Search

Monoclonal antibodies that recognize carbohydrate epitopes found in keratan sulfate glycosaminoglycan chains identified both intracytoplasmic and cell-surface carbohydrates of human keratinocytes. These carbohydrates were detected, using indirect immunoperoxidase methods, both in sections of paraffin-embedded tissues and in intact cultured keratinocytes. Of the seven anti-keratan sulfate monoclonal antibodies used in this study, five detected significant amounts of epitopes associated with keratinocytes. This indicates that only certain, specific types of keratan sulfate-like carbohydrates were expressed by these cells. The extent and localization of keratan sulfate-like carbohydrates appeared to be closely related to the differentiation status of cultured keratinocytes. These epitopes were very weakly expressed on surfaces of all monolayer keratinocytes, but flattened, suprabasal cells in high Ca++ cultures strongly expressed keratan sulfate-like carbohydrates on their surfaces. A much larger population of cultured keratinocytes expressed intracellular keratan sulfate-like carbohydrates identified by the same five antibodies that detected surface epitopes. In monolayer cells, keratan sulfate-like carbohydrates were predominantly found in a broad perinuclear zone. In addition, three of the five immunoreactive antibodies detected epitopes that appeared at cell boundaries, specifically at sites of close cell-to-cell contact. Thus, molecules bearing carbohydrates recognized by anti-keratan sulfate antibodies appear at developmentally important stages of keratinocyte differentiation, indicating that these carbohydrates may serve as markers for molecules important in the differentiation of human keratinocytes.

Monoclonal antibodies that specifically recognize epitopes on keratan sulfate glycosaminoglycans were used in this study to identify carbohydrate epitopes associated with many, but not all, types of epithelial cells. Immunoreactive cells included: keratinocytes, sebaceous gland cells, eccrine sweat gland duct cells, salivary gland excretory duct cells, colon adenocarcinoma cells, embryonic chick lung epithelial cells, embryonic chick mesonephric and metanephric kidney epithelial cells, and selected embryonic chick neural tube cells. Depending upon the type of epithelium, epitopes were located either within the cytoplasm or were located on cell surfaces. These epitopes were shared by cells from both human and chick tissues, indicating the absence of species specificity. Not all anti-keratan sulfate antibodies were equally effective in identifying epithelial-associated epitopes. One of the seven antibodies employed in this study failed to detect epitopes in almost all epithelial tissues studied. Of the remaining six antibodies, three were more effective than the others in recognizing epithelial-associated epitopes. These data indicate that carbohydrates that are typically associated with extracellular matrix can also be associated with epithelial cells, but in a form that is not necessarily related to extracellular matrix. These antibodies should prove to be useful in studies of the development of epithelial cells and tissues.

The location of different proteoglycan (PG) types in the developing atherosclerotic lesion was examined by the use of monoclonal antibodies directed toward specific epitopes on distinct PG types. Sections of aorta were prepared from young White Carneau pigeons fed an atherogenic diet to induce lesions rich in lipid-laden macrophages and from older pigeons that had naturally-occurring atherosclerotic lesions. Monoclonal antibodies (MAb) 3-B-3, 9-A-2, or 2-B-6 and 5-D-4, recognizing delta Di-6S generated from chondroitin 6-sulfate (C6S) PG; delta Di-4S generated from dermatan sulfate (DS) PG and from chondroitin 4-sulfate (C4S); and sulfated poly N-acetyllactosamine sequences common to keratan sulfate (KS), respectively, were used to localize PG types by indirect immunofluorescence. In normal aorta, C6S PG was localized primarily in the media and showed a fluorescent gradient (inner media greater than outer media greater than intima greater than adventitia). In the atherosclerotic plaque, major immunoreactivity was observed using MAb 9-A-2 or 2-B-6, whereas lesser amounts were observed with 3-B-3. Patterns of immunoreactivity differed; 9-A-2 or 2-B-6 appeared to be associated with cells whereas 3-B-3 appeared to be intercellular. Although normal aorta was negative for antibody 5-D-4, recognizing KS, atherosclerotic plaques were consistently positive for this antibody. The pattern of 5-D-4 reactivity appeared to be intercellular. Except for immediately below the lesion, no reactive product using 5-D-4 was observed in the media. No major differences in distribution of PG were observed between naturally-occurring or cholesterol-induced fibrous plaques. These results indicate that both 4-sulfated PG and a previously undescribed KS glycoconjugate are major components of the atherosclerotic lesion.

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.

Antibodies to corneal keratan sulfate proteoglycan (KSPG) were used to characterize the pattern of KSPG accumulation during differentiation of neural crest cells in the stroma of embryonic chick cornea. Immunohistochemistry with monoclonal antibody I22 to keratan sulfate found this KSPG antigen localized inside stromal cells at stage 29 (Day 6), ca. 12 hr after migration into the primary stroma. A 2- to 3-day lag then occurred before appearance of extracellular keratan sulfate, first seen on Day 9 (Stage 35) in the posterior stroma. Keratan sulfate antigen accumulated in a posterior to anterior direction during subsequent development. Uniform staining of the stroma for keratan sulfate did not occur until after Day 16. Among several tissues, only corneal stroma contained an extracellular matrix which stained for keratan sulfate, though intracellular staining of some cartilage cells was observed. Accumulation of KSPG antigens in developing cornea was measured in unfractionated guanidine extracts with a quantitative ELISA using three different antibodies against KSPG. Increases were first detected after Day 9 using monoclonal I22, and somewhat later with the other two antibodies. Assays with all three antibodies detected a sustained, exponential increase of KSPG throughout the 5 days prior to hatching. Keratan sulfate continued to accumulate after hatching, but an antibody with specificity to KSPG core protein, detected no relative increase in antigen after hatching. This suggests a modulation of KSPG primary structure late in development and after hatching. Overt differentiation of individual neural crest cells thus appears to begin ca. 12 hr after their arrival in the primary stroma; a lag of 2-3 days precedes active secretion of KSPG.

A panel of four separate monoclonal antibodies, all known to specifically recognize epitopes on keratan sulfate glycosaminoglycans, were employed in an immunocytochemical study of developing chick hind limbs. In addition, two monoclonal antibodies specific for epitopes on chondroitin/dermatan sulfate glycosaminoglycans were employed on equivalent sections to determine the degree of colocalization of keratan and chondroitin/dermatan sulfates. The spatial distributions of keratan sulfate and chondroitin/dermatan sulfate differed to some extent. In younger embryos, high extracellular concentrations of keratan sulfate occurred in joints and articular cartilages, with diminishing amounts being present in epiphyseal and diaphyseal regions. The high concentration of keratan sulfate in joints and articular cartilage corresponded to equally high concentration of chondroitin-6 sulfate. With advancing age, the above mentioned distribution was modified, most notably by increased amounts of keratan sulfate within diaphyseal regions. Finally, the use of four different anti-keratan sulfate monoclonal antibodies made it possible to compare keratan sulfate epitope expression. Differences in keratan sulfate epitopes were noted in some regions of bones, mostly in diaphyseal regions of younger bones and epiphyseal regions of older bones. This pattern of keratan sulfate expression suggests that different types of keratan sulfate may be present and their expression may be developmentally regulated.

We studied changes in subchondral bone and articular cartilage in an animal model of osteoarthrosis. In this model we applied repetitive impulsive loads to rabbits' knees. Their legs were held in short leg splints so the rabbits were unable to dampen the peak applied load with ankle flexion. After sacrifice, at 1 day to 6 weeks, we studied proximal tibial load-bearing cartilage histologically, biochemically, and with radioactive sulfate uptake. We also studied the subchondral bone under that cartilage histologically, histomorphometrically, with bone scan (99mTc pyrophosphate), and by tetracycline labeling. An increase in 99mTc labeling of the subchondral bone was the first reliable change observed. This was followed by an increase in tetracycline labeling, bone formation, and a decrease in porosity, which has been associated with relative stiffening of bone. Horizontal splitting and deep fibrillation of the overlying articular cartilage followed the early bone changes. All of these changes preceded changes in content and characterization of cartilage proteoglycans or increased chondrocyte activity as manifested by incorporation of radioactive sulfate. In this model the early bone changes preceded changes in the articular cartilage. The deep splitting of articular cartilage occurred prior to metabolic alteration of that tissue.

The carbohydrate determinants of keratan sulphate recognized by three monoclonal antibodies (5-D-4, 1-B-4 and MZ15) have been investigated by solid-phase radioimmunoassay using bovine corneal keratan sulphate as the immobilized reference antigen. The antibodies appeared highly specific for sulphated poly(N-acetyllactosamine) sequences, for their binding was strongly inhibited by preparations of keratan sulphate, but not by glycoproteins with non-sulphated poly(N-acetyllactosamine) sequences of I and i antigen types, a desulphated keratan sulphate hexasaccharide, an array of neutral and sulphated mono- and disaccharides and other glycosaminoglycans. Inhibition of binding assays using a series of structurally characterized sulphated di, tetra-, hexa-, octa- and decasaccharides, and partially characterized larger oligosaccharides, isolated from bovine corneal keratan sulphate after digestion with endo-beta-galactosidase (see preceding two papers in this journal) showed that the smallest oligosaccharide reactive with all three antibodies was the linear pentasulphated hexasaccharide, E-II although antibody 1-B-4 reacted with a tetrasulphated analogue. The heptasulphated octasaccharide, G-III, was more active; among the structurally characterized keratan sulphate oligosaccharides the nonasulphated decasaccharide, I-IV, was the most active. Thus, the hepta- and octasaccharide sequences, indicated by brackets below are proposed as candidate antigenic structures recognized by the three monoclonal antibodies. (Formula: see text). Antibody 5-D-4 differs from the other two antibodies in reacting relatively strongly with a minor oligosaccharide which chromatographs as a hexasulphated octasaccharide, G-I, and most strongly with a minor sulphated, linear dodecasaccharide, J-II, which has been partially characterized [Tang, P.W., Scudder, P., Mehmet, H., Hounsell, E. F. & Feizi, T., unpublished results] and may contain N-sulphated glucosamine residues.

Host-derived sulfated components that copurify and are physically associated with the envelope of Rauscher murine leukemia virions grown in JLS-V9 cells were characterized by digestion with chondroitinase ABC and chondroitinase AC II, as well as nitrous acid degradation. A dermatan-sulfate-chondroitin-sulfate copolymer and heparin or heparan sulfate wee shown to be associated with the virions. Competitive binding studies indicated a specificity of the virions for association with heparan sulfate. The physiological importance of the association is discussed.

Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.

Articular cartilage proteoglycans from an osteoarthritic mouse strain, STR/IN, were labeled in vivo with 35S-sulfate and characterized with respect to extractability, ability to aggregate, size of monomer and glycosaminoglycan chains, sulfation of glycosaminoglycans, relative amounts of chondroitin-4 sulfate and chondroitin-6 sulfate, and link proteins. The proportion of 35S-labeled proteoglycans extractable by 0.4M guanidine hydrochloride was the same in control and osteoarthritic animals. However, a greater proportion was extractable by 4M guanidine hydrochloride in the STR/IN animals as compared with the control mice. The ability of the 35S-proteoglycans to aggregate was comparable in controls and osteoarthritic mice, as judged by their exclusion on Sepharose CL-2B. Monomers from both controls and osteoarthritic animals eluted from Sepharose CL-2B with a KAV of 0.47. Glycosaminoglycans from control and osteoarthritic animals eluted from Sepharose CL-6B with a KAV of 0.63, and no differences in sulfation or chondroitin-4 sulfate content were found. Aggregates were immunoprecipitated with link protein-specific antiserum, and only link protein 2 was found in aggregates from control and osteoarthritic animals.

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.

Chondroitin, chondroitin 6-sulphate, chondroitin 4-sulphate and dermatan sulphate proteoglycans were immunolocalized by monoclonal antibodies applied to human muscle sections digested with chondroitinase. In normal muscle the 4 proteoglycans presented a different extracellular localization: unsulphated chondroitin sulphate (chondroitin) was present in the endomysium and around capillaries, chondroitin 6-sulphate in the basal membrane zone, chondroitin 4-sulphate in the vessel adventitia, in the endomysium around capillaries and, to a lesser degree, in the perimysium, dermatan sulphate in the perimysium and, to a lesser extent, in the vessel adventitia and in the endomysium around capillaries. The enlarged endomysium of pathological muscle contained chondroitin and chondroitin 4-sulphate. Chondroitin 6-sulphate and dermatan sulphate did not seem present in the increased connective tissue. No peculiar pattern was observed in the various neuromuscular diseases studied. The specific extracellular distribution, the different biochemical composition and the different ability to bind to other extracellular components suggest a different biological role of these compounds. Chondroitin 6-sulphate is a component of a highly specialized extracellular structure, namely basal membrane. Chondroitin and chondroitin 4-sulphate participate in the composition of actively changing extracellular matrix such as the endomysium in pathological muscle. On the other hand, dermatan sulphate is a constituent of the perimysium that is a more static extracellular structure.

Recent developments in preparatory procedures for electron microscopy and immunocytochemistry are presented as a background for the morphological diagnosis of bone dysplasia. With the new low temperature embedding techniques and the availability of monoclonal antibodies raised against the main matrix macromolecules, biopsies have proved to be an important complement to clinical and radiological workup and they may sometimes provide the final clue to the diagnosis.

A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of approximately equal to 65,000 (HA-BR65). N-terminal amino acids in the complex were selectively 14C-carbamylated. The resulting derivatized HA-BR65 was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a Mr by approximately equal to 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR65 to a polypeptide of approximately equal to 55,000 (HA-BR55) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with approximately equal to 109,000 (HA-BR109) as well as HA-BR65. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at Mr approximately equal to 120,000 (HA-BR120) and approximately equal to 140,000 (HA-BR140). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.

The aim of the present report is to call attention to the fact that corpora amylacea (CA) of the brain have similar biochemical and immunological characteristics as the connective tissue matrix. We have demonstrated that CA reacted positively toward a monoclonal antibody that specifically recognized the polysaccharide determinant on cartilage proteoglycans. Material strongly bound to concanavalin A was demonstrated in CA, blood vessel wall and cartilage and suggested high levels of mannose in these structures. These findings are in agreement with the known fact that both brain and cartilage contain keratan sulfate and high mannose glycoproteins. We propose that CA may result from accumulation of glycoconjugates normally present in the brain tissue matrix as the result of aging.

Methods of isolating the water insoluble 'link proteins' from preparations of bovine nasal cartilage proteoglycan aggregates have been investigated. Upon chromatography on Sepharose 4B in 0.1% sodium dodecyl sulfate, the 'link proteins' are found in a discrete, included peak, whereas the bulk of the proteoglycan emerges at the void volume. Some low molecular weight proteoglycan is associated with the 'link proteins'. An alternative procedure, i.e. chromatography on DEAE-cellulose in sodium dodecyl sulfate, has also been examined. In 0.1% sodium dodecyl sulfate, proteoglycan monomer is not absorbed by the column, whereas 'link proteins' are. Subsequently, the 'link proteins' with a minor fraction of the proteoglycan are eluted with 1.0% sodium dodecyl sulfate. Both procedures serve to separate the 'link proteins' from the bulk of proteoglycan present in an aggregate preparation, but additional steps are necessary to achieve homogeneity. Thus a 'link protein' preparation, fractionated from proteoglycan aggregate by equilibrium density gradient centrifugation under dissociative conditions, can be finally purified by chromatography on DEAE-cellulose in sodium dodecyl sulfate.

Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.

Chondroitin sulphate proteoglycans are widespread connective tissue components and chemical analysis of cartilage and other proteoglycans has demonstrated molecular speciation involving the degree and position of sulphation of the carbohydrate chains. This may, in turn, affect the properties of the glycosaminoglycan (GAG), particularly with respect to self-association and interactions with other extracellular matrix components. Interactions with specific molecules from different connective tissue types, such as the collagens and their associated glycoproteins, could be favoured by particular charge organizations on the GAG molecule endowed by the sulphate groups. So far, it has not been possible to identify and map chondroitins of differing sulphation in tissues, but we have now raised three monoclonal antibodies which specifically recognize unsulphated, 4-sulphated and 6-sulphated chondroitin and dermatan sulphate. These provide novel opportunities to study the in vivo distribution of chondroitin sulphate proteoglycans. We demonstrate that chondroitin sulphates exhibit remarkable connective tissue specificity and furthermore provide evidence that some proteoglycans may predominantly carry only one type of chondroitin sulphate chain.

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.

In this paper we report the use of immunological methods for specifically detecting and determining proteoglycan in cartilage and other connective tissues. Antibodies (polyclonal and monoclonal) have been raised against specific components of cartilage proteoglycan aggregates (i.e., proteoglycan monomer and link protein). Radioimmunoassay procedures and immunohistochemical procedures have been developed and used to demonstrate the occurrence of cartilage-like proteoglycan and link protein in bovine aorta. Similarly, immunofluorescent studies have been used to analyze proteoglycan distribution in skin. Using antibodies specific for chondroitin-4-sulfated proteoglycan, their presence was demonstrated in dermal connective tissue and connective tissue surrounding nerve and muscle sheaths. However, chondroitin-4-sulfated proteoglycan was completely absent in the epidermis of skin and areas surrounding invaginating hair follicles. These immunological procedures are currently being used to complement conventional biochemical analyses of proteoglycans found in different connective tissue matrices.