硫酸软骨素的提取和纯化分离技术

介绍了硫酸软骨素的来源、结构、性质及其生理功能,对硫酸软骨素的提取、纯化及分离技术进行了综述。     

猪喉软骨营养保健成分及清除自由基功能分析

详细分析了猪喉软骨氨基酸和矿物元素组分及含量,并用木瓜蛋白酶水解软骨,732阳离子树脂纯化粗多糖,首次分别探讨水解物肽和硫酸软骨素的体外自由基清除活性。结果发现软骨呈味氨基酸、钙、镁和铁元素丰富,水解物肽和硫酸软骨素多糖的自由基清除机理不同,水解物肽的羟基自由基和超氧自由基清除活性强于硫酸软骨素,DPPH自由基清除活性相反。     

TGF-β3-Induced Chondroitin Sulphate Proteoglycan Mediates Palatal Shelf Adhesion

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by β-d-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-β₃) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-β₃ is added to TGF-β₃ null mutant palatal shelves in culture. When chick palatal shelves (that do not adherein vivo nor express TGF-β₃, nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-β₃ is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-β₃.     

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.     

Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.     

Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the 1-3 and 1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking—(1e-4e) and (1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.     

The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle

The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.     

The use of Fourier‐transform infrared spectroscopy to characterize connective tissue components in skeletal muscle of Atlantic cod (Gadus morhua L.)

In the present study, Fourier‐transform infrared spectroscopy (FTIR) is investigated as a method to measure connective tissue components that are important for the quality of Atlantic cod filets (Gadus morhua L.). The Atlantic cod used in this study originated from a feeding trial, which found that fish fed a high starch diet contained relative more collagen type I, while fish fed a low starch (LS) diet contained relative more glycosaminoglycans (GAGs) in the connective tissue. FTIR spectra of pure commercial collagen type I and GAGs were acquired to identify spectral markers and compare them with FTIR spectra and images from connective tissue. Using principal component analysis, high and LS diets were separated due to collagen type I in the spectral region 1800 to 800 cm^?1. The spatial distribution of collagen type I and GAGs were further investigated by FTIR imaging in combination with immunohistochemistry. Pixel‐wise correlation images were calculated between preprocessed connective tissue images and preprocessed pure components spectra of collagen type I and GAGs, respectively. For collagen, the FTIR images reveal a collagen distribution that closely resembles the collagen distribution as imaged by immunohistochemistry. For GAGs, the concentration is very low. Still, the FTIR images detect the most GAGs rich regions.      

Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.     

基于工程化毕赤酵母一锅法合成硫酸软骨素A北大核心CSCD

硫酸软骨素(chondroitin sulfate,CS)是一种线性多糖,广泛应用于医疗和保健等领域。相比于传统动物组织提取法,微生物合成硫酸软骨素具有可控、易规模化放大等优势。为实现硫酸软骨素A(CSA)的高效合成,本研究首先通过整合软骨素合酶编码基因kfoC、kfoA以及UDP-葡萄糖脱氢酶编码基因tuaD至毕赤酵母GS115基因组中,构建了以甘油为唯一碳源发酵生产软骨素的毕赤酵母工程菌株。通过进一步优化软骨素合成途径,软骨素分批补料发酵水平达到2.6 g/L。在进一步整合表达软骨素-4-O-磺基转移酶的基础上,本研究通过向生产软骨素毕赤酵母工程菌株破碎液中添加3′-磷酸腺苷-5′-磷酰硫酸和软骨素-4-O-磺基转移酶,成功建立了CSA的一锅法生物合成体系。通过优化,最终实现0-40%不同磺酸化水平CSA的可控合成。本研究中CSA的一锅法生物合成体系操作简便、易放大,更适用于工业化大规模生产。本研究结果也为肝素等其他糖胺聚糖的合成提供了思路。