鹿茸硫酸软骨素蛋白聚糖的分离纯化研究

报道了用DEAE-纤维素(DE-23)离子交换柱层析从鹿茸二杠中分离、纯化及鉴定硫酸软骨素的方法.首先用适量蒸馏水浸泡鹿茸二杠并将其捣碎,离心取沉淀用盐酸胍浸提,浸提液对尿素液透析后经DEAE-纤维素(DE-23)离子交换柱层析,吸附大量的硫酸软骨素;再用含盐尿素溶液梯度洗脱、分离后,经软骨素酶消化及琼脂糖凝胶电泳, 与硫酸软骨素标准品比较,证实得到的物质为纯的硫酸软骨素蛋白聚糖,其得率约为48.77%.该方法使硫酸软骨素分离纯化一步完成,大大简化了纯化步骤.     

硫酸软骨素研究现状

硫酸软骨素是从动物软骨中提取的黏性多糖,在医药、化妆品和食品工业上有广泛的用途,市场前景广阔。本对硫酸软骨素的性质和用途做了简要的介绍,并阐述了近几年硫酸软骨素生产工艺的发展状况。     

硫酸软骨素快速提取法研究

采用先高温蒸煮后加稀碱与酶解相结合 ,并用氯仿反萃取的方法提取硫酸软骨素 ,与其它提取方法相比较 ,缩短了一半工艺流程 ,同时提高了纯度和提取率 ,减轻了碱法提取所带来的环境污染。     

膜分离技术在硫酸软骨素分离方面的应用

介绍了硫酸软骨素的生产工艺 ,应用膜分离技术对现有工艺进行了改进 ,使硫酸软骨素纯度达 95 .2 % ,收率提高3% ,并且简化了操作     

硫酸软骨素对慢性酒精中毒大鼠脑损伤的保护作用

目的:探讨硫酸软骨素对慢性酒精中毒脑损伤的作用及可能机制.方法:雄性 Wistar 大鼠60只随机分为6组,酒精模型组以剂量为8ml·kg-1·d-150%的酒精每天灌胃一次,纳洛酮药物组给予乙醇半小时后腹腔注射纳洛酮0.08mg·k-1·d-1,硫酸软骨素低、中、高剂量干预组在酒精模型组的基础上分别给予硫酸软骨素50、100、150mg·kg-1·d-1,空白对照组给予等体积的蒸馏水,持续2周;第三周把50%的酒精的剂量递增为12mg·kg-1·d-1,持续灌胃6周.在第八周末实验结束后取血,分离血清,留取脑组织.HE染色观察各组大鼠神经细胞的变化.生化测定各组大鼠血清及脑组织匀浆中谷胱甘肽过氧化物酶(GSH-PX)和超氧化物歧化酶(SOD)的活性以及脂质过氧物终未产物丙二醛(MDA);并测定脑皮质中β-内啡肽含量(β-EP).结果:模型组大鼠大脑皮质和海马区神经元数量明显减少,神经细胞排列紊乱.硫酸软骨素中剂量组大鼠大脑皮质和海马区神经细胞排列层次较清晰.与酒精组相比较,硫酸软骨素中剂量组大鼠血清和脑组织匀浆中MDA含量明显降低(P<0.01),脑皮质中β-内啡肽含量明显降低(P<0.01);GSH-PX含量及SOD活性显著升高(P<0.01).结论:硫酸软骨素对大鼠慢性酒精中毒脑损伤具有保护作用.     

硫酸软骨素对慢性酒精中毒氧化损伤的保护作用

目的:研究硫酸软骨素时慢性酒精中毒氧化损伤的保护作用.方法:60只Wistar大鼠随机分成六个组:空白组给予蒸馏水,酒精模型组给予50%的酒精8 ml·kg-1·d-1灌胃,纳洛酮组在给予酒精三十分钟后腹腔注射纳洛酮0.08mgkg-1·d-1,硫酸软骨素低、中、高剂量组在酒精模型组的基础上分别给予硫酸软骨素50,100和150mg·kg-1·d-1.两周后酒精的剂量增加到12mg·kg-1d-1.在第八周末,分离大鼠脑组织,观察大鼠神经细胞.用生物方法测定大鼠脑组织中GSH-PX、SOD、MDA以及Ache的活性.结果:模型组大鼠大脑皮质和海马区神经细胞的数量明显减少并且排列紊乱;和酒精模型组相比较,硫酸软骨素中剂量组大脑皮质和海马区神经细胞排列较整齐,酒精+Chondroitin组脑组织中MDA的含量和Ache降低(P＜0.01),GSH-PX的含量和SOD的活力均明显增加(P＜0.01).结论:硫酸软骨素时慢性酒精中毒氧化损伤具有保护作用.     

硫酸软骨素提取工艺的研究

硫酸软骨素是生物界广泛存在的酸性粘多糖,广泛存在于动物的器官软骨。以猪器官软骨为原料,采用碱提取和酶解相结合的方法提取硫酸软骨素,利用正交实验对碱提取过程中的温度,碱浓度以及提取时间等三个关键因素进行优化,并对硫酸软骨素成品的相关指标进行检测。正交实验结果表明,最佳的碱提取条件为：温度40℃、提取时间8h、碱提取浓度为0．15g／mL。在此条件下获得的碱提取液,通过酶解、脱色、醇沉、干燥,得到白色硫酸软骨素成品,总得率为20％,各项指标均符合国家部颁标准,达到出口要求。     

硫酸乙酰肝素蛋白聚糖的功能机制研究进展

硫酸乙酰肝素蛋白聚糖是由核心蛋白和与之相连的硫酸乙酰肝素糖链组成,广泛分布于细胞膜与细胞外基质中。其中多配体蛋白聚糖（syndecan）和糖基磷脂酰肌醇锚定蛋白聚糖（glypican）存在与细胞膜上,而串珠蛋白聚糖（perlecan）和组合蛋白聚糖（agrin）表达在细胞外基质中。该类蛋白在生理与病理历程中,如发育、伤口愈合、肿瘤发生发展、感染、免疫应答等过程中担任重要作用,这些功能是其核心蛋白和糖链共同作用的结果。概述硫酸乙酰肝素蛋白聚糖的功能及其机制研究进展,同时强调其在作为药物靶标和临床诊断研究中的应用。     

胶原蛋白X Ⅷ——基底膜硫酸类肝素蛋白聚糖

胶原蛋白XⅧ（COXⅧ）是一种十分保守的基底膜胶原蛋白成分,根据其蛋白质结构特点,它和COL XV一起构成MULTIPLEXINs胶原亚家族。同时,它还含有丰富的硫酸肝素糖链,是迄今发现的第三个基底膜硫酸肝素蛋白聚糖。COLXⅧ基因具有两个结构不同的启动子区,由此产生组织分布各异的不同变体,它们在胚胎发育中具有器官特异的调控功能,特别是在视网膜发育中,完整的COLXⅧα1链必不可少。     

对硫酸软骨素传统提取方法的改进

软骨中硫酸软骨素与蛋白质结合成蛋白多糖,并与胶原蛋白结合在一起。本文采用先高温蒸煮,后加稀碱与酶解相结合提取该药物,ＴＣＡ沉淀蛋白质后,高岭土吸附,再用氯仿连续反萃取,使产品质量达到优级纯,较其他方法缩短了原工艺流程,提高了纯度,减轻了碱－盐提取所带来的环境污染。     

The Role of Chondroitin Sulfate Proteoglycans in Nervous System Development

The orderly development of the nervous system is characterized by phases of cell proliferation and differentiation, neural migration, axonal outgrowth and synapse formation, and stabilization. Each of these processes is a result of the modulation of genetic programs by extracellular cues. In particular, chondroitin sulfate proteoglycans (CSPGs) have been found to be involved in almost every aspect of this well-orchestrated yet delicate process. The evidence of their involvement is complex, often contradictory, and lacking in mechanistic clarity; however, it remains obvious that CSPGs are key cogs in building a functional brain. This review focuses on current knowledge of the role of CSPGs in each of the major stages of neural development with emphasis on areas requiring further investigation:     

Chondroitin Sulfate and Chondroitin/Keratan Sulfate Proteoglycans of Nervous Tissue: Developmental Changes of Neurocan and Phosphacan

Abstract: We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached ～1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-β-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.     

Functions of Chondroitin Sulfate and Heparan Sulfate in the Developing Brain

Chondroitin sulfate and heparan sulfate proteoglycans are major components of the cell surface and extracellular matrix in the brain. Both chondroitin sulfate and heparan sulfate are unbranched highly sulfated polysaccharides composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and N-acetylglucosamine, respectively. During their biosynthesis in the Golgi apparatus, these glycosaminoglycans are highly modified by sulfation and C5 epimerization of glucuronic acid, leading to diverse heterogeneity in structure. Their structures are strictly regulated in a cell type-specific manner during development partly by the expression control of various glycosaminoglycan-modifying enzymes. It has been considered that specific combinations of glycosaminoglycan-modifying enzymes generate specific functional microdomains in the glycosaminoglycan chains, which bind selectively with various growth factors, morphogens, axon guidance molecules and extracellular matrix proteins. Recent studies have begun to reveal that the molecular interactions mediated by such glycosaminoglycan microdomains play critical roles in the various signaling pathways essential for the development of the brain.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.Chondroitin sulfates (CS)¹ are complex polysaccharides present at cell surfaces and in extracellular matrices. The polysaccharides belong to a subclass of glycosaminoglycans (GAGs) and are covalently linked to various core proteins to form CS-proteoglycans (CSPGs), each with differences in the protein structures and/or numbers of CS side chains. Apart from their structural role in cartilage, CSPGs contribute to the regulation of a diverse set of biological processes such as neurogenesis, growth factor signaling, angiogenesis, and morphogenesis (1–5). Although the molecular basis of CSPGs functions remains elusive, accumulating evidence suggests that the underlying activities relate to selective ligand binding to discrete structural variants of the polysaccharides. Thus, the current strategy for understanding the biological role of CSPGs aims to identify selective CS polysaccharide–ligand interactions. However, information on the number of CS-chains and their specific attachment site(s) on any given core protein is often scarce which limits our functional understanding of CSPGs.The biosynthesis of GAGs occurs in the endoplasmic reticulum and Golgi compartments and is initiated by the enzymatic addition of a beta-linked xylose (Xyl) to a Ser residue of the core protein. The sequential addition of two galactose residues (Gal) and a glucuronic acid (GlcA) onto the growing saccharide chain completes the formation of a tetrasaccharide linkage region (GlcAβ3Galβ3Galβ4XylβSer). This part of the biosynthesis is the same for CS and heparan sulfate (HS). However, for CS the biosynthesis continues with the addition of an N-acetylgalactosamine (GalNAcβ3), whereas HS biosynthesis continues with the addition of an N-acetylglucosamine (GlcNAcα4) (6). The CS-chains are thereafter elongated through the addition of repeating units of GlcA and GalNAc and are further modified by the addition of specifically positioned sulfate groups (7). Certain features of the core protein seem to influence if a certain Ser residue is selected for GAG attachment and whether CS or HS will be synthesized, but the selection mechanism is largely unknown. Sequence analysis of previously known GAG-substituted core proteins reveals that the glycosylated serine residues are usually flanked by a glycine residue (-SG-), and are associated with a cluster of acidic residues in close proximity (8). This motif may assist in the prediction of potential GAG-sites of core proteins; however, the use of such strategy is ambiguous because proteoglycans may also contain unoccupied motifs or motifs that are occasionally occupied (9).Glycoproteomics strategies have recently appeared that provide site-specific information of N- and O-glycans. Such strategies are typically based on a specific enrichment of glycopeptides and a subsequent analysis with nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) (10). By further developing this concept for proteoglycans (11), we have now analyzed CSPG linkage region glycopeptides of human samples, which enabled us to identify 13 novel human CSPGs in addition to 13 already established CSPGs. Urine and cerebrospinal fluid (CSF) samples were trypsinized and CS glycopeptides were enriched using strong anion exchange (SAX) chromatography. The CS chains were depolymerized with chondroitinase ABC, generating free disaccharides and a residual hexameric structure composed of the linkage region and a GlcA-GalNAc disaccharide dehydrated on the terminal GlcA residue (12). MS/MS analysis provided the combined sequencing of the residual hexasaccharide and of the core peptide.     

Heparan Sulfate Proteoglycans

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein–heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level.Heparan sulfate proteoglycans (HSPGs) are glycoproteins, with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains, a type of glycosaminoglycan (GAG) (Esko et al. 2009). Cells elaborate a relatively small set of HSPGs (∼17) that fall into three groups according to their location: membrane HSPGs, such as syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), the secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan, serglycin (Esko et al. 1985), which allowed functional studies in the context of a cell culture model (Zhang et al. 2006). A decade later, the first HSPG mutants in a model organism (Drosophila melanogaster) were identified (Rogalski et al. 1993; Nakato et al. 1995; Häcker et al. 1997; Bellaiche et al. 1998; Lin et al. 1999), which was followed by identification of mutants in nematodes, tree frogs, zebrafish, and mice (​and3).3). HS is evolutionarily ancient and its composition has remained relatively constant from Hydra to humans (Yamada et al. 2007; Lawrence et al. 2008).

Table 1.

Heparan sulfate proteoglycans

Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.