RESUMEN
Aggrecan, the major cartilage proteoglycan, is the macromolecular species primarily involved in the resiliency of cartilage tissue. Most aggrecan molecules occur in cartilage extracellular matrix as aggregates. Each aggregate has a supramolecular structure, with many highly anionic, brushlike aggrecan subunits noncovalently bound to a hyaluronan chain. To better examine the viscoelastic properties of aggrecan aggregate, contaminating subunits were removed by exclusion chromatography. At physiologic ionic strength, concentrated solutions of purified aggrecan aggregate exhibit predominantly elastic behavior at small shear strains. However, above a critical strain, gamma c, the shear moduli show a pronounced strain-softening transition, where the storage modulus decreases suddenly, and the loss modulus exhibits a maximum. At small stresses, the creep function is also highly elastic, exhibiting an equilibrium compliance and large recoverable compliance. When the stress is amplified, a discrete transition to viscous flow occurs at a yield stress sigma y. These nonlinear responses are similar to previous observations for close-packed colloidal suspensions of soft spheres, such as microgel or emulsion particles, for which a yield transition occurs when the stress and deformation are sufficient to move a particle past its neighbors.
Asunto(s)
Proteínas de la Matriz Extracelular , Proteoglicanos/química , Agrecanos , Animales , Cartílago/química , Bovinos , Elasticidad , Técnicas In Vitro , Lectinas Tipo C , Sustancias Macromoleculares , Tamaño de la Partícula , Proteoglicanos/aislamiento & purificación , Reología , Soluciones , ViscosidadRESUMEN
The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators of biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisade layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cáscara de Huevo/química , Matriz Extracelular/química , Proteoglicanos de Heparán Sulfato/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Cáscara de Huevo/inmunología , Matriz Extracelular/inmunología , Proteoglicanos de Heparán Sulfato/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Aves de Corral , Distribución TisularRESUMEN
Skin undergoes dramatic age-related changes in its mechanical properties, including changes in tissue hydration and resiliency. Proteoglycans are macromolecular conjugates of protein and carbohydrate (glycosaminoglycan) which are involved in these tissue properties. In order to examine whether age-related changes in skin proteoglycans may contribute to the age-related changes in the mechanical properties of skin, proteoglycans from human skin of various ages were extracted and analyzed. Samples were obtained from two different fetal ages, from mature skin, and from senescent skin. As a function of age, there is a decrease in the proportion of large chondroitin sulfate proteoglycans (versican) and a concomitant increase in the proportion of small dermatan sulfate proteoglycans (decorin). Based on reactivity with antibodies to various chondroitin sulfate epitopes, fetal versican differs from the versican found in older skin with respect to the chondroitin sulfate chains. Also, the decorin of fetal skin is slightly larger, while the decorin of older skin shows greater polydispersity in both its size and its charge to mass ratio. There are also age-related differences in the size and polydispersity of the core proteins of decorin. The most pronounced change in skin proteoglycans is the appearance in mature skin of a proteoglycan which is smaller than decorin, but which has the same amino terminal amino acid sequence as decorin. This small proteoglycan is abundant in mature skin and may be a catabolic fragment of decorin or an alternatively spliced form of decorin. In light of the known ability of decorin to influence collagen fibrillogenesis and fibril diameter, the appearance of this small decorin-related proteoglycan may have a significant effect on skin elasticity. The observation that proteoglycans in skin show dramatic age-related differences suggests that these changes may be involved in the age-related changes in the physical properties of skin.
Asunto(s)
Envejecimiento/metabolismo , Proteoglicanos/metabolismo , Piel/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Fenómenos Biomecánicos , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Decorina , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular , Feto/metabolismo , Humanos , Immunoblotting , Lectinas Tipo C , Peso Molecular , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , VersicanosRESUMEN
Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Sulfatos de Condroitina/inmunología , Dermatán Sulfato/inmunología , Glicosaminoglicanos/inmunología , Proteoglicanos/inmunología , Piel/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Especificidad de Anticuerpos , Unión Competitiva , Western Blotting , Bovinos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismo , Feto , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Piel/inmunologíaRESUMEN
Skeletal muscle development is a complex process in which cell migration and adhesion play important roles. Because these cellular activities involve cell surface and extracellular matrix molecules, proteoglycan analysis was performed for developing chick skeletal muscle. Proteoglycans are macromolecular conjugates of protein and carbohydrate found in the extracellular matrix and at the cell surface. In developing muscle, both in vivo and in vitro, there is a development-related progression from synthesis of primarily large proteoglycans at earlier stages to mainly small proteoglycans at later stages. This progression was demonstrated by radiolabeling developing muscle and extracting and characterizing the proteoglycans. The large proteoglycans synthesized earlier in myogenesis have been identified as the large chondroitin sulfate proteoglycan, versican. Among the small proteoglycans synthesized at later stages is the small dermatan sulfate proteoglycan, decorin. Immunolocalization of these proteoglycans shows that versican is initially present in pericellular locations around developing myotubes, whereas decorin is observed in the epimysium early in development, and then its distribution gradually spreads to also include the perimysium and endomysium. Studies of regenerating muscle show that there is a recapitulation of the embryonic pattern of proteoglycan synthesis, which, coupled with the results from embryonic muscle development, suggests a role for versican in some early aspect of myogenesis.
Asunto(s)
Pollos/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Proteoglicanos/biosíntesis , Animales , Carne , Fibras Musculares Esqueléticas/citología , Proteoglicanos/farmacologíaRESUMEN
The extracellular matrix of human fetal skin differs substantially from that of adult skin. Fetal skin contains sparse amounts of fibrillar collagen enmeshed in a highly hydrated amorphous matrix composed of hyaluronan and sulfated proteoglycans. Both fetal and adult skin contain two major interstitial proteoglycans that are extracted by chaotrophic agents and detergents. These are the large chondroitin sulfate proteoglycan versican and the small dermatan sulfate proteoglycan decorin. For this study, proteoglycans extracted from fetal and adult skin were compared on Western blots to determine the relative amounts of versican. Decorin present in the same samples provided an internal standard for these studies. Fetal skin differed from adult skin in that it contained a significantly higher proportion of versican than did adult skin. Immunohistochemical studies compared early-fetal with mid-fetal skin and found that versican was a significant component of the interstitial extracellular matrix at both of these stages of skin development. However, by the mid-fetal period, interstitial versican became restricted to the upper half of the dermis, although versican also continued to be highly expressed around hair follicles, glands, and vasculature in the lower half of the dermis. Fetal skin extracts differed from an adult skin extract by the presence of a 66-kDa protein immunologically related to versican and by the absence of a 17-kDa core protein of a proteoglycan related to decorin. Both of these molecular species may represent degradation products of their respective proteoglycans. Monoclonal antibodies which detect epitopes in native chondroitin sulfate glycosaminoglycan chains recognized versican extracted from fetal skin. However, the tissue distribution of these antigens did not entirely conform to that for versican core protein, suggesting that versican in different regions of the skin may be substituted with glycosaminoglycan chains with different microchemistries. The results of these studies indicate that human fetal skin is structurally different from adult skin in terms of both the distribution and the composition of the large, aggregating chondroitin sulfate proteoglycan versican.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Feto , Piel/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento , Western Blotting , Células Cultivadas , Decorina , Dermis/citología , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular , Fibroblastos/citología , Humanos , Inmunohistoquímica , Lectinas Tipo C , Peso Molecular , Proteoglicanos/inmunología , Proteoglicanos/metabolismo , Piel/embriología , Factores de Tiempo , VersicanosRESUMEN
The eggshell of the chicken is a useful model to study matrix components which affect biomineralization. As an extension of our previous immunohistochemical work which suggested the presence of dermatan sulfate proteoglycans in the mineralized region of the eggshell, a study was undertaken to characterize these molecules biochemically. After demineralization with HCl and extraction with 4 M guanidinium chloride containing protease inhibitors, the extract was partitioned by anion exchange chromatography. Step elution with 0.25 M and 1.0 M sodium chloride resulted in the generation of two fractions, both of which contain chondroitinase-sensitive proteoglycans with molecular weights estimated at 200,000 by gel electrophoresis. The proteoglycans in each fraction have core proteins with molecular weights of approximately 120,000 and glycosaminoglycans with average molecular weights of 22,000. Based on differential sensitivity to chondroitinase ABC and AC II, these glycosaminoglycans contain a small proportion of dermatan sulfate. The disaccharide compositions of these glycosaminoglycans differ for the proteoglycans eluted with 0.25 M and 1.0 M sodium chloride. Those eluted with lower sodium chloride are enriched in unsulfated chondroitin and have much more 4-sulfated than 6-sulfated disaccharides; those eluted with 1.0 M sodium chloride contain primarily 4-sulfated disaccharides, a small amount of 6-sulfated disaccharides, and less unsulfated disaccharides than the proteoglycans eluted with 0.25 M sodium chloride. The large difference in the proportions of unsulfated chondroitin may be the reason for the elution at different sodium chloride concentrations. Both of the anion exchange column fractions contain other proteins in addition to the proteoglycans. These proteins are not separated from the proteoglycans by a second anion exchange column or by molecular sieve chromatography under dissociative conditions. Of particular interest is the observation that the eggshell proteoglycans and their core proteins are recognized by a monoclonal antibody which recognizes an epitope on the core protein of avian versican. This suggests that, in spite of the large differences in the sizes of the core proteins of versican and the eggshell proteoglycans, these core proteins share some homology. Because anionic molecules are thought to be important regulators of biomineralization, and because preparations like those analyzed in this study have been shown to influence in vitro calcium carbonate crystallization, the eggshell proteoglycans may play a role in eggshell mineralization.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/química , Dermatán Sulfato/química , Cáscara de Huevo/química , Matriz Extracelular/química , Secuencia de Aminoácidos , Animales , Técnica de Desmineralización de Huesos , Pollos , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Dermatán Sulfato/aislamiento & purificación , Dermatán Sulfato/metabolismo , Cáscara de Huevo/metabolismo , Matriz Extracelular/metabolismo , Femenino , Datos de Secuencia MolecularRESUMEN
The objective of this study was to immunolocalize decorin and to assess changes as a result of pyridoxine (PN) deficiency in chick articular cartilage from femoral condyles. After maintenance on a normal diet for the first two weeks after hatching, 15 broiler chickens were deprived of this vitamin for 6 weeks. It was previously shown that the ankle joints of PN-deficient animals are swollen with effusions. They also present an abnormal gait, enlarged bony margins, and fissuring of the articular cartilages. Milder changes (no fissures) were also shown in the knee joints. Data from a previous study were suggestive that sulfated glycosaminoglycans are lost from the knee cartilage surface into synovial fluid. The current study was focused on the small proteoglycan, decorin, which coats the surface of collagen fibrils and may regulate their morphology. To examine decorin in normal and PN-deficient articular cartilage, a monoclonal antibody to an epitope on the protein core of decorin was used for immunohistochemical staining of tissue sections and for Western Blot analysis of cartilage extracts. Reduction of staining with the antibody was demonstrated in the tangential surface zone of PN-deficient cartilage, and Western Blot analysis showed reduced intensity of decorin bands compared to normal controls. These data suggest that a lack of decorin may play a role in the enlargement of collagen bundles in the tangential zone of PN-deficient articular cartilage as observed in a previous electron microscopic study.
Asunto(s)
Cartílago Articular/patología , Osteoartritis/etiología , Proteoglicanos/metabolismo , Deficiencia de Vitamina B 6/complicaciones , Animales , Western Blotting , Cartílago Articular/química , Pollos , Enfermedades del Colágeno/etiología , Decorina , Proteínas de la Matriz Extracelular , Inmunohistoquímica , Masculino , Proteoglicanos/análisis , Proteoglicanos/fisiología , Distribución Aleatoria , Deficiencia de Vitamina B 6/metabolismo , Deficiencia de Vitamina B 6/patologíaRESUMEN
Chondroitin sulfate proteoglycans, cell surface and extracellular matrix molecules in both neural and non-neural tissues, are highly regulated during normal development. Entire proteoglycan molecules may be either up-regulated or down-regulated, or only the chondroitin sulfate glycosaminoglycan portions of these molecules may be modified. Subtle changes in the chemistries of chondroitin sulfate chains can now be identified through the use of a panel of anti-chondroitin sulfate monoclonal antibodies. Each of these antibodies recognizes specific chemical structures which are non-randomly dispersed along the lengths of chondroitin sulfate chains. The location of individual epitopes within defined domains in these chains is demonstrated through controlled treatments of aggrecan with chondroitinase ABC, whereby portions of these chains are removed from the non-reducing terminal ends and where the remainder of the chains remains covalently attached to the core protein. In these situations, some epitopes, such as those recognized by antibodies CS-56 and 6C3, can be removed without loss of other epitopes, such as that recognized by antibody 4C3. The independent expression of individual epitopes is demonstrated by immunocytochemical analyses of developing skin appendages in embryonic chicks and fetal humans. These are sites where highly patterned morphogenetic movements result from epithelial-mesenchymal interactions. In both chicks and humans, some epitopes are constitutively expressed while others are strictly regulated in the mesenchymal portions of the developing skin appendages. These data strongly suggest that chondroitin sulfate proteoglycans, including their chondroitin sulfate chains, have important roles in regulating these epithelial mesenchymal interactions. Furthermore, these data underscore the significance of the aforementioned observation that individual epitopes are located in specific domains within chondroitin sulfate chains. The highly organized expression of chondroitin sulfate proteoglycans in the development of the central nervous system strongly argues for a similar role for these molecules in the organs that comprise this system.
Asunto(s)
Anticuerpos Monoclonales , Sulfatos de Condroitina/biosíntesis , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea , Embrión de Pollo , Condroitín Liasas/química , Sulfatos de Condroitina/química , Sulfatos de Condroitina/inmunología , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales , Epitelio/metabolismo , Epítopos , Humanos , Hidrólisis , Inmunohistoquímica , Proteoglicanos/biosíntesis , Piel/citología , Piel/embriología , Piel/metabolismoRESUMEN
The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Colágeno/análisis , Cáscara de Huevo/química , Animales , Pollos , Matriz Extracelular/químicaRESUMEN
Extracellular matrix development of chicken pectoral muscle was examined in the Low Score Normal (LSN) genetic muscle weakness and compared to both normal and avian muscular dystrophy (MD). At 20 days of embryonic development significant elevations were noted in LSN total glycosaminoglycan concentration and decorin, while at 14 days, LSN glycosaminoglycan and decorin levels were indistinguishable from the controls. Levels of a large skeletal muscle chondroitin sulfate proteoglycan (M-CSPG) appear to be unaffected. Morphologically, at 20 days, the extracellular matrix space between muscle fibers increased to a level characteristic to that observed in avian muscular dystrophy. At six weeks posthatch a marked increase in LSN collagen crosslinking relative to MD or control tissues was observed, while collagen concentration was not altered. By one year posthatch LSN collagen crosslink levels did not significantly differ from normal tissue. These data support the concept that the LSN muscle weakness is associated with changes in both proteoglycan and collagen characteristics.
Asunto(s)
Colágeno/metabolismo , Distrofia Muscular Animal/metabolismo , Músculos Pectorales/metabolismo , Proteoglicanos/metabolismo , Animales , Aves , Embrión de Pollo , Pollos , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Reactivos de Enlaces Cruzados , Decorina , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular , Femenino , Glicosaminoglicanos/metabolismo , Masculino , Debilidad Muscular , Músculos Pectorales/embriologíaRESUMEN
The avian eggshell is a natural biopolymer and mineral composite. It is a very useful model for biomimetic mineralization, since it is among the fastest forming hard tissues known. Isolated eggshell membranes, which were demineralized in vitro, were used to investigate the in vitro modulation of CaCO3 crystal deposition by organic matrix materials. Crystallization on the demineralized eggshell membrane occurred almost exclusively at the peripheries of residual calcium reserve assemblies, which contain a high concentration of sulfur. Similar structures are observed for eggshell membranes after natural demineralization. The characteristic rhombohedral crystal morphologies of the calcite crystals grown in this in vitro system are much less regular when grown in the presence of organic matrix or partially purified dermatan sulfate proteoglycans obtained from the eggshell. The effect of these macromolecules on the morphology and size of CaCO3 crystals is concentration-dependent. These studies indicate the complexity of the molecular and ionic interactions involved in the initiation and formation of the eggshell, with the focus on the role of the organic matrix.
Asunto(s)
Cáscara de Huevo/metabolismo , Animales , Biopolímeros , Carbonato de Calcio/metabolismo , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Colágeno/metabolismo , Cristalización , Dermatán Sulfato/metabolismo , Cáscara de Huevo/ultraestructura , Membranas/metabolismo , Membranas/ultraestructura , Microscopía Electrónica de Rastreo , Azufre/metabolismoRESUMEN
Developing skeletal muscle cells, as both myoblasts and myotubules, synthesize a distinctive large chondroitin sulfate proteoglycan. To probe the role of this proteoglycan in myogenesis, chick embryonic muscle cells in culture were treated with beta-D-xyloside, a compound which interferes with proteoglycan synthesis by acting as an artificial acceptor for glycosaminoglycan synthesis and thereby competing with the proteoglycan core protein. Analysis of the proteoglycans indicates that with increasing concentrations of beta-D-xyloside, synthesis of the chondroitin sulfate proteoglycan is inhibited, with concomitant massive synthesis of xyloside-linked chondroitin sulfate glycosaminoglycans. Xyloside does not appear to inhibit glycosaminoglycan attachment onto the small heparan sulfate and dermatan sulfate proteoglycans which are synthesized in the muscle cultures, even though, because of the mechanism of action of beta-xyloside, these proteoglycans should be affected. At submaximal concentrations of beta-xyloside, there is synthesis of both large chondroitin sulfate proteoglycans and xyloside-linked chondroitin sulfate. The xyloside-linked chondroitin sulfate chains have the same sulfation pattern as the core protein-bound skeletal muscle chondroitin sulfate (90% 6-sulfated isomer), but are much smaller (24,000 vs. 65,000 in molecular weight). The discrepancy in size but identify of sulfation indicates that, although sulfation takes place normally on either the core protein or the xyloside acceptor, termination of glycosylation occurs earlier for xyloside-initiated chondroitin sulfate. In spite of these dramatic effects on chondroitin sulfate proteoglycan synthesis, beta-xyloside elicits no observable effects on in vitro myogenesis. This suggests that the function served by the large chondroitin sulfate proteoglycan is not required in the more simplified environment of the muscle cultures.
Asunto(s)
Glicósidos/farmacología , Músculos/metabolismo , Proteoglicanos/biosíntesis , Animales , Células Cultivadas , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Cromatografía en Gel , Decorina , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular , Glicósidos/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/aislamiento & purificación , Cinética , Sustancias Macromoleculares , Peso Molecular , Músculos/efectos de los fármacos , Proteoglicanos/aislamiento & purificación , Factores de TiempoRESUMEN
Large, chondroitin sulphate-containing proteoglycans are synthesized by three prominent tissue in the embryonic chick limb. One of these proteoglycans is aggrecan, the phenotype-specific proteoglycan of cartilage. Another, PG-M, is produced by prechondrogenic mesenchymal cells. The third, M-CSPG, is made by developing skeletal muscle cells. While the carbohydrate components of PG-M and M-CSPG share some similarities, both of these proteoglycans clearly have different carbohydrate moieties from those of aggrecan. To compare these three proteoglycans at another level, their core protein structures were analysed in three ways: by the presence or absence of monoclonal antibody epitopes, by one-dimensional peptide display of the cyanogen bromide-cleaved core proteins and by electron microscopic imaging of the molecules. Monoclonal antibodies whose epitopes are present in aggrecan core protein were tested with core protein preparations from M-CSPG and PG-M. One of these, 7D1, recognizes both PG-M and M-CSPG, while another, 1C6, shows no reactivity for the non-cartilage proteoglycans. The absence of 1C6 reactivity is of interest, as its epitope is in a region of the aggrecan core protein known to have a functional homologue in the core proteins of PG-M and M-CSPG. The cyanogen bromide-fragmented peptide pattern of M-CSPG is the same as that of PG-M, and both are different from that of aggrecan. The aggrecan pattern has one prominent large band (molecular mass 130 kDa), some less prominent large bands (molecular mass 70-100 kDa) and several smaller bands. In contrast, the PG-M and M-CSPG patterns show no bands with molecular masses > 73 kDa, and the smaller bands (molecular mass < 40 kDa) have a different pattern to that of the smaller bands from aggrecan. The electron microscopic images of aggrecan show a core protein with one end having two globular regions separated by a short linear segment; adjacent to this is a long linear segment, which sometimes contains a third globular region at the end of the core protein opposite the end with the double-globe structure. M-CSPG and PG-M core proteins never show images with the double-globe structure. Instead, one end of the molecule has a single globular domain, and a second globular region is variably present at the opposite end of the core protein. Thus, by all three methods, the core proteins of PG-M and M-CSPG appear to be the same and both differ from the core protein of aggrecan.
Asunto(s)
Cartílago/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Mesodermo/metabolismo , Músculos/metabolismo , Animales , Anticuerpos Monoclonales , Western Blotting , Cartílago/embriología , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/ultraestructura , Cromatografía por Intercambio Iónico , Bromuro de Cianógeno , Microscopía ElectrónicaRESUMEN
Monoclonal antibodies have been developed that recognize epitopes in native chondroitin sulfate chains. One of these antibodies, CS-56, reportedly recognizes chondroitin 4- and 6-sulfates. However, this antibody, and four other anti-chondroitin sulfate antibodies, 4C3, 4D3, 6C3 and 7D4, do not recognize epitopes in chondroitin sulfate chains from Swarm rat chondrosarcoma proteoglycan, an indication that native chondroitin sulfate epitopes are more structurally complex than the standard 0-, 4-, and 6-sulfated disaccharide repeats that constitute the backbone of chondroitin sulfate chains. A series of limited chondroitinase digestions was performed on the large aggregating proteoglycan monomer extracted from embryonic chick chondrocyte cultures to identify the digestion parameters required to release the different native chondroitin sulfate epitopes. Some epitopes were more accessible to enzymatic digestion than other epitopes. The approximate location of epitopes was determined by measuring the size of undigested oligosaccharides retained on the core protein following a limited digestion, and correlating this with the level of immunoreactivity for the different antibodies. These analyses identified the locations of three different antigenic domains. Domain 1 resides at the linkage region and contains epitopes for two of the five antibodies, and a portion of the epitopes for a third antibody. Domain 2 lies in the interior of the chain and contains epitopes for three of the five antibodies. Domain 3 resides at the non-reducing terminus and does not contain epitopes for any of the anti-chondroitin sulfate antibodies used in this study. These results indicate that specific native chondroitin sulfate epitopes are non-randomly distributed within the linear framework of chondroitin sulfate chains.
Asunto(s)
Anticuerpos Monoclonales , Sulfatos de Condroitina/inmunología , Epítopos , Animales , Bovinos , Embrión de Pollo , Sulfatos de Condroitina/química , Condroitinasas y Condroitín Liasas/farmacología , Ensayo de Inmunoadsorción Enzimática , Ratas , TiburonesRESUMEN
There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.
Asunto(s)
Cáscara de Huevo/química , Matriz Extracelular/química , Animales , Anticuerpos Monoclonales , Pollos , Matriz Extracelular/inmunología , Glicosaminoglicanos/química , Glicosaminoglicanos/inmunología , InmunohistoquímicaRESUMEN
Chick embryonic skeletal muscle synthesizes three major types of proteoglycans: large chondroitin sulfate proteoglycans, small dermatan sulfate proteoglycans and small heparan sulfate proteoglycans. A monoclonal antibody has been raised which recognizes the small dermatan sulfate proteoglycan. Immunoblot analysis of a partially purified preparation of skeletal muscle proteoglycans indicates that the antibody reacts with a molecule which migrates with an estimated Mr of 100,000. Prior treatment of the proteoglycans with chondroitinase results in immunostaining of a species of estimated Mr 45,000. These values for the intact proteoglycan and its core protein suggest that the antibody is directed against a proteoglycan of the PG-II or decorin class. Immunohistochemistry indicates a widespread distribution of the proteoglycan, which is localized in connective tissue septa of skeletal and cardiac muscle, dermis, tendon, bone, perichondrium and cornea. Immunoblot analysis of the proteoglycan core proteins from these tissues demonstrates that the antibody recognizes the same 45,000-dalton band in each tissue. The widespread tissue distribution is also consistent with the antibody being directed against an epitope of PG-II. Neither the glycosaminoglycan chains nor N-linked oligosaccharides are required for reactivity and the antibody cross-reacts with other avian material, but not mammalian. This antibody, which has been designated CB-1, reveals developmental stage-specific changes in the deposition of PG-II in embryonic limb bud and skeletal muscle.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Pollos/inmunología , Proteoglicanos Tipo Condroitín Sulfato/inmunología , Dermatán Sulfato/inmunología , Proteoglicanos/análisis , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Embrión de Pollo , Condroitín Liasas/farmacología , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Tejido Conectivo/química , Tejido Conectivo/embriología , Decorina , Dermatán Sulfato/aislamiento & purificación , Proteínas de la Matriz Extracelular , Técnica del Anticuerpo Fluorescente , Humanos , Mamíferos/inmunología , Ratones , Músculos/química , Músculos/embriología , Especificidad de ÓrganosRESUMEN
In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas de la Matriz Extracelular/análisis , Matriz Extracelular/fisiología , Glicoproteínas/análisis , Glicosaminoglicanos/análisis , Músculos/embriología , Proteoglicanos , Agrecanos , Animales , Anticuerpos Monoclonales , Embrión de Pollo , Pollos , Matriz Extracelular/ultraestructura , Extremidades/embriología , Lectinas Tipo C , Músculos/citología , Músculos/fisiologíaRESUMEN
Recently, an assay for quantification of glycosaminoglycans has been reported based on precipitation with Safranin O (Lammi, M. and Tammi, M. (1988) Anal. Biochem. 168, 352-357). In this procedure, the precipitate which forms when the glycosaminoglycan or proteoglycan is mixed with the Safranin O is collected with a dot-blot apparatus onto a membrane filter. The intensity of the color in the dots is measured densitometrically and is proportional to the amount of glycosaminoglycan or proteoglycan in the sample. This report describes a modification of the densitometric Safranin O assay which allows its use as a spectrophotometric assay. For this, the precipitates are solubilized in cetylpyridinium chloride and the absorbance determined for the resulting solutions. As with the densitometric method, guanidinium chloride diminishes the color intensity. However, the color is stable, even after solubilization, for at least one week. The precipitates collected from as much as 10 micrograms of material can be solubilized in as little as 100 microliters of cetylpyridinium chloride, so that increased sensitivity may be obtained if the solubilized precipitate is measured in a microcuvet. Thus, solubilization with cetylpyridinium chloride allows use of the Safranin O assay for glycosaminoglycans and proteoglycans even when a densitometer is unavailable.
Asunto(s)
Glicosaminoglicanos/análisis , Espectrofotometría/métodos , Cetilpiridinio/química , Precipitación Química , Sulfatos de Condroitina , Guanidina , Guanidinas/química , Heparitina Sulfato/análisis , Fenazinas/análisis , Solubilidad , Análisis EspectralRESUMEN
Chondrocytes of different ages synthesize proteoglycans which have structural differences in both the chondroitin sulfate and keratan sulfate glycosaminoglycans. In order to ascertain whether age-dependent differences also occur in the core protein, the chick limb bud mesenchymal cell culture system was utilized to analyze newly synthesized proteoglycan core protein from undifferentiated mesenchymal cells (day 1 and 2), newly differentiated cartilage (day 4), mature cartilage (day 8), and senescent cartilage (day 16). The core protein synthesized at various times was identified by radiolabeling with [3H]leucine and [35S]sulfate immediately prior to extraction and purification. The sizes of the various core protein preparations were compared by electrophoresis on a 3% polyacrylamide gel after partial deglycosylation with chondroitinase AC and keratanase. The proteoglycans from day 4, 8, and 16 cultures each give rise to a single band of approximately 475,000 daltons. The proteoglycans from day 1 and 2 cultures also give rise to the 475,000 dalton band, but each contains several other components which produce a smear of high molecular weight material on the gel. The monomer proteoglycans were incubated with cyanogen bromide and the resultant peptides separated by electrophoresis on a 5-17.5% polyacrylamide gel. The peptide displays of core proteins synthesized on days 4, 8 and 16 are virtually identical in terms of the number and electrophoretic distribution of the core protein peptides. In contrast, proteoglycan core proteins from day 1 and day 2 cultures give rise to peptide displays which resemble those from older cultures in some respects but have distinct features as well. The absence of structural variation in the newly synthesized proteoglycan core proteins from cartilage of different ages suggests that the age-related changes in the structure of the intact proteoglycans result from differences in the glycosaminoglycan biosynthetic machinery rather than alterations in the acceptor molecule (i.e., the core protein).