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 Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures

1979 ◽  
Vol 179 (1) ◽  
pp. 35-45 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Antigenic Site ◽  
Trypsin Digestion ◽  
Antigenic Determinants ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Before And After ◽  
Hyaluronic Acid Binding

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide ‘maps’ prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.

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 Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

1982 ◽  
Vol 203 (3) ◽  
pp. 683-689 ◽  
Author(s):  
H D Keiser ◽  
J B Adlersberg ◽  
H M Steinman
Keyword(s):  
Biochemical Characterization ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Exchange Chromatography ◽  
Equilibrium Density ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Sepharose 4B

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Radioimmunoassay of the molecules and the substructures

1980 ◽  
Vol 187 (3) ◽  
pp. 687-694 ◽  
Author(s):  
J Wieslander ◽  
D Heinegárd
Keyword(s):  
Hyaluronic Acid ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Relative Content ◽  
Binding Region ◽  
Rich Region ◽  
Link Protein ◽  
Specific Radioimmunoassay ◽  
Cartilage Proteoglycans ◽  
Hyaluronic Acid Binding

Antibodies specifically reacting with the link proteins, the hyaluronic acid-binding region and chondroitin sulphate-peptides were used to design specific radioimmunoassay procedures. The sensitivity of the method used for the link protein was about 20 ng/ml, and the other two components could be determined at concentrations of about 2 ng/ml. The radioimmunoassay procedures were tested by using proteoglycan subfractions or fragments thereof. The procedures used to quantify link protein and hyaluronic acid-binding region showed no cross-interference. Fragments of trypsin-digested proteoglycan monomers still reacted in the radioimmunoassay for hyaluronic acid-binding region. Subfractions of proteoglycan monomers separated according to size had a gradually higher relative content of the hyaluronic acid-binding region compared with both chondroitin sulphate-peptides and uronic acid, when the molecules were smaller. The proteoglycans therefore may contain a variably large chondroitin sulphate-rich region, which has a constant substitution with polysaccharide side chains.

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 A chondroitin sulphate proteoglycan from human cultured glial and glioma cells. Structural and functional properties

1984 ◽  
Vol 221 (3) ◽  
pp. 845-853 ◽  
Author(s):  
B Norling ◽  
B Glimelius ◽  
A Wasteson
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Glioma Cells ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans ◽  
Structural And Functional Properties

A chondroitin sulphate proteoglycan capable of forming large aggregates with hyaluronic acid was identified in cultures of human glial and glioma cells. The glial- cell- and glioma-cell-derived products were mutually indistinguishable and had some basic properties in common with the analogous chondroitin sulphate proteoglycan of cartilage: hydrodynamic size, dependence on a minimal size of hyaluronic acid for recognition, stabilization of aggregates by link protein, and precipitability with antibodies raised against bovine cartilage chondroitin sulphate proteoglycan. However, they differed in some aspects: lower buoyant density, larger, but fewer, chondroitin sulphate side chains, presence of iduronic acid-containing repeating units, and absence (less than 1%) of keratan sulphate. Apparently the major difference between glial/glioma and cartilage chondroitin sulphate proteoglycans relates to the glycan rather than to the protein moiety of the molecule.

scholarly journals Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans

1986 ◽  
Vol 236 (1) ◽  
pp. 71-75 ◽  
Author(s):  
T T Glant ◽  
K Mikecz ◽  
P J Roughley ◽  
E Buzás ◽  
A R Poole
Keyword(s):  
Core Protein ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Cartilage Proteoglycan ◽  
Age Related ◽  
Age Dependent ◽  
Old Adult ◽  
Cartilage Proteoglycans

Monoclonal antibodies were prepared that recognize different age-related epitopes on proteoglycan subunits of high buoyant density isolated from human epiphysial and articular cartilages. Antibody EFG-4 (IgG1) recognizes a proteinase-sensitive segment associated with the core protein. Antibody BCD-4 (IgG1) reacts with keratan sulphate bound to core protein. Both epitopes are minimally expressed in foetal cartilage and increase with age after birth to become maximally expressed in adult cartilage by about 30 years of age. In contrast, monoclonal antibody alpha HFPG-846 (IgM) recognizes a core-protein-related epitope that is maximally expressed in young foetal cartilage, declines up to birth and thereafter and is almost absent after about 30 years of age. Antibody alpha HFPG-846 was used to isolate by immuno-affinity chromatography two subpopulations of proteoglycan subunits from a 16-year-old-human cartilage proteoglycan subunit preparation. Only the antibody-unbound population showed a significant reaction with antibodies EGF-4 and BCD-4. The amino acid and carbohydrate compositions of these proteoglycan fractions were different, and one (antibody-bound) resembled those of foetal and the other (antibody-unbound) resembled those of adult proteoglycans isolated from 24-27-week-old-foetal and 52-56-year-old-adult cartilage respectively. These observations demonstrate that human cartilages contain at least two chemically and immunochemically distinct populations of proteoglycans, the proportions and content of which are age-dependent. It is likely that these populations represent the products of different genes, though their heterogeneity may be compounded by the result of different post-translation modifications.

scholarly journals Separation and characterization of two populations of aggregating proteoglycans from cartilage

1985 ◽  
Vol 225 (1) ◽  
pp. 95-106 ◽  
Author(s):  
D Heinegård ◽  
J Wieslander ◽  
J Sheehan ◽  
M Paulsson ◽  
Y Sommarin
Keyword(s):  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Rich Region ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Lower Mobility ◽  
Cartilage Proteoglycans

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 × 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 × 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.

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