Size Of Chondroitin Sulfate Chains Research Articles

ラット卵巣か粒膜細胞におけるプロテオグライカンの生合成に与えるｐ‐ｎｉｔｒｏｐｈｃｎｙｌ‐ｘｙｌｏｓｉｄｅの影響―Ｇｏｌｇｉ装置の糖合成機能に関する解析―

Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [³⁵S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [³⁵S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [³⁵S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.

Versican undergoes specific alterations in the fine molecular structure and organization in human aneurysmal abdominal aortas.

Versican is the major matrix proteoglycan in aortic wall and participates in various biological functions of the tissue. In the present study the molecular characteristics of versican isolated from normal human aorta as well as those of versican expressed in aneurysmal aortic tissue were examined. Versican was isolated by combined anion-exchange and gel permeation chromatography and was further characterized by high-performance liquid chromatography, polyacrylamide gel electrophoresis and immunoblotting. In both tissues versican is exclusively substituted with chondroitin sulfate chains, in contrast to other human tissues where both chondroitin and dermatan sulfate chains are attached onto versican core proteins. Except for the significant decrease in the concentration of versican in the aneurysmal tissue, this PG undergoes specific alterations in the aneurysmal tissue. The molecular size of versican isolated from diseased tissue is decreased with a simultaneous increase in the ratio of glycosaminoglycan to protein in this tissue. The latter reflect the extensive fragmentation of versican in the diseased tissue and most probably the generation of shorter peptides enriched to glycosaminoglycan chains. Although the size of chondroitin sulfate chains is identical in both versican preparations, a significant increase in the percentage of 6-sulfated disaccharides is observed in chondroitin sulfate chains of versican in aneurysmal aortas, which is accompanied by decrease in 4-sulfated and non-sulfated units.

Notochordal Cells Interact with Nucleus Pulposus Cells: Regulation of Proteoglycan Synthesis

The disappearance of notochordal cells is correlated with early degenerative changes in the intervertebral disc. With increased disc degeneration there is a marked decrease in proteoglycan synthesis, resulting in loss of mechanical function. One possible mechanism for the decrease in proteoglycan synthesis is the loss of notochordal cells from the tissue. In this study, nucleus pulposus cells cocultured with notochordal cells exhibit an increase in proteoglycan synthesis. Interestingly, purified notochordal cells synthesize little proteoglycan as observed by [35S]sulfate incorporation into proteoglycans. The observed increase in proteoglycan synthesis does not appear to be dependent on cell–cell contact; rather it is the result of soluble factor(s) produced by notochordal cells. Finally, no difference in chondroitin sulfate chain size in notochordal-stimulated nucleus pulposus cells was observed which is consistent with an up-regulation in aggrecan core protein synthesis. These results are consistent with canine breeds where notochordal cells persist into adult age and disc degeneration is not observed. This suggests notochordal cells play a vital role in maintaining disc integrity.

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.

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro.

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.

Isolation and characterization of proteoglycans synthesized in ovo by embryonic chick cartilage and new bone.

Cartilage proteoglycans have been well characterized in a number of developing systems, both in vitro and in vivo, but the newly synthesized molecules have been analyzed only from culture material. Because of potential culture artifacts, an attempt was made to characterize the proteoglycans newly synthesized in ovo in chick embryo sternum, tibial epiphysis, and tibial shaft. These in ovo synthesized proteoglycans share many structural features with chick proteoglycans synthesized by chondrocytes in culture including average monomer size, chondroitin sulfate chain size, keratan sulfate chain size, and the ability to aggregate with hyaluronic acid. Moreover, the newly synthesized in ovo proteoglycans, notably those of the tibial epiphysis, display reproducible changes in their structure as a function of embryonic age. These changes correlate with similar changes documented for chick cartilage proteoglycans synthesized in culture. Finally, the proteoglycans synthesized in ovo in the day 17 tibial shaft include, in addition to cartilage proteoglycans, one proteoglycan which seems to be characteristic of bone.

Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [ 3H]Serine incorporation was maximally inhibited by 3 × 10 −4 m puromycin but unaffected by 10 −5 m puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.

Polydispersity of proteoglycans synthesized by chondrocytes from the Swarm rat chondrosarcoma.

The population of proteoglycan monomers in aggregates was purified from chondrocyte cultures after labeling with either [3H]serine and [35S]methionine or [3H]serine and [35S]sulfate. Digestion of the monomers labeled with [3H]serine and [35S]methionine with trypsin indicated that serine was enriched (approximately 80%) in the chondroitin sulfate attachment region of the core protein, while methionine was enriched (approximately 60%) in the hyaluronic acid-binding region. Sepharose CL-2B chromatography and velocity gradients were used to isolate monomer subfractions which differed in molecular size. The 3H/35S ratios for the subfractions from monomers labeled with [3H]serine and [35S]methionine were nearly constant, indicating that the core protein lengths were constant regardless of the size of the monomer. Conversely, the 3H/35S ratios for subfractions of monomers labeled with [3H]serine and [35S]sulfate increased significantly with decreasing sizes. Chondroitin sulfate chains in subfractions were released by alkaline borohydride treatment. The mean molecular weights of the chondroitin sulfate chains decreased from approximately 18,500 in the largest subclass of monomers to approximately 12,500 in the smallest. [3H]Serine-labeled monomer subfractions were digested with papain to determine the proportion of serine residues substituted with chondroitin sulfate. The distribution of label was constant for greater than 90% of the monomers from the largest to the smallest. The results indicate that, for at least 90% of the newly synthesized monomers that are able to aggregate, variation in chondroitin sulfate chain size is the only contributing factor to polydispersity in their molecular size.

The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma.

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.

Maturation-related changes in proteoglycans of fetal articular cartilage

Proteoglycans of the articulating and growing zones of maximum and minimum contact of bovine fetal articular cartilage were studied and compared to proteoglycans of immature calf and adult steer. During fetal maturation, localized changes were observed as early as the second trimester of fetal life but were restricted to the most superficial zones. Proteoglycans extracted from the growing zones were purified by density-gradient ultracentrifugation. The majority of proteoglycan monomers were able to interact with endogenous hyaluronate to form aggregates. Monomers had, at all fetal stages, similar elution profiles on Sepharose 2B and similar ratios of chondroitin sulfate chains/keratan sulfate chains/ O-glycosidically linked oligosaccharides. Keratan sulfate chains were of similar size at all stages, but chondroitin sulfate chain size decreased markedly with fetal maturation. In the first and second trimesters of fetal life, the proteoglycans were poorly substituted with glycosaminoglycans. A major increase in the absolute number of glycosaminoglycans and oligosaccharides attached to core protein was detected during the third trimester of fetal life. No further changes in substitution occurred in early postnatal life. Enzymatic digestion of proteoglycan monomer demonstrated that the increase in substitution with keratan sulfate occurred to the same extent in the main polysaccharide attachment region and in the keratan sulfate-rich region.

Changes in the sulfated proteoglycans synthesized by “aging” chondrocytes. II. Organ-cultured vertebral columns.

Sulfated proteoglycans synthesized by intact embryonic chick vertebral cartilages maintained in organ culture were characterized by labeling the vertebral chondrocytes with [35S]sulfate. More than 95% of the sulfated macromolecules synthesized by the cartilages are retained in their extracellular matrix. Sucrose velocity sedimentation gradient analyses revealed that the intact cartilages synthesized primarily proteoglycan monomers typical of cartilage and also small amounts of a smaller sized proteoglycan. The continuously varying size distribution of monomers which characterize a population of proteoglycans, could, in part, be attributed to heterogeneity of other chondroitin sulfate chain size. Changes in the proteoglycans synthesized by intact cartilages were analyzed between 6 h and 8 days of organ culture: (i) monomers decreased in molecular size with age in organ culture; (ii) the decrease in monomer size could be partially attributed to a shortening of their chondroitin sulfate chains; (iii) the 6S/4S disaccharide composition of the chondroitin sulfate chains also changed with culture age, from 1.8 to 0.3. This change was shown to be independent of the concomitant decrease in monomer size. These changes are similar to those which occur in proteoglycans synthesized by articular cartilages from chickens of increasing age. This cartilage culture system is likely to be a more useful model for studying changes which accompany aging and/or senescence in cartilage in vivo than the more commonly studied dispersed chondrocyte cultures.

Changes in the sulfated proteoglycans synthesized by “aging” chondrocytes. I. Dispersed cultured chondrocytes and in vivo cartilages.

Structural and chemical changes in the sulfated proteoglycans synthesized by "aging" cultures of dispersed chondrocytes were compared to those synthesized by freshly excised, intact cartilage explants in organ culture from chickens of various ages. In vitro, chondrocytes isolated from embryonic chick vertebral cartilages synthesized a proteoglycan monomer characteristic of cartilage (type IV). Monomers synthesized between 3 days and 6 weeks in culture: (i) showed a decrease in average molecular size, which could be correlated, in part, with a decrease in the average size of the chondroitin sulfate chains; (ii) were able to interact with hyaluronic acid to form proteoglycan aggregates regardless of size; (iii) maintained a relatively constant 6S/4S disaccharide ratio of 2.2 for chondroitin sulfate; and (iv) maintained a constant keratan sulfate composition of 5 to 10%. Sulfated proteoglycans synthesized by freshly excised embryonic tibia cartilaginous head and by tibial articular cartilages of 1- and 6-year-old chickens were compared. With age: (i) the type IV monomer size decreased; (ii) the chondroitin sulfate chain size decreased; (iii) the type IV monomers retained their ability to interact with hyaluronic acid; (iv) the 6S/4S disaccharide ratio decreased from 1.7 to 0.6; (v) the keratan sulfate composition increased from 7 to 40%. Although monomer sizes decrease in both the culture and explant systems, in other respects the differences are considerable. These findings suggest that the changes in sulfated proteoglycans observed in subcultured monolayers of chondrocytes must be interpreted with caution when attempting to relate them to the changes that occur in aging in vivo chondrocytes.