The comparative study of the IR spectra of proteoglycans]

The IR spectra of sodium salt hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan-sulfate, protein-chondroitin-keratan-sulfate and aggregates of proteoglycans of the hyaline cartilage, heparin fractions containing 3 and 4 residues of sulfuric acid per dimer of polymer were obtained. It was shown that comparative analysis of IR spectra of the proteoglycans makes it possible to identify the 1150 cm-1, 1125 cm-1.     

The hydration of proteoglycans of bovine cornea

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a disaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

The hydration of proteoglycans of bovine cornea.

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage

The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4-sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage.     

Composition of glycosaminoglycans in human pancreatic cancer

Five glycosaminoglycans were isolated from tryptic digestion of both cancerous and normal tissues of the human pancreas and were assayed by determining the carbohydrate content of materials. Separation of these five polymers was achieved by Dowex 1-X2 column chromatography and fractionation with Benedict's solution. They were identified as hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate, respectively. The total amount of glycosaminoglycans in cancer tissue increased in comparison to the controls. The increase in tissue content of glycosaminoglycans was accompanied by increases in chondroitin-4-sulfate and chondroitin-6-sulfate levels.     

Disaccharide analysis of glycosaminoglycans synthesized by cardiac myxoma cells in tumor tissues and in cell culture

We analyzed the main disaccharide units of glycosaminoglycans synthesized by cardiac myxoma cells in vivo and in cell culture using high-performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labeling. Cardiac myxoma tissues contained large amounts of chondroitin-6-sulfate (46%) and hyaluronic acid (32%), along with some chondroitin-4-sulfate (13%), chondroitin (6%), and much less dermatan sulfate (3%). Cultured cardiac myxoma cells synthesized mainly chondroitin-6-sulfate. The abundant glycosaminoglycans in myxoma tissues may make up the characteristic friable gelatinous matrix which is favorable for embolism and tumor cell growth.     

Alterations in epidermal sulfated proteoglycan production following topical application of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate to mouse skin.

Topical application of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin causes marked changes in epidermal cell growth and differentiation. In the present studies we characterized the production of sulfated proteoglycans in the epidermis following treatment with TPA since these macromolecules are important structural and functional components of the tissue. We found that 35S-sulfate was readily incorporated into mouse epidermal proteoglycans. Sepharose CL-4B column chromatography revealed one major peak of sulfated proteoglycans in this tissue (Kav = 0.4-0.5). Approximately 65% of these proteoglycans were heparan sulfate and 10-20% chondroitin sulfate. Using specific monoclonal antibodies and flow cytometry, we found that the epidermal cells produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate. Within 24 hr of application of TPA to mice, an increase in glycosaminoglycan content of the epidermis was observed. This was associated with a decrease in 35S-sulfate uptake into the tissue. Although TPA had no effect on the size or relative distribution of the epidermal sulfated proteoglycans, an increase in chondroitin-4-sulfate expression was observed in treated skin. Changes in the production of proteoglycans following TPA treatment may underlie structural alterations that occur in the epidermis during tumor promotion.     

Proteoglycans and collagenase in hypertrophic scar formation.

The collagen fibers of the nodules and whorl-like figures in hypertrophic scars are "coated" with proteoglycans, mainly chondroitin-4-sulfate. The latter was shown to prevent collagenase from breaking down collagen. This suggests that the presence of great amounts of chondroitin-4-sulfate in hypertrophic scars may contribute to the overabundance of collagen deposition which is characteristic of this abnormal healing process.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: Relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage.

From the carbohydrate-protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4-sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondroitinase ABC, and subsequent beta-elimination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500-MHz 1H-NMR spectroscopy. The nonsulfated compound (A) had the following conventional structure: delta GlcA(beta 1-3)-GalNAc(beta 1-4)GlcA(beta 1-3)Gal(beta 1-4)Xylol, where GlcA, delta GlcA and GalNAc are glucuronic acid; 4,5-unsaturated glucuronic acid and 2-deoxy-2-N-acetylamino-D-galactose, respectively. The other compounds were sulfated derivatives of compound A. Two monosulfated compounds (B and C) had an ester sulfate on C4 or C6 of the GalNAc residue, respectively and the disulfated compound (D) had two ester sulfate groups, namely, one on C4 of the GalNAc and the other on C4 of the Gal residue substituted by GlcA. The molar ratio of A/B/C/D was 0.21:0.16:0.36:0.27. The compound containing Gal-4-O-sulfate was previously isolated by us in the form of a sulfated glycoserine [delta GlcA(beta 1-3)GalNAc(4-O- sulfate)(beta 1-4)GlcA(beta 1-3)Gal(4-O-sulfate)(beta 1-3)-Gal(beta 1- 4)Xyl beta 1-O-Ser] from the carbohydrate-protein linkage region of rat chondrosarcoma chondroitin-4-sulfate proteoglycans [Sugahara K., Yamashina, I., DeWaard, P., Van Halbeek, H. & Vliegenthart, J.F.G. (1988) J. Biol. Chem. 263, 10,168-10,174]. The discovery of this structure in the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans from nontumorous cartilage indicates that it is not a tumor-associated product but rather a physiological biosynthetic product since it represents a significant proportion. The biological significance of this structure is discussed in relation to glycosaminoglycan biosynthesis.     

Characterization of the chondroitin sulfate produced by B16 mouse melanoma cells

The mucopolysaccharides produced by B16 mouse melanoma cells have been isolated in milligram quantities from the spent media in which the cells were grown in the presence of 2-amino-2-deoxy-d-glucose-t and ³⁵S]-sulfate. The mucopolysaccharides obtained by precipitation with cetylpyridinium chloride from the Pronase digest of the media were further purified by gel filtration, ion-exchange chromatography, and treatment with nucleases. The major components were identified as chondroitin-4-sulfates by identification of the hexosamine as 2-amino-2-deoxy-d-galactose, and by digestibility with hyaluronidases, chondroitinase AC, and chondro-4-sulfatase. The o.r.d. curve and i.r. spectra of these components also confirmed their similarity to chondroitin-4-sulfate from cartilage. The molecular weight of the polysaccharide chains was estimated to be in the range 90,000–120,000 by sedimentation equilibrium analysis.     

Chondroitin sulfate synthesis by mouse embryonic, extraembryonic, and teratoma cells in vitro

Sulfated glycosaminoglycan (GAG) synthesis by primary cultures of embryo, yolk sac, and trophoblast was compared with synthesis by the same tissues in utero. In general, the in vivo and in vitro results were in good agreement. As was the case in vivo, the three tissues synthesized chondroitin-4-sulfate and chondroitin-6-sulfate (but no dematan sulfate) at characteristic ratios.Cultured embryos are already capable of synthesizing chondroitin sulfates, primarily chondroitin-4-sulfate, before, or at, the 64-cell stage. During the attachment and initiation of outgrowth stages, blastocysts synthesize more chondroitin-6-sulfate than chondroitin-4-sulfate. Thereafter, progressively more chondroitin-4-sulfate is synthesized so that the 4:6 ratio increases, resembling that of trophoblast cells.Blastocyst-derived cell lines and teratoma cell cultures were also studied. One blastocyst-derived line, MB4, synthesized GAG with a pattern similar to that of yolk sac, which it resembles biochemically in other respects as well. The GAG profile of MB2, a parietal endoderm-like cell line resembled neither that of embryo, yolk sac, nor trophoblast cells. Embryonal carcinoma (undifferentiated teratoma) cells had a chondroitin sulfate pattern different from that of most of the other cultures.     

Effect of ascorbic acid on secreted proteoglycans from rabbit articular chondrocytes

Addition of ascorbic acid (25, 50 100 micrograms/ml) to cultures of rabbit articular chondrocytes did not change the total amount of proteoglycans produced. However, it induced an increased retention of these macromolecules in the pericellular fraction. The size of the proteoglycan subunits and the length of glycosaminoglycan chains, released in the medium, were not modified on exposure to ascorbic acid (25 micrograms/ml). On the other hand, the rate of non-sulfated chondroitin was increased 2.5-fold, whereas chondroitin-4-sulfate was depressed 1.5-fold.     

The amino-terminal region of a proteochondroitin core protein, secreted by aortic smooth muscle cells, shares sequence homology with the pre-propeptide region of the biglycan core protein from human bone

Smooth muscle cells, isolated from rat and bovine aortae and grown in vitro, synthesize chondroitin sulfate proteoglycans which are secreted into the growth media. Analysis of metabolically [35S]-labeled macromolecules, employing ion-exchange chromatography, revealed a single peak of radioactivity, upon elution with a linear salt gradient. Treatment of the material with enzymes that specifically degrade chondroitin sulfate demonstrated that chondroitin-4-sulfate was the predominant species isolated from rat smooth muscle cells and that chondroitin-4-sulfate and dermatan sulfate were the predominant species isolated from bovine aortic smooth muscle cells. Treatment of the native proteoglycans with chondroitinase ABC and subsequent SDS-PAGE analysis of the digestion products resulted in the appearance of a band with an apparent molecular weight of 45,000. Electrotransfer of the core protein to Immobilon-P membrane and gas phase sequencing of the amino-terminal region revealed striking homology between the core proteins of the rat and bovine proteochondroitin with the pre-propeptide region of human bone biglycan.     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.     

Analyses of glycosaminoglycans in human lung cancer

Studies were conducted on the total amount of glycosaminoglycans and glycosaminoglycan composition in adenocarcinoma tissue of human lung. The glycosaminoglycans were prepared by exhaustive proteinase digestion of adenocarcinoma tissue from human lungs and of lung tissue without pulmonary diseases taken at autopsy as a control. The glycosaminoglycan classes were characterized by biochemical, enzymatic, and electrophoretic methods. The presence of heparin, which has until now not been found in lung cancer tissue, was demonstrated on both carcinoma and control tissues. The levels of whole glycosaminoglycans were markedly increased in cancer tissue compared to the controls. The classes of glycosaminoglycans which increased in lung carcinoma tissue were predominantly chondroitin-4-sulfate and chondroitin-6-sulfate. Both hyaluronic acid and heparin were slightly increased in cancer tissue.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

Recombinant human thrombomodulin(csa+): a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate

The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.