Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.     

Comparative studies on human and bovine nasal cartilage proteoglycan complex components

Summary Human and bovine nasal proteoglycan complex components were prepared in parallel. Some molecular properties of human and bovine proteoglycan subunits (PGS) were compared. Two major human link-like proteins have been characterized and purified; their compositions and molecular weights were very similar to those observed for the previously described bovine link proteins.     

Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.     

Dilute solution properties of proteoglycan fractions from bovine nasal cartilage

Fresh proteogycans (adult bovine nasal cartilage) isolated from the densest portion of a dissociative density gradient had a weight-average molecular weight of ca. 10⁶ in 4M guanidine hydrochloride (GdnHCI) by light scattering. Fractions of such material obtained by elution with 4M GdnHCI from 2% agarose gel, both normal and cross-linkd, has proteoglycan subunit molecular weights ranging from 0.8 to 2.6 × 10⁶ and root-mean-square radii ranging from 35 to 52 nm in the same solvent. The protein molecular weight per proteoglycan subunit was about 1.2 × 10⁵ and that of keratan sulfate about 1.8 × 10⁵, both independent of total molecular weight. A random-flight “graft copolymer” model having uniform side chains of chondroitin sulfate (40 disaccharides) and keratan sulfate (15 disaccharides) and a random-coil polypeptide back bone was used to estimate the unperturbed radius, whihc was about 19 nm for a mol wt of 1.5 × 10⁶. Experimental light-scattering data for fractions were fitted very well by theoretical curves for the particale scattering factor for both linear and appropriate branched polymers. Examination of coil expansion on the basis of perturbation calculations for branched polymer models suggested that expansion did not account for the experimentally observed radii in terms of unperturbed radii calculated from the model. A possible explanation is that substantial local stiffening of the polypeptide chain due to substitution of side-chain clusters increases the unperturbed radii. The intrinsic viscosity [η] is 4M GdnHCI ranged from 120 to 180 ml/g, and could be interpreted in terms of th eequivalent sphere model; the Flory number has approximately its normal value for flexible linear polymers. The treatment of the sedimentation coefficient by this is less successful, since the Man delkern-Flory parameter β apparently increases with increasing molecular weight; average value are similar to those for flexible polymers, but the variation in β makes this method useful only for rough estimation of molecular weight of proteoglycans. Molecular weights of purified proteoglycans are the same in 0.2M NaCI as in 4M GdnHCI, while crude preparations gave higher molecular weights in 0.2M NaCI, probably because of association due to incomplete removel of “linking” proteins.     

Small-angle x-ray scattering of bovine nasal cartilage proteoglycan in solution

The proteoglycan subunit (PGS) from bovine nasal cartilage was examined in water and in 0.15 N LiCl by small-angle x-ray scattering (SAXS). The molecular weight of 2.5 × 10⁶ and the radius of gyration, R_g = 493 Å, in 0.15 N LiCl, obtained by SAXS, are in good agreement with values reported by others for similar preparations. Values of the radius of gyration of the cross section, mass per unit length, and persistence length of the PGS are also reported. The low value of intrinsic viscosity ([η]) found in 0.15 N LiCl, and a comparison of the experimental distance distribution function to that of the theoretical distance distribution function for sphere, suggest that the PGS in salt solution approaches spherical symmetry. The much higher value of [η] in water suggests a prolate ellipsoid of low axial ratio.     

Electron microscopic studies on guinea pig rib cartilage

Summary The guinea-pig rib cartilage consists of chondrocytes dispersed in an intercellular substance composed of collagen fibrils, often characteristically cross-striated, and polygonal granules. Electron-dense membrane-bounded matrix vesicles are also observed intercellularly, especially in the central, partly calcified zone of the cartilage. With respect to their location in a cross-section of the rib, the chondrocytes differ in size, shape and intracellular fine structure. Thus, three separate types of cells are recognized. Peripheral chondrocytes have a flattened shape and are largely occupied by the nucleus. In the cytoplasm, the granular endoplasmic reticulum is the most extensive organelle. Intermediate chondrocytes are oval or round in shape. The endoplasmic reticulum and the Golgi complex are both prominent. Mitochondria and membrane-bounded cytoplasmic dense bodies are more numerous than in the peripheral cells. The ground cytoplasm often contains a few lipid droplets. In the central chondrocytes, such droplets sometimes fill the entire cytoplasm. Concomitantly, the nucleus is usually completely heterochromatic and the cells are therefore regarded as being metabolically inert.After preparations including ruthenium red staining en bloc, the general stainability of the chondrocytes is decreased. Intracellularly, positive ruthenium red staining of granular material within the Golgi vacuoles are to be observed. Extracellularly, the matrix granules are stained with this polyvalent, cationic dye. Extraction of the cartilage with 4 M guanidine-HCl removes all matrix granules and about 70% of the proteoglycans, measured as hexosamine, from the tissue. It is concluded that the matrix granules contain proteoglycan complexes.Financial support was received from the Swedish Medical Research Council (proj. no. 12X-3355), the Swedish Cancer Society (proj. no. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Harald and Greta Jeansson Foundation, the C. B. Nathhorst Foundation, and from the funds of Karolinska Institutet.The skilled technical assistance of Mrs. Eva Lundberg and the secretarial assistance of Mrs. Inger Åhrén are gratefully acknowledged. The authors are indebted to Dr. S. Lohmander for helpful suggestions during the progress of the work.     

The subunits of chemically treated proteoglycan isolated from bovine nasal cartilage

1. The light fraction of the proteoglycan of bovine nasal cartilage was split by treatment with 0.1m-hydrochloric acid in acetone. The products were separated by gel filtration on 4% agarose and two retarded fractions were detected and isolated. These two fractions were found to have a Stokes radius of 134 and 47 A respectively, as determined by calibration of the column against proteins of known hydrodynamic volumes. 2. The 47 A fraction had a protein content of 4% and a glucosamine/galactosamine ratio 1:23. The 134 A fraction had a protein content of 20% and a glucosamine/galactosamine ratio 1:4.8. 3. The results of the viscometric studies on both fractions suggested that the 134 A fraction alone exhibited the property of undergoing reversible pH-dependent aggregation with a transition point at pH4.9. 4. It was concluded that these fractions could represent subunits of the native cartilage proteoglycan.     

Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan.

Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.     

Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions.

Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a 'beaded' structure and a contiguous filamentous 'tail' at one end and others which appeared as round 'blobs', some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the 'blob-like' appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.     

Studies on the polydispersity and heterogeneity of cartilage proteoglycans. Identification of 3 proteoglycan structures in bovine nasal cartilage.

1. Three chondroitin sulphate components were isolated from adult bovine nasal cartilage after treatment with alkaline NaB3H. Average molecular weights of 13000, 18 600 and 28 000 were obtained for chondroitin sulphate species representing 10, 52 and 38% (w/w) of the total chondroitin sulphate respectively. Each chondroitin sulphate pool has a narrow molecular-weight distribution. 2. A proteoglycan subunit preparation, isolated from one nasal cartilage by extraction and density-gradient fractionation in dissociative solvents, partitioned on a CSCl density gradient according to size and composition. Variation of proteoglycan molecular weight across the gradient was directly related to the average chondrotin sulphate chain length, which in turn reflected the relative proportion of the three chondroitin sulphate pools in each proteoglycan fraction. Consideration of proteoglycan molecular parameters, compositions and behaviour on sedimentation leads to a proposal that nasal cartilage contains 3 distinct proteoglycan pools, each of which has a constant number of chondroitin sulphate side chains of different average molecular weight. 3. Molecular-weight distribution parameters for these proteoglycan preparations indicate that all serine residues on the protein core capable of initiating chondroitin sulphate biosynthesis are occupied and that proteoglycan polydispersity results directly from the polydispersity of the attached chondroitin sulphate component.     

Identification of proteoglycan from salmon nasal cartilage

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207 bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine–glycine, found in mammalian aggrecans were observed in salmon, however, random serine–glycine (or glycine–serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after β-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.     

The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage.

A preparation containing the link proteins may be obtained from bovine nasal cartilage by extraction with 4 M guanidine hydrochloride and by equilibrium density gradient centrifugations of the extract as commonly employed in the isolation of proteoglycan monomers. In the present paper, protein-rich proteoglycans have been removed from such a preparation to give purified link proteins by chromatography on Sepharose CL-6B in 1% sodium dodecyl sulfate. The individual link proteins, which in order of increasing electrophoretic mobility are termed link proteins 1, 2, and 3, have been separated and isolated in a subsequent preparative gel electrophoresis step. The link proteins present in largest amount, link proteins 1 and 2, have essentially the same amino acid compositions, and following partial digestion with the V8 protease from Staphylococcus aureus and analytical electrophoresis in sodium dodecyl sulfate, their peptide patterns closely resemble each other. Therefore,it is probable that link proteins 1 and 2 are structurally similar. Link protein 1 contains more carbohydrate than link protein 2 (9.5% and 3.0%, respectively) and it is suggested that the major difference between them is in carbohydrate content.     

Isolation and chemical characterization of two distinct "link proteins" from bovine nasal cartilage proteoglycan complex

Bovine nasal cartilage proteoglycan aggregates have been dissociated and separated by dissociative density gradient centrifugation into proteoglycan sub-units and "link fraction". The latter contained mainly the two "link proteins" as shown by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two "link proteins" were then separated by preparative gel electrophoresis under dissociative conditions. Molecular weight and amino acid composition of both proteins are presented.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.