scholarly journals Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1978 ◽  
Vol 175 (3) ◽  
pp. 913-919 ◽  
Author(s):  
Dick Heinegård ◽  
Stefan Lohmander ◽  
Johan Thyberg
Keyword(s):  
Electron Microscopy ◽  
Hyaluronic Acid ◽  
Electron Micrographs ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Side Chain ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Central Filament ◽  
Proteoglycan Aggregates

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.

scholarly journals Domain structure of cartilage proteoglycans revealed by rotary shadowing of intact and fragmented molecules

1984 ◽  
Vol 224 (1) ◽  
pp. 331-333 ◽  
Author(s):  
H Wiedemann ◽  
M Paulsson ◽  
R Timpl ◽  
J Engel ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Side Chain ◽  
Keratan Sulphate ◽  
Molecular Electron ◽  
Attachment Region ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans ◽  
Rotary Shadowing

The rotary-shadowing technique for molecular electron microscopy was used to study cartilage proteoglycan structure. The high resolution of the method allowed demonstration of two distinct globular domains as well as a more strand-like portion in the core protein of large aggregating proteoglycans. Studies of proteoglycan aggregates and fragments showed that the globular domains represent the part of the proteoglycans that binds to the hyaluronic acid, i.e. the hyaluronic acid-binding region juxtapositioned to the keratan sulphate-attachment region. The strand-like portion represents the chondroitin sulphate-attachment region. Low-Mr proteoglycans from cartilage could be seen as a globule connected to one or two side-chain filaments of chondroitin sulphate.

scholarly journals Dermatan sulphate proteoglycans from sclera examined by rotary shadowing and electron microscopy

1987 ◽  
Vol 242 (3) ◽  
pp. 761-766 ◽  
Author(s):  
N P Ward ◽  
J E Scott ◽  
L Cöster
Keyword(s):  
Electron Microscopy ◽  
Chondroitin Sulphate ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Cartilage Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Rotary Shadowing

Two dermatan sulphate-containing proteoglycans from bovine sclera were examined by rotary shadowing and electron microscopy, and the results were compared with previous biochemical findings. Both the large iduronate-poor proteoglycan (PGI) and the small iduronate-rich proteoglycan (PGII) possessed a globular proteinaceous region. Whereas PGI had a branched extension from the globular region, with five to eight side chains attached to it, PGII had only a single tail, which was of glycosaminoglycuronan. PGII aggregated via globular-region interactions, which were much diminished by reduction and alkylation. PGI aggregated via side chains and globular-region interactions. Although a few PGI aggregates were large, and similar to the hyaluronan-cartilage proteoglycan aggregates [Weidemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333], hyaluronan did not cause enhanced aggregation. PGII is very similar in shape to the small cartilage chondroitin sulphate proteoglycan, whereas PGI somewhat resembles the large cartilage chondroitin sulphate proteoglycan, although with many fewer glycosaminoglycan side chains, and probably only one globular region as opposed to two in the cartilage proteoglycan.

scholarly journals Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage

1981 ◽  
Vol 199 (1) ◽  
pp. 17-29 ◽  
Author(s):  
S Björnsson ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Cell Cultures ◽  
Low Ph ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans ◽  
Free Monomer

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.

Analysis of the intermediate size proteoglycans from the developing chick limb buds11Presented in part at 72nd Annual Meeting of the American Society of Biological Chemists, St Louis, Missouri, U.S.A. 31 May to 4 June, 1981.

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 61-74
Author(s):  
Nagaswamisri Vasan
Keyword(s):  
Chondroitin Sulphate ◽  
Binding Capacity ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Limb Bud ◽  
Side Chain ◽  
Intermediate Size ◽  
Proteoglycan Aggregates ◽  
American Society ◽  
Hyaluronic Acid Binding

Limb-bud proteoglycans are heterogeneous molecules which vary in their chemical and physical properties with development. This report describes proteoglycan intermediates (PG-I) that predominate in stage-34 limbs, and compares them with proteoglycan aggregates (PG-A) in stage-38 limbs. We analysed proteoglycans and their components extracted with guanidinium chloride by subjecting them to density gradient centrifugation, molecular sieve chromatography, electrophoretic separation, and selective enzymatic degradation. PG-I and PG-A have similar chondroitin sulphate composition, amino sugars, chondroitin sulphate side-chain length, glycoprotein link factors, and hyaluronic acid binding capacity, and both cross react with antisera prepared against cartilage-specific chick sternal proteoglycans. However, PG-I has lower molecular weight, lower buoyant density, and fewer chondroitin sulphate side chains on the protein core. The PG-I in the developing limb can be considered a mixture of smaller aggregates and cartilage-specific large monomers in which the former predominate.

scholarly journals Proteoglycans of hyaline cartilage: Electron-microscopic studies on isolated molecules

1975 ◽  
Vol 151 (1) ◽  
pp. 157-166 ◽  
Author(s):  
J Thyberg ◽  
S Lohmander ◽  
D Heinegård
Keyword(s):  
Hyaline Cartilage ◽  
Chondroitin Sulphate ◽  
Costal Cartilage ◽  
Side Chain ◽  
Gel Chromatography ◽  
Electron Microscopic ◽  
The Core ◽  
Central Filament ◽  
Microscopic Studies ◽  
Chondroitin Sulphate Chain

Proteoglycan monomers from guinea-pig costal cartilage, bovine nasal and bovine tracheal cartilage were observed in the electron microscope after being spread in a monomolecular layer with cytochrome c. The proteoglycan molecule appeared as an extended central core filament to which side-chain filaments were attached at various intervals. The molecules from the three sources displayed great ultrastructural similarities. On average, the core filament was about 290 nm long, there were about 25 side-chain filaments per core filament, the side-chain filaments were about 45 nm long, and the distance between the attachment points of the side-chain filaments to the core filament was about 11 nm. With regard to the overall size of the molecules, no evidence of distinct subpopulations was obtained. Good correlation was found between ultrastructural data for the proteoglycan molecules and chemical data obtained by enzyme digestions and gel chromatography. Together these data strongly support the interpretation of the electron-microscopic pictures as indicating a central filament corresponding to the protein core and side-chain filaments corresponding to the chondroitin sulphate chain clusters of the proteoglycan monomers.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species

1981 ◽  
Vol 199 (1) ◽  
pp. 81-87 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Limb Bud ◽  
Binding Region ◽  
Human Cartilage ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycans ◽  
Rabbit Articular Cartilage ◽  
Hyaluronic Acid Binding

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.

scholarly journals Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

1982 ◽  
Vol 203 (3) ◽  
pp. 691-698 ◽  
Author(s):  
Harold D. Keiser
Keyword(s):  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Antigenic Determinants ◽  
Density Gradient Ultracentrifugation ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Immunoprecipitation Reaction ◽  
Hyaluronic Acid Binding

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.

scholarly journals Extended and globular protein domains in cartilage proteoglycans

1987 ◽  
Vol 245 (3) ◽  
pp. 763-772 ◽  
Author(s):  
M Paulsson ◽  
M Mörgelin ◽  
H Wiedemann ◽  
M Beardmore-Gray ◽  
D Dunham ◽  
...  
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Globular Domain ◽  
Protein Core ◽  
Nasal Cartilage ◽  
Cdna Sequences ◽  
Multidomain Structure ◽  
Variable Extent ◽  
Cartilage Proteoglycans

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3′-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].

scholarly journals Cartilage proteoglycan aggregate formation Role of link protein

1981 ◽  
Vol 197 (3) ◽  
pp. 669-674 ◽  
Author(s):  
A Franzén ◽  
S Björnsson ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Dry Weight ◽  
Aggregate Formation ◽  
Binding Region ◽  
Link Protein ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.

scholarly journals Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates

1995 ◽  
Vol 307 (2) ◽  
pp. 595-601 ◽  
Author(s):  
M Mörgelin ◽  
M Paulsson ◽  
D Heinegård ◽  
U Aebi ◽  
J Engel
Keyword(s):  
Electron Microscopy ◽  
Core Protein ◽  
Gel Filtration ◽  
Spatial Arrangement ◽  
Link Protein ◽  
Original Length ◽  
Central Filament ◽  
Proteoglycan Aggregates ◽  
Rotary Shadowing

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.

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