no effect on uncharged polysaccharides such as dextran or Ficoll. Furthermore, it seems unlikely that the primary effect could be caused by increased hydration due to increased water activity since centrifugation in Cs 2SO 4 (which reduces the density of nucleic acids by increasing their hydration) leads to substantial decreases in density of all polysaccharides including the uncharged dextran and Ficoll. Although the density of proteins, such as albumin and lysozyme, is not appreciably affected by guanidinium ion; proteoglycan and isolated keratan sulfate fractions of high protein content are markedly decreased in density by guanidinium ion. Such density shifts of the components of proteoglycan must be considered in any attempt to interpret experiments which study disaggregation phenomena in CsCl/guanidinium chloride gradients.

Effects of TSH on the chemical composition of orbital tissue in guinea pigs.

Female guinea pigs were injected either with saline, with thyroxine, or with varying doses of TSH. Following a single ip injection of Na2 (35S)O4, the content of 35S was determined for the Harderian gland, ventral lacrimal gland, and serum. Specific glycosaminoglycan fractions were isolated from the Harderian gland using successive columns of cetylpyridinium chloride-cellulose and ECTEOLA, and were assayed for levels of hexosamine and of 35S. The following results were obtained: (1) TSH increased the 35S content of the serum, the Harderian gland, and the ventral lacrimal gland, while T4 had no effect; (2) TSH increased the proportion of total tissue 35S in chondroitin sulfate and decreased it in keratan sulfate; (3) the hexosamine content of the Harderian gland was increased by TSH, but not by T4; (4) TSH increased hexosamine levels in the hyaluronic acid, chondroitin sulfate, and glycoprotein fractions of the Harderian gland, and decreased them in the keratan sulfate fraction. We conclude that in experim...

Long term observations on the articular cartilage and autologous costal cartilage transplanted to osteochondral defects on the femoral head. A histological, autoradiographic (35S-sulphate) and microchemical (glycosaminoglycan) study in adult rabbits.

In 20 adult rabbits autologous costal cartilage was transplanted to an osteochondral defect on the femoral head. After 1, and 11/2 years the tissue of the transplant area and the articular cartilage from the operated and non-operated joints were studied. The histological and autoradiographic (35S-sulphate) study showed that the articular surface and the bulk of the transplant area consisted mainly of viable cartilaginous tissue showing signs of degeneration to varying degree. Also, fibrous tissue was found on the articular surface of the transplant area. The quantitative microchemical study of glycosaminoglycans, hydroxyproline and calcium was made on microdissected freeze-dried material. The total hexosamines and the keratan-sulphate and/or glycoprotein fraction were lower in the transplant than in the articular cartilage from the operated joints while the contrary was true for chondroitin sulphate. The articular cartilage from the operated joints showed a reduction of total hexosamines and of all glycosaminoglycan fractions compared to the non-operated joints. These changes were especially pronounced in samples showing signs of degeneration in Azur-Astained, freeze-dried sections. It is suggested that in early and slight superficial articular cartilage degeneration the glycoprotein and/or keratan-sulphate fraction is reduced while chondroitin-sulphate remains relatively unaltered.

Glycosaminoglycans of the rat incisor pulp

1. 1. The continuously growing, dentinogenically active rat incisor is a suitable model for the biochemical as well as morphological study of the process of hard tissue deposition. Thus the glycosaminoglycans (acid mucopolysaccharides) of the rat incisor dental pulp have been isolated, separated, quantitated and characterized by means of a cetylpyridinium chloride (CPC)-cellulose micro-column technique. 2. 2. The total amount of hexosamine was found to be 4.8 μg/mg tissue dry weight. The dry weight of this loose connective tissue was 10% of the tissue wet weight. 3. 3. Four hexosamine-containing fractions were isolated from the CPC-cellulose columns: one glycoprotein- and keratan sulphate-containing fraction, one hyaluronate fraction, one fraction containing predominantly 4-sulphated chondroitin sulphate, and one fraction with predominantly 6-sulphated chondroitin sulphate. In maxillary incisor dental pulps the combined chondroitin sulphate fractions accounted for 75%, and the hyaluronate fraction for 12%, of the total hexosamine. This is consistent with earlier results obtained using electrophoretic methods. 4. 4. By means of epichlorohydrin triethanolamine (ECTEOLA)-cellulose chromatography, a minor keratan sulphate fraction could be found. 5. 5. The hexosamines in the hyaluronate and combined chrondroitin sulphate fractions were separated on Dowex 50W-X8 columns. In the former only glucosamine was found. In the latter galactosamine dominated, but a small amount of glucosamine (less than 10%) was also found. 6. 6. A “neutral MgCl 2 elution profile” indicated that the molecular weight rangr of the chondroitin sulphates was 4·10 4−7·10 4. 7. 7. An “acid MgCl 2 elution profile” for the combined chondroitin sulphate fractions was also obtained. This suggested a variation in the sulphate content of the chondroitin sulphate.

Glycosaminoglycans in Human Comeal Buttons Removed at Keratoplasty

Microfractionation and analysis of individual human corneal buttons were performed on both normal corneas and on corneas removed at keratoplasty from eyes with keratoconus or swollen, opaque corneas. Keratoconus buttons were found to have a relative increase in the glucosaminoglycan (keratan sulfate) fraction and a significant decrease in the galactosaminoglycan (chon-droitin sulfate) fraction. There was decreased sulfation of galactosaminoglycans in keratoconus buttons. Both the glucosamino glycans and the galactosaminoglycans were decreased in some swollen opaque corneas. In these corneas a highly sulfated glycosaminoglycan was recovered not present in normal corneas, which was comparable in many ways to dermatan sulfate.

Microchemical studies on glycosaminoglycans and calcium in autologous costal cartilage transplanted to an osteochondral defect on the femoral head of adult rabbits.

In adult rabbits, a transplant was made to an osteochondral defect on the femoral head measuring 3×4×3 mm of autologous costal cartilage which had first been implanted intramuscularly for 4–5 weeks and fresh autologous costal cartilage. After 6 different observation times between 2 and 52 weeks the animals were killed. The original material, the tissue in the transplant area on the articular surface and the articular cartilage above the “tidemark” were analysed in each individual animal, after microdissection of freeze-dried sections of these structures, by a quantitative microchemical technique for their content and composition of glycosaminoglycans and for their calcium content. The hexosamine content (% per organic dry weight) was generally higher in the transplants than in the articular cartilage at observation times of 2 and 6 weeks, after which time it was similar. The distribution of the different glycosaminoglycan fractions was different in the transplants compared with the articular cartilage. Thus the glycoprotein and/or keratan sulphate fraction was smaller in the transplant than in the articular cartilage, while the opposite held for the chondroitin sulphate. With increasing observation times, some resemblance became evident between the transplant and articular cartilage with respect to the relative size of these fractions. The solubility profiles of the chondroitin sulphate were similar. The calcium content was generally higher in the transplant than in the articular cartilage, after 12 weeks it showed a pronounced decrease in comparison with the original material. The chemical findings indicate that the cartilage in the transplant area did not undergo any marked degenerative changes.

Studies on the incorporation of (U-14C)glucose and (35S)sulphate into the acid glycosaminoglycans of neonatal rat skin.

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.

Fractionation and quantitative determination of keratan sulfate using cetylpyridinium chloride and ECTEOLA - cellulose

1. 1.The cetylpyridinium chloride-cellulose method does not permit an accurate determination of keratan sulfate in tissue digest, due to the fact that both keratan sulfate and glycopeptides are soluble in excess cetylpyridinium chloride. 2. 2. For this reason a modification of this technique has been developed. By extraction with iso-amyl alcohol cetylpyridinium chloride is removed from an aqueous solution of keratan sulfate and glycopeptides. The water phase is subsequently subjected to fractionation on ECTEOLA-cellulose to yield one glycopeptide and one keratan sulfate fraction. 3. 3. The combined cetylpyridinium chloride and ECTEOLA procedures have been applied to the estimation of keratan sulfate in artificial mixtures as well as in tissue digests.

Sialic acid in the keratan sulfate fraction from whale cartilage

From whale nasal cartilage a keratan sulfate fraction was obtained in good yield by ethanol fractionation on a cellulose column. The keratan sulfate fraction was further fractionated on Dowex 1 into at least four fractions having various ratios of sulfate to hexosamine from 0.75 to 1.8. All the fractions obtained contained various amounts of galactosamine, sialic acid, mannose, fucose and amino acids, in addition to glucosamine and galactose. The sialic acid content of the whale keratan sulfate fraction was remarkably high in contrast with corneal keratan sulfate. From whale keratan sulfate the sialic acid was isolated and identified as N-acetylneuraminic acid. The results of pronase digestion and treatment with alkali suggested the presence of the sialic acid in the carbohydrate moiety, not in the alkali-labile peptide moiety of the keratan sulfate fraction. The sialic acid was easily liberated by neuraminidase digestion, which indicates that the sialic acid is attached to the non-reducing end of the carbohydrate moiety.

Biochemical Heterogeneity of the Corneal Glycosaminoglycans

A procedure has been developed for the extraction and purification of the corneal glycosaminoglycans under mild conditions and without the use of proteolytic enzymes. Nearly 85% of the stromal glycosaminoglycans were solubilized by homogenization of the fresh tissue with water and 0.1 m potassium carbonate at 4°C in a VirTis homogenizer. Proteins not associated with methods the glycosaminoglycans were removed by passing the extracts through SE-Sephadex columns. The keratan sulfates were then separated from the chondroitin sulfates by treatment with ammonium sulfate, and each of the fractions was then chromatographed separately on DEAE-Sephadex. Chemical analyses showed that the two populations of stromal glycosaminoglycans are highly heterogeneous. The four species of chondroitin sulfate differed from one another mainly in sulfate content, with the ratio sulfate/galactosamine varying from 0.43 to 0.89. The principal amino acids in the chondroitin sulfate-peptide molecule were glutamic acid, serine, glycine and aspartic acid. Seven species of keratan sulfate, with ratios of sulfate/glucosamine varying from 0.92 to 1.70, were isolated. Only one of the fractions, representing approximately 16% of the total population, was undersulfated. All the others contained one or more moles of sulfate for each mole of amino sugar. The various keratan sulfate fractions were also heterogeneous with respect to neutral sugar content in that four of them contained approximately equimolar amounts of galactose and glucosamine while in the other three, the ratio of galactose/glucosamine was approximately 1.5. The principal amino acid in all of the keratan sulfate fractions was aspartic acid.

Keratan sulfate fractions from bovine and human tissues

Keretan sulfate fractions having various sulfate contents have been obtained from different tissue sources. Highly sulfated fractions of keratin sulfate have been obtained from embryonic and adult bovine intervertebral tissues, as well as from adult human intervertebral tissues. Under-sulfated keratan sulfate fractions were isolated only from bovine corneal stroma and from bovine nasal-septum cartilage. All tissues produced fractions showing relatively wide variations of sulfate content. Increase in keratan sulfate content of bovine intervertebral tissues was noted during embryonic development, although this concentration was one-half or less that of adult bovine tissues. Acid mucopolysaccharide content of embryos in the last trimester corresponded to those found in adult bovine intervertebral tissues.