Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

大肠杆菌表达的重组人骨形成蛋白-7的复性

带有pBV221-hBMP-7的E．coli表达得到的rhBMP-7以不溶的包涵体形式存在,用高浓度的变性利溶解后,经过DEAE-FF纯化,得到高纯度的目的蛋白,达95％以上。分别用尿素浓度梯度降低法、添加促复性剂及人工分子伴侣法对蛋白质进行复性,并通过不同方法对复性结果进行比较。Western blot中辉度扫描结果显示,GSH／GSSG法样品二聚体／单体比例为79．5／20．5,尿素浓度梯度降低法二聚体／单体比例为73．6／26．4,表明GSH／GSSG法复性样品溶液上清中含较高比例的蛋白质二聚体。根据不同复性样品对NIH3T3细胞ALP活性影响大小的比较结果,氧化还原剂最有助于二聚体的形成,蛋白质活性最高。     

Isolation of proteoglycans from human articular cartilage.

Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.     

Effect of elcatonin on osteoinduction by recombinant human bone morphogenetic protein-2

To evaluate the effect of elcatonin on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), 5 microg of rhBMP-2 was implanted into intramuscular sites of rats. For 14 days after the implantation, elcatonin was administered intraperitoneally with total dosage of 80 U, 8 U, and 0.8 U, respectively. For the control group, only physiological saline was administered. At 21 days after implantation, the area of the oval shadow in the radiologic findings depended on the elcatonin dose and the amount of trabecular bone and the number of osteoblasts observed in the histologic findings depended on the dosage of elcatonin. The values of ALP activity and Ca content also showed an elcatonin dose dependency. These results suggested that elcatonin is effective in enhancing osteoinduction by rhBMP-2 within the dose range of this study, and that elcatonin has an anabolic effect on osteoblasts in addition to an antiresorptive effect.     

The proteoglycans of bovine nasal cartilage and human articular cartilage: a sedimentation equilibrium analysis

Cartilage proteoglycan was isolated from bovine nasal septum and fractionated according to buoyant density after dissociative CsCl density gradient centrifugation. Gel-exclusion chromatography showed that hyaluronic acid was present in fractions of density lower than 1.69 g/mL. The molecular weight, assessed by sedimentation equilibrium analysis, of the proteoglycan present in the fractions with density > 1.69 g/mL, which appeared chromatographically homogeneous and constituted 54% of the preparation, ranged from 1.0 to 2.6 × 10⁶ for v = 0.55 cm³ g^?1. Carbodiimide-induced modification of the carboxyl groups by methylamine resulted in a reduction of the molecular weight to 0.74 – 1.25 × 10⁶. An analogous reduction in molecular weight was obtained after equilibration of this proteoglycan fraction with hyaluronic acid oligomers containing five disaccharide units. Since both procedures are known to cause inhibition of the interaction between proteoglycans and hyaluronic acid, it is suggested that this lower molecular-weight range represents the true degree of polydispersity of the sub-units of hyaline cartilage proteoglycan constituting this fraction, while the higher values obtained for the intact proteoglycan are the result of the presence of hyaluronic acid in the sample. The molecular-weight range of the whole proteoglycan subunit preparation, assessed after carboxyl group modification, was 0.5–1.2 × 10⁶. Apparently normal and abnormal cartilage was excised from single human osteoarthrosic femoral heads. Proteoglycans extracted by 4M guanidine hydrochloride were isolated after dissociative density gradient centrifugation and subjected to carboxyl group modification. Preparations from normal tissue exhibited molecular-weight averages ranging from 5 to 9 × 10⁵. A molecular-weight reduction was observed with proteoglycans isolated from abnormal areas.     

Effect of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days -2 to 0, -2 to 7, and -2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration.     

Delayed aggregation of proteoglycans in adult human articular cartilage

The biosynthesis and macromolecular organization of proteoglycans was studied in explants of adult human articular cartilage. In a series of pulse-chase experiments, labelling with (³⁵S)sulphate, it was shown that the proteoglycan monomer is synthesized as a precursor that has a low affinity for hyaluronic acid. These findings suggest a possible mechanism by which the rate of incorporation of proteoglycans into the extraceHular matrix may be controlled.     

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.     

Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

To improve recombinant human bone morphogenetic protein-2 (rhBMP-2) yield, cell lines stably expressing hBMP2 were cultured in the presence of polyarginine peptide IND-1 and showed up to 6-fold increase in the yield of mature BMP-2. Repeated addition of IND-1 to cell cultures consistently improved BMP-2 yields over 53 days without affecting cell growth and viability. Investigation of its mechanism of action showed that IND-1 inhibited pro-protein convertase (PC) activity when incubated with cell lysates. However, when intact cells were cultured with IND-1, no change in cellular PC activity was observed. Furthermore, knockdown of furin (a prototypical member of the PCs) in cells did not affect their BMP-2 yields, suggesting furin/PC inhibition is unlikely the mechanism by which IND-1 enhances BMP-2 yields. IND-1 as a medium additive thus enhances BMP-2 production in mammalian cell expression systems.     

Recombinant human bone morphogenetic protein-2 stimulates differentiation in primary cultures of fetal rat calvarial osteoblasts

Molecular mechanisms of lipid synthesis and their controls in hepatic stellate cells are not known. We have previously proposed that, in contrast to other fat storing cells, hepatic stellate cells are not involved in energy storage, but they represent a particular cell population specialized in storage of lipid-soluble substances, the major one being probably retinol. In agreement with this hypothesis, induction of the lipocyte phenotype in stellate cells is not under the control of insulin, but responds to retinoids and other molecules that modify the gene expression program in these cells. In the present study we have monitored the activity of the two major enzymes involved in lipid synthesis during the induction of the lipocyte phenotype in hepatic stellate cells: glycerol-3-phosphate dehydrogenase (GPDH) that mediates the de novo lipid synthesis, and lipoprotein lipase that mediates incorporation of plasma lipids. In early stages of lipocyte induction, both pathways of lipid synthesis are activated. When lipocytes have already constituted the lipid droplets, lipoprotein lipase pathway is downregulated, while GPDH activity remains high. Adult liver has been reported to lack lipoprotein lipase, but under stress, lipase activity was detected around and at the surface of the intrahepatic vasculature. We have now shown that the lipase activity can be induced in the hepatic stellate cells, located in the Disse's space. The high lipoprotein lipase activity under acute induction of lipocyte phenotype, followed by the low activity under conditions of metabolic equilibrium, are in compass with the increased activity of this enzyme under stress, and its low activity in adult liver parenchyma under normal conditions.     

离子交换色谱法对大肠杆菌表达的人重组骨形态发生蛋白-7的纯化

重组大肠杆菌高量表达重组人骨形态发生蛋白-7(rhBMP-7),每升培养液约得到湿菌体3g,其中目的蛋白约占菌体总蛋白量的40％。裂解离心,用低浓度变性剂洗涤初步纯化包涵体,上清中无目的蛋白损失;将包涵体溶解于高浓度变性剂溶液中,目的蛋白纯度提高到60％;然后在不同条件下用离子交换色谱法对变性状态下的蛋白质进行纯化,绝大部分杂蛋白被除去,目的蛋白纯度达95％以上;改变条件,可以减少rhBMP-7损失;用Western blot对目的蛋白进行特异性鉴定。     

Age-related changes in the chemical composition of bovine articular cartilage. The structure of high-density proteoglycans

Proteoglycans were extracted from the articular cartilage of foetal, calf and adult bovine metacarpal–phalangeal joints with 4m-guanidinium chloride. After extraction, the high-density proteoglycans (PG-I fractions) were prepared by sedimentation in two sequential CsCl-density-gradient procedures [Swann, Powell & Sotman (1979) J. Biol. Chem. 254, 945–954]. The PG-I fractions from foetal, calf and adult tissues accounted for 75%, 52% and 46% respectively of the extracted components. The glucosamine, galactose, N-acetylneuraminic acid and protein contents increased with age. The overall amino acid compositions of PG-I fractions were similar. Fractionation of PG-I-fraction samples on a Bio-Gel A-50m column indicated that the molecular weight decreased with age. The PG-I fractions were specifically ³H-labelled by treatment with galactose oxidase followed by reduction with NaB³H₄. The ³H radioactivity was incorporated into both galactose and galactosamine residues of different carbohydrate side chains. The elution profiles of alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples on a Sepharose 6B column showed that the molecular weights of chondroitin sulphate chains were 13500, 12000 and 10500 in foetal, calf and adult tissues respectively. Fractionation of the alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples and ³H-labelled calf and adult PG-I-fraction samples on a Bio-Gel P-10 column showed that there was an inverse relationship between the low-molecular-weight O-linked oligosaccharides and the higher-molecular-weight sialic acid-containing constituents at different ages. The oligosaccharide components of foetal, calf and adult PG-I-fraction samples represented 79%, 69% and 36% respectively of the total sialic acid content of the proteoglycans. Similarly in the ³H-labelled calf and adult samples 75% and 30% of the total radioactivity were present in the oligosaccharide components respectively. Digestion with chondroitinase AC-II and infrared analyses showed that the PG-I-fraction F and C samples contained primarily chondroitin 4-sulphate chains whereas PG-I-fraction sample A was 6-sulphated. These studies show that the major proteoglycans (PG-I fractions) in the articular cartilage of foetal, calf and adult animals differ in the content, types and structure of the chondroitin sulphate, keratan sulphate and oligosaccharide constituents. These changes in proteoglycan structure reflect the gross age-related changes in the chemical composition of the tissue.     

Correlated metabolism of proteoglycans and hyaluronic acid in bovine cartilage organ cultures

Proteoglycans exist in cartilage as complexes in which many proteoglycan molecules are bound to a central filament of hyaluronic acid. Many studies have investigated changes taking place in proteoglycan monomer structure during cartilage catabolism usually under the assumption that hyaluronic acid is a relatively inert metabolic component of the complex. In this paper we present organ culture data supporting a new hypothesis that the catabolism of proteoglycans and hyaluronic acid are coordinately regulated by chondrocytes. The data indicates that: 1) newly synthesized hyaluronate and proteoglycan maintain a nearly constant ratio, almost identical to that existing for the total chemical amounts of these two components in cartilage tissue; 2) these two components are catabolized with virtually identical kinetics; and 3) this catabolic relationship in vitro reflects the loss of hyaluronate and proteoglycans from native, undissociated aggregates as isolated from the tissue. We conclude that hyaluronate catabolism is an integral part of the overall mechanism of proteoglycan resorption in cartilage and that further understanding of this process may be key to the elucidation of the regulatory pathways for proteoglycan resorption in health and disease.     

An oligosaccharide component in proteoglycans of articular cartilage.

Two types of sialic acid-containing component are released from articular cartilage proteoglycan monomer (D1) treated with 0.05 M NaOH containing 1 M NaBH4. The smaller component, which has not been described before, contains galactosamine, glucosamine, galactose and sialic acid (Molar ratio 1:1:1:2). It is eluted from ECTEOLA-cellulose with low molarity (0.4 M) sodium formate and has Kav of 0.70 on Bio-gel P30. Its presence on the proteoglycan monomer was demonstrated at all stages of foetal and adult life.     

Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations

A prokaryotic expression system has been used to produce recombinant human bone morphogenetic protein-2 (rhBMP-2). However, low rhBMP-2 yields and protein loss during purification and renaturation are the hurdles in the clinical application. Previous studies have indicated that variables such as temperature, host cell, salt concentration, and culture time affect the final rhBMP-2 yield. The optimization of these conditions in an Escherichia coli culture yielded 28.258 mg of rhBMP-2 per liter of culture. To reduce rhBMP-2 loss during purification and renaturation, we performed purification before renaturation in the prokaryotic expression system instead of using the traditional renaturation-before-purification approach. rhBMP-2 was separated on a Sephacryl S-300 HR column and eluted from a DEAE-Sepharose Fast Flow column. The collected protein was refolded by dialysis with urea buffer, which was followed by dialysis with ultrapure water. The purified rhBMP-2 dimer significantly increased alkaline phosphatase (ALP) activity and osteogenic activity in the femoral muscle and showed the same level of bone-forming activity as natural BMP-2. This optimized procedure for expression and renaturation of rhBMP-2 has potential clinical applications.     

Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro.

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.     

Differences in the rates of aggregation of proteoglycans from human articular cartilage and chondrosarcoma.

Pieces of adult human articular cartilage and chondrosarcoma were incubated in the presence of [35S]sulphate. After continuous or pulse-change incorporation of radioactivity, proteoglycans were extracted with 4.0 M-guanidinium chloride, purified by equilibrium density-gradient centrifugation and fractionated by gel chromatography. A comparison of the results suggests that the formation of stable aggregates occurs at a lower rate in articular cartilage than in chondrosarcoma.