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Biomedical subjects

A H Plaas

Publications and source records attributed to A H Plaas.

At least 19 recordsLinked to original sources
PubMed2001

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy.

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.

Acetylglucosamine↗
PubMed2001

Proteoglycans and glycosaminoglycan fine structure in the mouse tail tendon fascicle.

The isolated mouse tail tendon fascicle, a functional and homogenous volume of tendon extracellular matrix, was utilized as an experimental system to examine the structure function relationships in tendon. Our previous work using this model system demonstrated relationships between mean collagen fibril diameter and fascicle mechanical properties in isolated tail tendon fascicles from three different groups of mice (3-week and 8-week control and 8-week Mov13 transgenic) K.A. Derwin, L.J. Soslowsky, J. Biomech. Eng. 121 (1999) 598-604. These groups of mice were chosen to obtain tendon tissues with varying collagen fibril structure and/or biochemistry, such that relationships with material properties could be investigated. To further investigate the molecular details of matrix composition and organization underlying tendon function, we report now on the preparation, characterization, and quantitation of fascicle PGs (proteoglycans) from these three groups. The chondroitin sulfate/dermatan sulfate (CS/DS)-substituted PGs, biglycan and decorin, which are the abundant proteoglycans of whole tendons, were also shown to be the predominant PGs in isolated fascicles. Furthermore, similar to the postnatal maturation changes in matrix composition previously reported for whole tendons, isolated fascicles from 8-week mice had lower CS/DS PG contents (both decorin and biglycan) and a higher collagen content than 3-week mice. In addition, CS/DS chains substituted on PGs from 8-week fascicles were shorter (based on a number average) and richer in disulfated disaccharide residues than chains from 3-week mice. Fascicles from 8-week Mov13 transgenic mice were found to contain similar amounts of total collagen and total CS/DS PG as age-matched controls, and CS/DS chain lengths and sulfation also appeared normal. However, both decorin and biglycan in Mov13 tissue migrated slightly faster on sodium dodecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE) than the corresponding species from 8-week control, and biglycan from the 8-week Mov 13 fascicles appeared to migrate as a more polydisperse band, suggesting the presence of a unique PG population in the transgenic tissue. These observations, together with our biomechanical data [Derwin and Soslowsky, 1999] suggest that compensatory pathways of extracellular matrix assembly and maturation may exist, and that tissue mechanical properties may not be simply determined by the contents of individual matrix components or collagen fibril size.

Animals↗
PubMed2001

Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-beta-galactosidase digestion products.

Many tissues contain glycoproteins and proteoglycans, which are substituted with N-or O-linked keratan sulfate, a glycosaminoglycan in which the lactosamine (-galbeta1,4glcNAc-) disaccharide backbone is variably modified by sulfation, fucosylation, and sialylation. We report here a rapid, sensitive, and quantitative procedure for obtaining a complete disaccharide compositional analyses for keratan sulfates after FACE separation of products generated by hydrolysis of the glycosaminoglycans with B. fragillis keratanase II and E. freundii endo-beta-galactosidase. Seven digestion end products are separable in a single electrophoretic step using Monosaccharide composition gels. These are: the unsulfated disaccharide, glcNAcbeta1,3gal, the fucosylated trisaccharide, galbeta1,2[fucalpha1,3]glcNAc6S, the mono- and disulfated disaccharides, galbeta1,4glcNAc6S or gal6Sbeta1,4glcNAc6S from the chain interior, and the sialylated mono- and disulfated trisaccharides neuAalpha2,3galbeta1,4glcNAc6S or neuAalpha2,3gal6Sbeta1,4glcNAc6S from the nonreducing terminus. FACE analyses also revealed the presence of a contaminant beta-galactosidase activity in keratanase II enzyme preparations which cleaves the disaccharide, galbeta1,4glcNAc6S to its constituent monosaccharides, gal and glcNAc6S. It was particularly prominent at enzyme concentrations > 2 mU per nmole substrate glcNH(2) or after prolonged digestion times (> 12 h), and was not inhibitable by thiogalactosides or N-acetyl-lactosamine. As these monosaccharide products would not be detectable using the commonly described analytical methods for KS hydrolase products, such as (1)H-NMR and HPLC analyses, our data illustrate that the FACE procedure represents an improved approach for accurate compositional microanalyses of corneal and skeletal keratan sulfates, especially applicable to experimentation involving small amounts (1-2 microg) of this glycosaminoglycan.

Acetylglucosaminidase↗
PubMed2000

Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44.

Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44. The binding of versican to L- and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A cross-blocking study indicated that L- and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L- and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.

Animals↗
PubMed2000

Proteoglycan metabolism during repair of the ruptured medial collateral ligament in skeletally mature rabbits.

The metabolism of the chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) decorin and biglycan is markedly altered during short-term (3-6 weeks) and long-term (40 weeks-2 years) repair of surgically ruptured medial collateral ligaments from mature rabbits. A PG-rich extracellular matrix accumulates in injury gaps by 3 weeks postsurgery and extends into tissue regions containing the original ligaments, and elevated PG levels remain apparent up to 2 years postinjury. CS/DS PGs were prepared from such ligaments and identified after SDS-polyacrylamide gel electrophoresis by Alcian blue staining or immunoblotting. In normal ligaments, decorin is the most abundant proteoglycan (accounting for approximately 80% of the total); the remainder is biglycan and a large PG, possibly versican. In repairing ligaments, decorin is barely detected, but instead a large proteoglycan and abundant amounts of biglycan accumulate. Biglycan is present in two forms in repairing ligaments, and they can be separated on SDS-PAGE into 200- and 140-kDa forms. The slower migrating species is absent in normal ligaments and may represent a different glycoform (containing either a single or two short chondroitin/dermatan sulfate chains) of biglycan. Alteration in PG expression and posttranslational processing during medial collateral ligament repair are similar to those reported for repair and scar formation of other connective tissues. The accumulation of biglycan observed here may interfere with proper collagen network remodeling and may lead to persistent inflammatory and matrix turnover processes, thus preventing restoration of a long-term functional ligament tissue.

Animals↗
PubMed1999

Sulphation heterogeneity in the trisaccharide (GalNAcSbeta1, 4GlcAbeta1,3GalNAcS) isolated from the non-reducing terminal of human aggrecan chondroitin sulphate.

We report here the isolation and sulphation isomer analyses of trisaccharides GalNAcS(beta1,4)GlcA(beta1,3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for 1 h at 37 degrees C with 30 micro-units of endo-chondroitinase ABC per microgram of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-filtration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroitinase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4Sbeta1,4GlcAbeta1,3GalNAc4S, GalNAc4Sbeta1,4GlcAbeta1,3GalNAc6S, GalNAc4,6Sbeta1,4GlcAbeta1, 3GalNAc4S and GalNAc4,6Sbeta1,4GlcAbeta1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee cartilages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide, suggesting that the sulphation of the last disaccharide might not have a direct effect on the specificity of chondroitin sulphate terminal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equivalent to that in the last repeating disaccharides at the non-reducing terminus, suggesting that neither chondroitin 4-sulphotransferase nor chondroitin 6-sulphotransferase shows preferential activity near the chain terminus.

Adolescent↗
PubMed1998

Glycosaminoglycan sulfation in human osteoarthritis. Disease-related alterations at the non-reducing termini of chondroitin and dermatan sulfate.

Chondroitin lyase products of aggrecan and small proteoglycans from normal and osteoarthritic cartilages were analyzed for chain internal Deltadisaccharides and terminal mono- or disaccharides. Chondroitin and dermatan sulfate chains from arthritic cartilages were of essentially normal size and internal sulfation but had significantly altered sulfation of the terminal residues. Whereas in normal cartilage, approximately 60% of terminal GalNAc4S was 4, 6-disulfated, it was reduced to approximately 30% in osteoarthritic cartilage. This is most likely due to a lower terminal GalNAc4, 6S-disulfotransferase activity and reveals that metabolic changes in osteoarthritis can affect this distinct sulfation step during chondroitin and dermatan sulfate synthesis. GlcAbeta1,3GalNAc6S-, the mimotope for antibody 3B3(-), was present on approximately 8 and approximately 10% of chains from normal and osteoarthritic cartilages, respectively. 3B3(-) assayed by immunodot blot was within the normal range for most osteoarthritic samples, with only 5 of 24 displaying elevated reactivity. This resulted not from a higher content of mimotope, but possibly from other structural changes in the proteoglycan that increase mimotope reactivity. In summary, chemical determination of sulfation isomers at the non-reducing termini of chondroitin and dermatan sulfate provides a reliable assay for monitoring proteoglycan metabolism not only during normal growth of cartilage but also during remodeling of cartilage in osteoarthritis.

Adult↗
PubMed1998

Age-related changes in the structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage.

Bovine articular cartilage fibromodulin has been isolated from animals aged 3 months to 8 years, and the attached keratan sulphate (KS) chains digested with keratanase II. The oligosaccharides generated have been reduced, examined by high-pH anion-exchange chromatography and their structures identified by comparison with standards. It has been shown that in fibromodulin from young articular cartilage, the KS chains do not possess either non-reducing terminal (alpha2-6)-linked N-acetylneuraminic acid or fucose (alpha1-3)-linked to sulphated N-acetylglucosamine residues. However, an age-related increase has been observed in the abundance of both (alpha2-6)-linked N-acetylneuraminic acid and (alpha1-3)-linked fucose, neither of which is found in KS isolated from non-articular cartilage, irrespective of the age of the source. Interestingly, the KS chain length remains constant as a function of age, which possibly relates to a role in collagen fibril assembly. In addition, no significant age-related changes were identified in levels of galactose sulphation.

Acetylglucosaminidase↗
PubMed1998

Changes in sulfation patterns of chondroitin sulfate in equine articular cartilage and synovial fluid in response to aging and osteoarthritis.

OBJECTIVES: To determine effects of aging on sulfation of chondroitin sulfate (CS) in articular cartilage and synovial fluid from normal equine middle carpal joints, and to determine whether CS compositional analysis can be used to assess alterations in proteoglycan turnover in degenerative cartilage obtained from horses with carpal osteochondral fractures. SAMPLE POPULATION: Carpal articular cartilage and synovial fluid from 44 cadavers with normal joints and from 16 Thoroughbred racehorses during routine carpal arthroscopic surgery. PROCEDURE: After papain/chondroitinase digestion of cartilage, CS disaccharides (unsulfated disaccharide delta Di0S, and monosulfated disaccharides delta Di4S and delta Di6S) were quantified by capillary zone electrophoresis. The CS was purified from synovial fluid chondroitinase digested, and analyzed. The CS nonreducing terminal residues, N-acetylgalctosamine (galNAc) or glucuronic acid adjacent to a 4-sulfated or 6-sulfated galNAc, were quantified. RESULTS: In cartilage, the delta Di6S-to-delta Di4S ratio increased with age; in degenerative cartilage, this ratio was not significantly different from the normal value. Percentage of delta Di0S decreased with age and was significantly higher in degenerative than in normal cartilage. The galNAc4S and galNAc4,6S represented > or = 96% of the terminal residues. There was a significant decrease in 6-sulfation of the terminal residues in degenerative cartilage. CONCLUSIONS: 6-Sulfation of internal and terminal CS residues increased with age. Cartilage degeneration in racehorses was accompanied by deposition of CS chains with altered sulfation patterns, in normal and diseased joints of horses > 2 years old, synovial fluid CS was not indicative of cartilage CS and may represent turnover products of a subpopulation of proteoglycan within the matrix.

Aging↗
PubMed1997

Inhibition of cartilage degradation and changes in physical properties induced by IL-1beta and retinoic acid using matrix metalloproteinase inhibitors.

Bovine cartilage explants were treated with 100 ng/ml recombinant human interleukin-1beta (IL-1beta) or 1 microM all-trans retinoic acid (RA) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, samples cultured with IL-1beta or RA were treated with 4 microM recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) or a synthetic metalloproteinase inhibitor (L-758,354) to inhibit this degradation. Treatment with IL-1beta or RA each resulted in >90% GAG loss after 8 days in culture. Addition of TIMP or L-758,354 to the culture media inhibited IL-1beta-induced loss of tissue GAG by 40 and 65%, respectively, and inhibited RA-induced GAG loss by 35 and 65%, respectively. Analysis of degradation products in the culture media using a G1 antibody indicated that IL-1beta- and RA-treated plugs released 68-kDa fragments of aggrecan, corresponding to a segment of the aggrecan core protein from the G1 domain to the C-terminus NITEGE, consistent with "aggrecanase" activity. Release of the G1 fragment was inhibited by treatment with L-758,354. Both IL-1beta and RA induced significant loss of hyaluronan from cartilage explants after 8 days of exposure and HA loss was also inhibited by addition of L-756,354 to the culture media. IL-1beta, but not RA, induced a significant increase in swelling ratio (wet weight in 0.01 M NaCl normalized to wet weight in DMEM) after 8 days in culture, consistent with degradation of the collagen network, and the increase in tissue swelling was inhibited by treatment with TIMP-1 or L-758,354. Exposure to IL-1beta or RA resulted in significant changes in cartilage physical properties including streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient after 8 days in culture, and these changes were inhibited by 40-90% by exposure to TIMP and by 50-90% by exposure to L-758,354. Measurement of dynamic streaming potential showed that changes due to treatment with IL-1beta alone were highly dependent in compression frequency, with dramatic changes seen at high frequency prior to changes in mechanical properties, and little initial change seen at low frequency. Streaming potential and equilibrium modulus of explants treated with RA decreased to 10% of their initial values after 8 days in culture, but decreased to only 40 and 90%, respectively, when treated with RA plus TIMP-1.

Aggrecans↗
PubMed1997

Chemical and immunological assay of the nonreducing terminal residues of chondroitin sulfate from human aggrecan.

Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcAbeta1,3GalNAc4S and GlcAbeta1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were approximately 50% 4,6-disulfated in aggrecans from adults (22-72 years of age). Second, the terminal disaccharide GlcAbeta1,3GalNAc4S was on approximately 7% of chains on aggrecan from fetal through 15 years of age, but on only approximately 3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAbeta1,3GalNAc6S, approximately 9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(-) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.

Adolescent↗
PubMed1996

Compression of cartilage results in differential effects on biosynthetic pathways for aggrecan, link protein, and hyaluronan.

The differential effects of static compression and recovery from compression on biosynthesis and biosynthetic pathways of aggrecan, link protein, and hyaluronan were assessed. During compression, biosynthesis of aggrecan and link protein were inhibited to approximately 25 and approximately 40%, respectively, of free-swelling control levels. In marked contrast, hyaluronan synthesis was unaffected by static compression. After release from 12-h 50% static compression, aggrecan synthesis remained inhibited for up to 2.5 days; however, link protein synthesis completely recovered to free-swelling control levels within 8 h after release. Hyaluronan synthesis remained at control levels after release of compression. During compression, aggrecan core protein pool size was decreased, whereas the rate of processing into the proteoglycan form remained essentially the same as in free swelling control tissue. Four hours after release from compression, aggrecan core protein pool size remained small and the rate of intracellular processing of aggrecan had become slower than that of free swelling control tissue. Due to the altered core-protein processing kinetics, fewer but longer chondroitin sulfate chains were added to the core proteins. Sulfation was not markedly altered. The differential effects of static compression and release on the biosynthesis of aggrecan, link protein, and hyaluronan are similar to the changes in the biosynthetic pathways that are affected in response to IL-1 treatment, suggesting that the response to static compression is not a general inhibition of cellular activity, but appears to be part of a specific transduction mechanism.

Aggrecans↗
PubMed1996

Proteoglycans of human rotator cuff tendons.

Rotator cuff and biceps tendons that appeared grossly normal were procured from adult cadavers without a history of shoulder problems. These tendons were analyzed for the amount and type of glycosaminoglycan, type of proteoglycan, and histology. When compared with the distal/tensional region of biceps tendon, the glycosaminoglycan content of supraspinatus, infraspinatus, and subscapularis tendons was 2.5-fold higher and the glycosaminoglycan content of the proximal/compressed region of biceps tendon was 3-fold higher. The ratio of hyaluronic acid to chondroitin sulfate/dermatan sulfate in all three cuff tendons was approximately 1. Rotator cuff tendons contained large proteoglycan similar to aggrecan, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel migration elution from Sepharose CL-4B, and content of both chondroitin sulfate and keratan sulfate chains. Both decorin and biglycan were also present, as demonstrated by migration in sodium dodecyl sulfate-polyacrylamide gels and core protein immunoreactivity. In contrast decorin was the only proteoglycan prominent in distal/tensional regions of biceps tendon. Histological analysis showed layers of loosely organized alcian blue-stained material running between the longitudinal collagen fiber bundles. The proteoglycan content of rotator cuff tendons was similar to fibrocartilage in tendons that have been subjected to compressive loads in situ. This suggests that cells of normal adult rotator cuff tendons have adapted to loads distinct from pure tension. However, the histological organization did not resemble mature fibrocartilage. The increased amount of proteoglycan in rotator cuff tendons may serve to separate and lubricate collagen bundles as they move relative to each other during normal shoulder motion.

Adult↗
PubMed1996

Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine.

Sulfated glycosaminoglycans such as chondroitin sulfate are composed of three structural domains, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a nonreducing terminal, and these domains may confer specific functions on particular chain populations. We report here a new and highly sensitive method for the detection and quantitation of all nonreducing terminal residues and internal disaccharides obtained by chondroitinase ABC or ACII digestion of aggrecan chondroitin sulfate. The procedure involves a quantitative reductive animation of the reducing ends of sulfated mono- and disaccharide chondroitinase products with 2-aminopyridine and boranedimethylamine. All derivatized saccharides can be separated and quantitated by fluorescence in a single chromatographic step on an AS4A anion exchange column, eluted with a gradient (0-500 nM) of sodium trifluoroacetate. The reproducibility and stability of the derivatisation, together with the sensitivity of the chromatography system, allowed for routine quantitation in the range of 3-500 pmol of reducing group (corresponding to about 1.5-250 ng of disaccharide or 0.75-125 ng of monosaccharide). Moreover, the fluorescence yield (fluorescence area units per pmol of reducing group) was virtually identical for all saccharides analyzed. Application of this method to an analysis of aggrecan purified from calf epiphyseal cartilage and from rat chondrosarcoma chondrocyte cultures allowed a precise identification and quantitation of the internal disaccharides and the nonreducing terminal structures, together with an estimation of the number average molecular weight of CS chains in these aggrecan preparations.

Aminopyridines↗
PubMed1995

Glycosaminoglycan addition to proteoglycans by articular chondrocytes--evidence for core protein-specific pathways.

The intracellular compartmentalization of enzyme activities involved in the elongation and sulfation of glycosaminoglycans on aggrecan, decorin, and fibromodulin was investigated using brefeldin A, a compound with known inhibitory action on normal vesicular transport and secretion of macromolecules. Treatment of bovine chondrocyte cultures with the compound resulted in greater than 98% inhibition of Na35SO4 incorporation into macromolecules, whereas [3H]leucine or [3H]glucosamine continued at 60-70% of the levels measured in control cultures. The release of newly synthesized products into the medium was also decreased markedly by brefeldin A to 7 and 2% of control levels for [3H]leucine- and [3H]glucosamine-labeled macromolecules, respectively. Analysis of [3H]-glucosamine-labeled products in these cultures showed that synthesis of sulfated glycosaminoglycans (chondroitin/dermatan sulfate and keratan sulfate) was inhibited in response to brefeldin A, whereas hyaluronan synthesis was essentially unaffected. Significant amounts of elongated chondroitin continued to be synthesized in the presence of brefeldin A. Immunoprecipitation of [3H]leucine-labeled decorin, aggrecan, and fibromodulin from cells showed that aggrecan and fibromodulin were not substituted with glycosamino-glycans, whereas all decorin molecules synthesized under these conditions were substituted with chondroitin. The results suggest that in articular chondrocytes, elongation of the glycosaminoglycan chains on decorin, but not their sulfation, occurs in a Golgi compartment unaffected by disruption of vesicular core protein transport. This is in contrast to glycosaminoglycan elongation and sulfation on aggrecan and fibromodulin, where both processes apparently occur in the trans-Golgi network, which becomes inaccessible to these core proteins in the presence of brefeldin A. The results further suggest that in brefeldin A-treated cells decorin is contained in a discrete ER-Golgi compartment separated from aggrecan; this compartment is accessible to p-nitrophenyl-beta-D-xylosides, since beta-xylosides become elongated with chondroitin even in the presence of brefeldin A.

Aggrecans↗
PubMed1995

Large and small proteoglycans of osteoarthritic and rheumatoid articular cartilage.

OBJECTIVE: To identify characteristic changes in large aggregating (aggrecan) and small proteoglycan (PG) populations in articular cartilages during osteoarthritis (OA) and rheumatoid arthritis (RA). METHODS: Aggrecan populations in guanidine extracts of femoral condylar cartilages of 46 OA and 8 RA patients who underwent total knee arthroplasty, as well as of 2 fetuses and 6 normal adults, were separated in agarose-polyacrylamide composite gels. Small PGs (biglycan, decorin, and fibromodulin) in the same extracts were analyzed in 12% polyacrylamide gels. Gels were stained or electrophoretically transferred and probed with antibodies to aggrecan epitopes and to small PGs. Epitope contents of the samples were also compared by inhibition radioimmunoassay. RESULTS: There were significant differences found among normal and diseased samples in their electrophoretic mobilities, band distributions, and antibody staining. OA and especially RA samples were heavily degraded, lacked certain aggrecan populations, and contained fewer keratan sulfate and chondroitin-6-sulfate epitopes compared with normal samples. Levels of chondroitin-4-sulfate and "fetal-type" epitopes were elevated in the OA samples compared with the normal ones. More core proteins of small PGs were found in diseased than in normal cartilages, but they were more heterogeneous in size and glycosaminoglycan substitution. CONCLUSION: There is extensive degradation of both large and small PGs in diseased cartilages, but a repair process does exist, especially in OA cartilages. Chondrocytes of diseased cartilages are able to synthesize fetal-type aggrecans. Small PGs are glycosylated differently in diseased cartilages than in normal ones.

Adult↗
PubMed1995

Structure of chondroitin sulfate on aggrecan isolated from bovine tibial and costochondral growth plates.

The structure of chondroitin sulfate on aggrecan isolated from the rib and proximal tibial growth plates of bovine fetuses was investigated, and the previously reported increase in the hydrodynamic size of chondroitin sulfate chains between the reserve and hypertrophic zones of the rib was confirmed in the tibial growth plate. Superose 6 gel chromatography, calibrated for chondroitin sulfate chain length by monosaccharide analysis, showed that the average molecular mass of chondroitin sulfate in the reserve and maturing zones of both growth plates was 21,600 and 30,400, respectively. Determination by capillary zone electrophoresis of the disaccharide composition of chains following chondroitinase digestion showed that delta Di-0S, delta Di-4S, and delta Di-6S together accounted for more than 98% of the disaccharides in the digests from all zones of both growth plates; delta disulfated and delta trisulfated disaccharides were not detected. Furthermore, this analysis revealed a gradient in chondroitin sulfate composition from the reserve to the hypertrophic zone, characterized by a marked increase in the content of delta Di-6S (from about 32% to about 52%) and a marked decrease in the content of delta Di-4S (from about 53% to about 35%). Moreover, this altered pattern of sulfation was detected on chains of all sizes in the hypertrophic zone, suggesting that a proportion of the reserve zone aggrecan might be removed and replaced with aggrecan rich in chondroitin-6-sulfate synthesized during the proliferative and maturation stages of the resident chondrocytes. These data are discussed in relation to the biosynthetic mechanisms that control chondroitin sulfate chain length and sulfation on aggrecan and their modification during chondrocyte proliferation, maturation, and hypertrophy in the growth plate.

Aggrecans↗