Showing papers on "Keratan sulfate published in 1985"

Quantification of keratan sulfate in blood as a marker of cartilage catabolism.

Abstract: We have developed an enzyme-linked immunosorbent-inhibition assay which makes use of a monoclonal antibody specific for keratan sulfate to quantify keratan sulfate present as single chains in adult human serum. In adults hospitalized with conditions not thought to affect the turnover of keratan sulfate-containing tissues, the serum levels varied from individual to individual (53-1,009 ng/ml) but did not show significant differences with respect to age, sex, or disease category. There were no significant differences between the serum levels of adult hospitalized patients and those of nonhospitalized normal adults. In contrast, the concentration of keratan sulfate in the sera of children aged 5-12 was significantly higher. No keratan sulfate was detected in the sera of 3 adult patients with macular corneal dystrophy, an inherited disorder of the cornea. This may indicate that individuals with macular corneal dystrophy have no keratan sulfate-containing proteoglycans in their cartilage. Adult patients with osteoarthritis have significantly higher concentrations of circulating keratan sulfate. This suggests that the assay could prove valuable in monitoring increased cartilage catabolism in joint diseases.

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans.

Abstract: Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate.

Abstract: We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.

Isolation and characterization of glycosaminoglycans in human plasma.

Abstract: We have described methodology for the isolation and quantitation of glycosaminoglycans present in human plasma. Plasma glycosaminoglycans can be quantitatively adsorbed on a DEAE-Sephacel ion exchanger and eluted with a salt gradient as two groups: a low-charge fraction and a high-charge fraction. The low-charge fraction consists of chondroitin sulfate with a low sulfate content and the high-charge fraction consists of heparan sulfate, chondroitin sulfate, and keratan sulfate (type I). We have determined the plasma concentration of each of these glycosaminoglycans in six normal human subjects. We have established that none of the glycosaminoglycans in plasma are covalently linked to plasma proteins. All are isolated as complexes with plasma proteins in noncovalent linkages. The glycosaminoglycans in the low-charge fraction are bound with high affinity to a single plasma glycoprotein by a lectin-type bond that can be disrupted by a simple glycoside. The high-charge fraction contains three major proteins and several minor proteins associated with the glycosaminoglycans by both lectin-type and ionic bonding. The plasma proteins associated with glycosaminoglycans represent less than 0.5% of the total plasma proteins. Little is known about the physiologic role of the plasma glycosaminoglycans as components of metabolic processes. Because glycosaminoglycans have been implicated in lipid metabolism and atherosclerosis, we tested all of these compounds, isolated in free form, on the in vitro hydrolysis of triglycerides by lipoprotein lipase. Plasma heparan sulfate stimulated the rate of this reaction severalfold. All other plasma glycosaminoglycans were inactive. Thus, plasma heparan sulfate may play an important role in plasma lipoprotein metabolism.

Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye

Abstract: The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non-human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate electrophoresis For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical The human TM contains hyaluronic acid (HA), chondroitin-4-sulfate and/or 6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 290% HA, 141% CS, 215% DS, 203% KS, and 150% HS The cynomolgus monkey trabecular GAGs include: 128% HA, 143% CS, 152% DS, 421% KS, and 156% HS

Ultrastructural histochemistry of the surface lamina of normal articular cartilage.

Abstract: Two collagen-poor, ultramicroscopic layers are described at the surface of canine articular cartilage. They are distinguished by staining with an electron-dense cationic dye, Cupromeronic Blue, in a critical electrolyte concentration technique and by digestion with testicular hyaluronidase. The superficial layer, approximately 50 nm thick, stained at low electrolyte concentrations but failed to stain in conditions specific for sulphated glycosaminoglycans. It was hyaluronidase-resistant and may be either glycoprotein or protein in nature. The deeper layer, 100-400 nm thick, stained positively at electrolyte concentrations specific for sulphated glycosaminoglycans but not in conditions specific for keratan sulphate. It was removed by hyaluronidase digestion. This layer probably represents a chondroitin sulphate-rich proteoglycan. These surface layers may be important in the lubrication of the articular surface and in the permeability and compression resistance of the superficial cartilage zone.

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

Abstract: The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Monoclonal antibodies to proteokeratan sulfate of rabbit corneal stroma.

Abstract: Hybrldomas were developed that secreted monoclonal antibodies against two proteokeratan sulfates (PKS) from rabbit corneal stroma. A total of 28 antibodies were isolated, all of the IgG type with k light chains. All were found to react with both PKSs. As judged by immunohistochemical staining, none of them reacted with scleral or eonjunctival components, nor with sections of skin, but all reacted with nasal cartilage. When tested by enzyme-linked immunosorbent assays, against components of the proteoglycans, all of the antibodies reacted with keratan sulfate-peptide (isolated from papain digests of PKS or of cartilage proteoglycan), and all but two reacted with oligosaccharide-containing protein cores (prepared by keratanase treatment of PKS). Reactivity with cores was probably due to residual portions of the keratan sulfate chains since the endogenous oligosaccharide-peptides (non-sulfated, non-keratan sulfate oligosaccharides isolated after papain digestion of PKS) were not active. None of the antibodie...

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Abstract: Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.

Influence of Constituents of Proteoglycans on Type II Collagen Fibrillogenesis

Abstract: Summary Turbimetry was used to study the influence of glycosaminoglycans of cartilage proteoglycans on type II collagen fibrillogenesis. The monosaccharides, D-glucuronate, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and D-galactose, all decreased the rate of the fibril formation. D-glucuronate had the strongest effect. The influence of chondroitin sulphate on type II collagen fibrillogenesis depended on the pH of the final solution, the length of the chondroitin sulphate chains and the concentration of chondroitin sulphate. At pH = 7.3 all chondroitin sulphate preparations decreased the rate of fibrillogenesis, while at pH = 6.9 and lower Fibrillogenesis was stimulated by chondroitin sulphate (whale/shark), chondroitin 4-sulphate (whale) and chondroitin 6-sulphate (shark). The chain length of these three appeared to be longer than the chain length of chondroitin sulphate (human) and chondroitin sulphate oligosaccharides (whale/shark). At high concentrations (more than 3 mg/ml) fibril formation was less strongly retarded by keratan sulphate (human) than by chondroitin sulphate. At low concentrations a slight stimulation was observed in the presence of keratan sulphate. Glycosaminoglycans did not bind to collagen fibrils. At 0.5 mg/ml chondroitin 4-sulphate had a large solubilizing effect on fibrils compared to chondroitin 6-sulphate. Fibrillogenesis of type II collagen is in many aspects not comparable with fibrillogenesis of type I collagen.

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

Abstract: The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.

Reversible Effects of Retinoic Acid on Glycosaminoglycan Synthesis during Differentiation of HL-60 Leukemia Cells

Abstract: Glycosaminoglycans (GAGs) play an important role in cell-cell and cell-substratum interactions, and undergo specific changes during neutrophil development. Previous studies (Luikart, S. D., Maniglia, C. A., and Sartorelli, A. C. Cancer Res., 44: 2907–2912, 1984) have shown that both dimethyl sulfoxide and 4-β-phorbol-12-β-myristate-13-α-acetate decreased GAG production by a hypoxanthine-guanine phosphoribosyl transferase-deficient clone of HL-60 promyelocytic leukemia cells prior to the appearance of a mature myeloid or monocytoid phenotype. To expand these investigations further, GAGs were analyzed by cetylpyridinium chloride precipitation and DEAE-Sephacel ion-exchange chromatography after labeling of parental HL-60 cultures with [ 35 S]sulfate and d-[ 3 H]glucosamine for 6 h, following treatment with 1 µm all- trans retinoic acid (RA). Chondroltin sulfate represented the major GAG species produced, although endo-β-galactosidase-sensitive undersulfated macromolecules which possibly might be keratan sulfate, were also identified. GAG production decreased over a time period of 144 h in culture. RA treatment reduced the amount of radiolabeled cell-associated GAGs by 50% after 48, 96, and 144 h of exposure. In contrast, commitment to myelocytic maturation of the majority ( i.e. , approximately 60%) of the cells occurred between 72 and 96 h of RA treatment. Concurrently with the appearance of mature granulocytic cells, two-thirds of the radiolabeled GAGs were recovered from the medium, compared to one-third in untreated cultures, a phenomenon that resulted in an overall alteration in the distribution of GAGs. When RA was removed by washing after either 48 h ( i.e. , precommitment to differentiation) or 96 h ( i.e. , postcommitment to differentiation), a 1.5- to 3.5-fold increase in GAG production was noted 48 h later; this increase was unrelated to the medium change or to alterations in cell cycle distribution. The amounts of endo-β-galactosidase-sensitive macromolecules were unaltered. Thus, although 1 µm RA inhibited the synthesis of chondroitin sulfate by HL-60 leukemia cells, this inhibition was reversible by removal of the drug and appeared to be unrelated to the commitment to myelocytic maturation.

Proteoglycans and Glycosaminoglycans of Normal and Strontium Rachitic Epiphyseal Cartilage

Abstract: An analytical protocol for the analysis of proteoglycans and proteins extracted from small sections of cartilage has been applied to the upper and lower parts of the epiphyseal growth plate of normal rats and rats with strontium-induced rickets. Only one polydisperse proteoglycan population was found in each of the four tissue portions. In strontium-induced rachitic animals the aggregability of proteoglycan monomers with hyaluronate was considerably increased in the lower part of the growth plate. The proteoglycans in the upper portion of the epiphysis contained somewhat less of chondroitin sulfate and keratan sulfate both in normal and strontium-induced rachitic rats. The chondroitin sulfate chains were somewhat larger in samples from strontiuminduced rachitic rats compared with controls while in all groups about 90 per cent of the galactosamine in chondroitin sulfate was sulfated at position four. The findings of an altered composition of proteoglycans in strontium-induced rickets demonstrate that strontium not only prevents the mineral growth but may also induce the chondrocytes to produce a matrix with a different macromolecular composition.

Proteoglycans and Glycosaminoglycans of Epiphyseal Cartilage in Florid and Healing Low Phosphate, Vitamin D Deficiency Rickets

Abstract: The composition of proteoglycans and glycosaminoglycans in two portions of the rat epiphyseal growth cartilage in florid and healing low phosphate, vitamin D deficiency rickets was studied. The concentration of both chondroitin sulfate and keratan sulfate was reduced with about 25 per cent in the lower (mineralizing) part of the growth plate in healing rickets compared to the lower (hypertrophic) part in florid rickets. In all samples about 85 per cent of the chondroitin sulfate chains had a sulfate ester group in the fourth position. Both in florid and healing rickets only one main population of polydisperse proteoglycans was found. In florid rickets the capacity to form aggregates with hyaluronic acid was the same in the upper and lower parts of the growth plate. With the onset of mineralization in healing rickets, however, a substantial decrease in this aggregability was demonstrated. These results give further support to the hypothesis that proteoglycans and, especially, proteoglycan aggregates, play a role in the mineralization process.

Intralysosomal formation and metabolic fate of N-acetylglucosamine 6-sulfate from keratan sulfate.

Abstract: The physiological relevance of the ability of β-N-acetylhexosaminidase A to liberate N-acetylglucosamine 6-sulfate from polymeric keratan sulfate was investigated. Upon intravenous injection into rats of[35S]sulfate-labeled proteokeratan sulfate up to 25% of the radioactivity excreted with the urine were identified as N-acetyl-glucosamine 6-sulfate. Within 24 h, however, excretion of inorganic sulfate rose at the expence of the sulfated monosaccharide. Upon incubation in vitro of liver lysosomes from rats treated with proteokeratan sulfate, inorganic sulfate and minor amounts of sulfated monosaccharide were found in the incubation fluid. Cultured rat peritoneal macrophages ingested proteokeratan sulfate with a clearance rate of 6–9 μg × h−1× mg cell protein−1 and degraded it rapidly. Inorganic sulfate but not N-acetylglucosamine 6-sulfate was delivered to the culture medium. During a chase period the amount of intracellular N-acetylglucosamine 6-sulfate fell, and a corresponding amount of sulfate could be found extracellularly. Significant amount of N-acetylglucosamine 6-sulfate were only found in the culture medium when the cells were challenged with zymosan. These results suggest that N-acetylglucosamine 6-sulfate is a physiological intermediate during the degradation of keratan sulfate, but is usually hydrolyzed intralysosomally by N-acetylglucosamine-6-sulfate sulfatase. Genetic deficiency of the sulfatase in humans therefore results in excessive excretion of the sulfated amino sugar but not of keratan sulfate.

Corneal Proteoglycan Biosynthesis and Macular Corneal Dystrophy

Abstract: Proteoglycans are constitutive components of all connective tissues. They consist of a protein core containing covalently attached glycosaminoglycan and oligosaccharide side chains. Antibodies have been prepared against proteoglycans isolated from a variety of tissues including cornea (Hassell et al, 1982), bone (Fisher e t al, 1983), basement membrane (Hassell e t al, 1980b), and cartilage (Poole et al, 1980). These antibodies are usually directed against the protein core of the proteoglycans and have been useful for studying the synthesis of the core protein (Kimura et al, 1981; Bumol and Reisfeld, 1982) and identifying related proteoglycans. For example, some antibodies against the large chondroitin sulfate proteoglycan from cartilage react with a large dermatan sulfate proteoglycan from aorta (Gardell et al, 1980; MangkarnkanokMank et al, 1981) and a large chondroitin sulfate proteoglycan from bone (Fisher et al, 1983). Thus, these proteoglycans, although from different tissues, could be derived from the same gene product or, probably from very related gene products. The proteoglycans likely vary mainly in post translational modifications (i.e. glycosaminoglycan and oligosaccharide additions).

Proteoglycans and Glycosaminoglycans in Cartilage from the Brachymorphic (bm/bm) mouse

Abstract: The brachymorphic (bm/bm) mouse is a disproportionate dwarf with a disturbance of the endochondral growth of the skeleton. Rib cartilage from 25-day-old affected animals and their normal siblings was analyzed for its contents and composition of proteoglycans. In addition to the previously reported undersulfation of chondroitin sulfate, it was demonstrated that one of the two types of aggregating proteoglycan and possibly the small ones are decreased in bm/bm costal cartilage, both in the growth region and in the remaining part. The molecular defect of the bm/bm condition is known to affect the synthesis of 3-phosphoadenosine 5-phosphosulfate (Sugahara and Schwartz, Proc. Natl. Acad. Sci. USA 76: 6615-6618, 1979). The above finding therefore suggests the existence of feedback mechanisms for the regulation of proteoglycan synthesis, whereby the undersulf ation of glycosaminoglycans would result in decreased synthesis or increased turnover of certain proteoglycan subpopulations. Analysis of the glycosamino glycan side chains indicated that mouse rib cartilage contains small amounts of keratan sulfate of extremely small size. The affected and control tissues, however, seemed to contain equal amounts of both glucosamine and galactosamine.

The histochemistry of urea-unmasked glycosaminoglycans int he skin of the eel, Anguilla japonica

Abstract: In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.

Phosphorylation of proteoglycans. Identification of phosphorylation sites in chondroitin sulfate-rich region of core protein.

Abstract: Serine residues, which are the sites of phosphorylation in proteoglycans, were demonstrated to be located on chondroitin sulfate-containing peptides. These peptides appeared to be derived primarily from the chondroitin sulfate-rich region of the proteoglycan core protein. The localization of phosphate moieties in the chondroitin sulfate-containing peptides was observed in all experiments. The phosphate moieties were retained on chondroitin sulfate-containing peptides after the protein core was treated with either papain or trypsin. Two phosphopeptide preparations, derived from chondroitin sulfate-containing peptides of proteoglycan subunits, were extensively purified. These 2 phosphopeptide preparations were shown by carbohydrate analysis to be free of keratan sulfate-containing peptides or peptides from the hyaluronic acid binding region of the core protein. One of the phosphopeptide preparations had a phosphate: serine molar ratio of 0.40. This indicated that nearly one-half of the serine residues were phosphorylated rather than glycosylated. Peptides derived from the core protein that contained keratan sulfate had no phosphate moieties.