Structural characterization of human alveolar bone proteoglycans

Proteoglycans were extracted from EDTA-demineralized human alveolar bone under dissociative conditions using 4 M guanidinium chloride in the presence of protease inhibitors. The extract was further purified by anion-exchange chromatography on DEAE-Sephacel, using a step-wise salt gradient. The proteoglycan-rich fraction was analysed for carbohydrate, protein and amino acid composition and molecular size by SDS-PAGE. Glycosaminoglycan content was determined by cellulose acetate electrophoresis after proteolysis. The sulphate isomers of the glycosaminoglycans were confirmed by Fouriertransformed infra-red spectroscopy. Two chondroitin sulphate-proteoglycan species were identified with molecular weights of 79 and 55–65 kDa, respectively. The core proteins had molecular weights of 49 kDa for both proteoglycans, with the amino acid content rich in glycine, leucine, glutamate and aspartate. The chondroitin sulphate chains were mainly as the 4-sulphate isomer forms although low but detectable amounts of 6-sulphate isomer were also present.     

The acidic macromolecules in rabbit cortical bone tissue

Rabbit cortical bone powder was extracted with EDTA solutions at neutral pH and the soluble constituents fractionated by DEAE-cellulose chromatography. The acidic fractions obtained were further investigated by gel chromatography and chemical analysis. Two classes of proteoglycans were present; one containing chondroitin sulphate and the other containing material resembling keratan sulphate mixed with a smaller amount of chondroitin sulphate. Sialoglycoproteins were detected by specific chemical introduction of tritium label into the sialic acid residues of the acidic glycoprotein fraction. Following sodium dodecyl sulphatepolyacrylamide gel electrophoresis and fluorography, the presence of numerous distinct sialoglycoprotein components was demonstrated.     

硫酸软骨素蛋白多糖与牙周炎的相关性

本实验旨在研究硫酸软骨素蛋白多糖与牙周炎的相关性。采用免疫组织化学方法,对牙周炎引起的硫酸软骨素蛋白多糖变化进行研究。结果显示硫酸软骨素(DS、C4S、C6S)广泛分布于上皮下结缔组织、牙周韧带和血管周围,在牙槽骨局限于哈佛氏管和骨小腔内壁。牙周炎时随着组织溶解破坏,其染色反应减弱消失,说明硫酸软骨素是牙周组织的基本成分,与牙周炎关系密切。     

Effect of p-nitrophenyl-xyloside on the biosynthesis of proteoglycan in rat ovarian granulosa cells--analyses of glycosaminoglycan synthesis in the Golgi apparatus

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.     

Occlusal hypofunction causes changes of proteoglycan content in the rat periodontal ligament

The biological functions of proteoglycans and glycosaminoglycans are closely associated with mechanical stress on the tissue. In order to reveal the relationship between proteoglycans in the periodontal ligament and mechanical stress such as occlusal stimuli, occlusal hypofunction of rat unilateral mandibular molars was induced by extraction of the opposing first, second and third maxillary molars. Immunohistochemical analyses were performed using antibodies for chondroitin sulfate, decorin, biglycan, heparan sulfate and keratan sulfate, and hyaluronic acid-binding protein. Chondroitin sulfate, observed more strongly in the cervical side than in the apical side of the periodontal ligament of the unextracted sides of mandible, and uniformly present in the extracellular matrix of the periodontal ligament, decreased significantly from 1 wk post-extraction of the antagonists, with a decrease in thickness and disarrangement in fibrous components. Decorin core protein, uniformly present in the periodontal ligament of the unextracted sides, decreased as early on as 2 d post-extraction. Heparan sulfate, mainly localized on the cell surface of vascular endothelial cells and osteoclastic cells as well as in the extracellular matrix of the unextracted sides, decreased significantly in association with the decreased number of blood vessels and osteoclastic cells as early on as 2 d post-extraction. Biglycan, keratan sulfate and hyaluronic acid, uniformly distributed in the periodontal ligament of the unextracted sides, showed little change after the extraction. These results demonstrate that occlusal hypofunction causes tissue remodeling of the periodontal ligament, with a significant decrease of chondroitin sulfate, decorin and heparan sulfate.     

The soft tissue cover of the mandibular condyle

Age-related changes of the composition of the extracellular matrix of the soft tissue cover of the mandibular condyle (STC), especially of the large proteoglycans, have been investigated. Proteoglycans were extracted from the STC of neonatal, juvenile and adult domestic pigs, fractionated by density gradient centrifugation and analyzed by electrophoresis/Western blotting.Experiments revealed firstly that a large CS/KS proteoglycan (aggrecan) is an essential constituent of the STC at all ages. This proteoglycan is required for nutrition of avascular tissues, and age-related changes in its average size and substitution with KS (keratan sulfate) may be a response to altered functional loading and tissue architecture of the STC. Secondly it was shown that a large CS/DS (chondroitin sulfate/dermatan sulfate) proteoglycan characterized by a doublet of core proteins at 200 and 250 kDa, thereby resembling perlecan, is present in the tissue of adults, but not of neonates and juveniles. Thirdly a large CS/DS proteoglycan characterized by core proteins at 350, 450 and 550 kDa, thereby resembling versican, was present in juveniles. It was detectable only weakly in neonates and not in adults. Results of core protein analysis were confirmed by results of agarose gel electrophoresis/Western blotting of the undigested proteoglycans isolated directly from the tissue extracts.Versican is believed to destabilize cell-matrix interactions required for cell proliferation and differentiation. In this context, presence of versican-like proteoglycans in the STC of growing individuals and its disappearance in adults appears to be related to the growth potential of the mandibular condyle.     

Glycosaminoglycans of human cementum

The glycosaminoglycans in human cementum have been studied. Following proteolytic digestion of guanidine/EDTA and collagenase extracts of cementum, glycosaminoglycans were isolated and then separated by cellulose acetate membrane electrophoresis. After specific elimination by enzymatic and chemical treatments the glycosaminoglycans were identified as hyaluronic acid, chondroitin sulfate and dermatan sulfate. Neither heparan sulfate nor keratan sulfate were observed. Quantitation of the glycosaminoglycans in both extracts revealed chondroitin sulfate to represent the major species present. Hyaluronic acid was observed predominantly in the guanidine/EDTA extract while dermatan sulfate was a quantitative minor component of both extracts.     

Modification of the proteoglycans of rat incisor dentin – predentin during in vivo fluorosis

Proteoglycans (PGs) were isolated from the dentin–predentin of fluorotic and control rat incisor teeth using demineralization in EDTA, followed by extraction with 4 M guanidinium chloride in the presence of protease inhibitors. Differences in the behaviour of fluorotic and control PG were evident during purification by anion exchange chromatography on Q-Sepharose and MONO-Q interfaced with fast protein liquid chromatography. The PG from fluorotic teeth exhibited a more anionic profile, due to changes in glycosaminoglycan characteristics, since no apparent differences were evident between the respective core proteins, both of which were 45 kDa. The constituent glycosaminoglycan chains of fluorotic dentin were of lower molecular size and showed the additional presence of dermatan sulfate and heparan sulfate by comparison to non-fluorotic controls, where only chondroitin-4-sulfate was detected.     

A quantitative chemical study of glycosaminoglycans in the articular disc of the bovine temporomandibular joint

Glycosaminoglycans were prepared from the disc by digestion with papain. The disc contained 5% of its dry weight as glycosaminoglycan. Fractionation by ion-exchange chromatography, followed by precipitation at varying concentrations of ethanol, together with chemical and enzymatic analyses, showed this glycosaminoglycan to consist of approx. 5% hyaluronic acid, 14% dermatan sulphate, 79% chondroitin sulphate and 2% keratan sulphate. Disaccharides obtained from the chondroitin sulphate were 75% 6-sulphated, 21% 4-sulphated and 4% non-sulphated. Chemical analysis showed a low average degree of sulphation of chondroitin sulphate. Immunohistochemical staining of sagittal sections of the disc showed chondroitin sulphate to be distributed throughout, whereas dermatan sulphate (as proteodermatan sulphate) appeared to be concentrated in the periphery.     

Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis . This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0–200 ng ml⁻¹ of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P.  gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.     

Human gingival glycosaminoglycans in cyclosporin-induced overgrowth

Glycosaminoglycans in normal and cyclosporin‐induced gingival overgrowth were extracted by papain digestion and purified by Mono Q‐FPLC chromatography. The purified glycosaminoglycans were analyzed by agarose gel electrophoresis and by the pattern of degradation products formed by chondroitin lyases on HPLC chromatography. Our results on the glycosaminoglycan composition showed presence of chondroitin 4‐ and 6‐sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in both normal gingiva and cyclosporin‐induced gingival overgrowth. The total and relative amounts of glycosaminoglycans were similar between normal and overgrown gingiva. This suggests that the glycosaminoglycan composition is not changed in cyclosporin‐induced gingival overgrowth. Our present biochemical results conflict with histochemical and biosynthetic data previously reported by other groups. Those studies suggested that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin induced comparably high levels of these compounds in in vitro cultures of gingival fibroblasts. Therefore, these discrepant results suggest that a cyclosporin‐induced increase on gingival glycosaminoglycans still remains an open question. The implications of these conflicting results are discussed.     

Synthesis of proteoglycans in rat mucosal keratinocytes

The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [³⁵S] sulfate or D-[1-³H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the ³⁵S-laballed
glycosaminoglycans showed that heparan sulfate was the major component (∼90%) and that small amounts (∼10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-³H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [³⁵S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a K_av of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Chemical and immunochemical characteristics of proteoglycans in bovine gingiva and dental pulp

Proteoglycans were extracted with 4 M guanidinium chloride at 6 degrees C and purified by ion-exchange chromatography and precipitation with cetyl-pyridinium chloride. Chromatography on Sepharose CL-4B under dissociating conditions separated larger (PG1) and smaller (PG2) proteoglycans. Gingival PG2, by virtue of its amino-acid composition and the exclusive presence of L-iduronate-rich dermatan sulphate, was a proteodermatan sulphate (PDS) with a similar molecular weight to periodontal-ligament PDS. Reaction with four monoclonal antibodies to bovine skin PDS confirmed the relationship between these small proteoglycans and that of skin. Their glycoprotein cores, liberated by digestion with chondroitinase ABC, were similar in size (mol. wt = 55,000 by SDS-gel electrophoresis). Pulp PG2 had a small amount of PDS but the main component contained D-glucuronate-rich sulphated galactosaminoglycans. Similar galactosaminoglycans, which included chondroitin sulphate, characterized the larger proteoglycans of gingiva and pulp; significant amounts of L-iduronic acid-rich dermatan sulphate or heparan sulphate were not present.     

Glycosaminoglycan patterns in gingival proteoglycans of rat with age.

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.     

Molecular size distribution analysis of human gingival glycosaminoglycans in cyclosporin- and nifedipine-induced overgrowths

Glycosaminoglycans are thought to accumulate in formative lesions like drug-induced gingival overgrowth. Recent evidences, however, suggest that the amounts of glycosaminoglycans are comparable in overgrown and healthy gingiva. Besides, alterations in the size distribution of glycosaminoglycan molecules isolated from phenytoin-induced overgrown samples have also been suggested. Therefore, we sought to determine possible differences in molecular size distribution of gingival glycosaminoglycans in other types of drug-induced overgrowths. Purified gingival glycosaminoglycans from healthy and cyclosporin- and nifedipine-induced overgrown gingival tissues were analyzed by agarose gel electrophoresis and their molecular-size distribution was evaluated by both gel filtration chromatography and polyacrylamide gel electrophoresis. Our results on the gingival glycosaminoglycan composition showed presence of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in all types of gingival tissues examined. In addition, hyaluronic acid was predominantly of a large size eluting near to the void volume of a Superose-6 column, while the sulfated glycosaminoglycans were mainly composed of low molecular size glycosaminoglycans. Our results show no differences in the molecular-size distribution of hyaluronic acid and sulfated glycosaminoglycans among healthy and drug-induced overgrown gingival tissues.     

Metabolism of heparan sulfate proteoglycans in Drosophila cell lines]

Proteoglycans with glycosaminoglycan side chains are found on the cell surface and in the extracellular matrix. Recently, it has been demonstrated that they are widely distributed in invertebrates, as well as in vertebrates. In the fruit fly, Drosophila melanogaster, core proteins of heparan sulfate proteoglycans (HSPGs), including division abnormally delayed (dally), dally-like, perlecan, and syndecan, have been identified by genetic analyses. Biochemical studies of the glycosaminoglycan structure in Drosophila have also found the presence of chondroitin sulfate, in addition to heparin sulfate; however, its core protein remains to be identified. Functional studies of HSPG in Drosophila have shown that dally and dally-like (members of glypican) are required for Wingless (a member of the Wnt family) signaling, and they are also implicated in affecting signaling of Decapentaplegic (a member of the transforming growth factor-beta/bone morphogenetic protein superfamily). Further evidence that HSPGs play important roles in these cellular signaling processes has been established from studies of genetic mutants defective in biosynthetic enzymes of HSPGs, such as sugarless (a homologue of UDP dehydrogenase), sulfateless (a homologue of HS N-deacetylase/N-sulfotransferase), and tout-velu (a homologue of Ext encoding HS polymerase). In this paper, the metabolism of Drosophila HSPGs was investigated to further clarify their biological functions, in two Drosophila cell lines: S 2 cell and Kc cell, using metabolic labeling techniques in combination with various pulse-chase protocols.     

Studies on matrix components relevant to structure and function of the temporomandibular joint]

Proteoglycans (PGs), or their component glycosaminoglycan (GAG) chains, have long been recognized as small in quantity but as significant components for many connective tissues. The temporomandibular joint (TMJ) disc and synovial fluid were rarely investigated in this regard. This research was aimed at the biochemical analysis of the composition of collagen, GAGs and PGs in the normal human and porcine TMJ discs, and of PGs in the synovial fluid. 1. The collagen type analysis by gel electrophoresis revealed that the human and porcine TMJ discs were composed only of type I collagen. 2. The human TMJ disc contained 69.9% chondroitin sulfate, 24.5% dermatan sulfate, 5.6% hyaluronic acid and a trace amount of keratan sulfate, and the porcine TMJ disc, 5.8%, 91.4%, 2.8% and a trace amount, respectively. Immunohistochemical staining of the functional parts of the TMJ disc showed a universal distribution of these GAGs. 3. PGs were extracted from the TMJ disc with 4 M guanidine chloride and separated by ion-exchange and gel-filtration chromatography. A high molecular weight PG resembling the cartilage PG and two low molecular weight PGs having a core protein of Mr 40 K were isolated. 4. PGs suggested to be derived from the matrix of other tissues were found in the abnormal human synovial fluid. From these results it was suggested that the TMJ disc was thought not to be fibrocartilage but a fibrous tissue adapted against to the compressive and tensile force.     

The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid

Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfute was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate M_r 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated from inflamed tissue was mostly a small molecular weight species which elutccl in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.     

Analysis of chondroitin sulfate isomers in the periodontium of the monkey using high-performance liquid chromatography

Glycosaminoglycan (GAG) was extracted from monkey periodontium, consisting of gingiva, periodontal ligament, alveolar bone and cementum, and from dental pulp and dentin by digestion with Pronase E. Unsaturated disaccharide isomers formed by chondroitinase AC digestion from chondroitin sulfate were labeled with dansylhydrazine and analyzed by high-performance liquid chromatography. These tissues showed different molar ratios of the unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lowest in the dental pulp, followed by the gingiva, periodontal ligament, dentin, alveolar bone, and cementum, in that order. It was greater in the calcified than in the uncalcified tissues.     

Proteoglycans and glycosaminoglycans in phenytion-induced gingival overgrowth

Proteoglycans and glycosaminoglycans in normal gingival and phenytoin-induced gingival overgrowth were studied by gel electrophoresis and HPLC methods after extration with guanidinium hydrochloride and subsequent cesium chloride gradient centrifugation. The results showed that normal gingival. The relative collagen content was decreased in the phenytoin lesion. These results are in agreement with our revious stereological study, which reported an accumulation of the non-collagenous matrix chondroitin sulfates Containing non-sulfated, 4-sulfated and 6-sulfated disaccharide units, dermatan sulfate, hyaluronic acid and presumably also heparan sulfate in both normal gingival. and phenyton-induced gingival overgrowth. The increased amounts of PGs seen in the PHT lesion were associated with an increase mainly in chondroitinase sensitive glycosaminoglycans of high molecular weight and with a relative increase in iduroinc acid content. This study is consistent with the view that the PHT-lesion represents a tissue with an altered composition of the connective tissue.