硫酸软骨素蛋白聚糖在脑发育中的作用

蛋白聚糖 (ＰＧ)是一种或多种糖胺聚糖链 (ＧＡＧ)和核心蛋白共价结合形成的复合物 ,硫酸软骨素蛋白聚糖 (ＣＳＰＧ)即核心蛋白与硫酸软骨素 (ＣＳ)链共价交连的一类蛋白聚糖 ,不同的核心蛋白与ＣＳ链相连形成不同的ＣＳＰＧ。聚集蛋白聚糖家族 (ａｇｇｒｅｃａｎｆａｍｉ ｌｙ) ,磷酸蛋白聚糖 (ｐｈｏｓｐｈａｃａｎ) ,神经蛋白聚糖Ｃ(ｎｅｕｒｏｇｌｙＣ)是哺乳动物脑发育中的 3种经典的ＣＳＰＧ。其它如星形软骨素蛋白聚糖(ａｓｔｒｏｃｈｏｎｄｒｉｎ) ,饰胶蛋白聚糖 (ｄｅｃｏｒｉｎ) ,睾丸蛋白聚糖 (ｔｅｓｔｉｃａｎ) ,细胞蛋白聚糖 …     

Cloning and Characterization of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase from a Marine Bacterium

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886–27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17–65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications.     

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.     

Monoclonal Antibody #5-2-26 Recognizes the Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.     

Iduronic Acid in Chondroitin/Dermatan Sulfate: Biosynthesis and Biological Function

The ability of chondroitin/dermatan sulfate (CS/DS) to convey biological information is enriched by the presence of iduronic acid. DS-epimerases 1 and 2 (DS-epi1 and 2), in conjunction with DS-4-O-sulfotransferase 1, are the enzymes responsible for iduronic acid biosynthesis and will be the major focus of this review. CS/DS proteoglycans (CS/DS-PGs) are ubiquitously found in connective tissues, basement membranes, and cell surfaces or are stored intracellularly. Such wide distribution reflects the variety of biological roles in which they are involved, from extracellular matrix organization to regulation of processes such as proliferation, migration, adhesion, and differentiation. They play roles in inflammation, angiogenesis, coagulation, immunity, and wound healing. Such versatility is achieved thanks to their variable composition, both in terms of protein core and the fine structure of the CS/DS chains. Excellent reviews have been published on the collective and individual functions of each CS/DS-PG. This short review presents the biosynthesis and functions of iduronic acid-containing structures, also as revealed by the analysis of the DS-epi1- and 2-deficient mouse models.     

Monoclonal Antibody Tor 23 Recognizes a Determinant of a Presynaptic Acetylcholinesterase

A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent-treated electroplax homogenates and insoluble in high-salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.     

A Chondroitin/Dermatan Sulfate Form of CD44 Is a Receptor for Collagen XIV (Undulin)

Collagen XIV, a fibril-associated collagen with interrupted triple helices, is expressed in differentiated soft connective tissues and in cartilage. However, a cellular receptor for this protein has not been identified. Here we show that human placental collagen XIV, isolated by a mild and simple two-step method, serves as adhesive protein for a variety of mesenchymal and some epithelial cells. Cell adhesion could be inhibited by preincubation of the collagen XIV substrate with heparin or with the chondroitin/dermatan sulfate proteoglycan decorin and by pretreatment of cells with chondroitinase ABC or heparinase III, suggesting a cell membrane proteoglycan as receptor. Affinity chromatography of¹²⁵I-labeled fibroblast cell surface proteins on collagen XIV–Sepharose yielded a chondroitin/dermatan sulfate proteoglycan with a molecular mass of 97–105 kDa after chondroitinase ABC digestion and of 60–70 kDa after further treatment withN-glycosidase F. The eluates contained also some high-molecular-weight material that was susceptible to digestion with heparinase but no detectable integrins. Immunoprecipitation with a specific monoclonal antibody identified the prominent chondroitin/dermatan sulfate proteoglycan as a member of the CD44 family. The interaction between collagen XIV and cells appears to be finely tuned, since matrix-associated glycosaminoglycans, and particularly proteoglycans like decorin, could compete with cells for the binding site(s) on collagen XIV under physiological conditions.     

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope

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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.     

Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K_av shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a K_av shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K_av of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.The synthesis and physical properties (size and charge) of proteoglycans are altered under some pathological conditions such as cancer (1), spinal cord injury (2), atherosclerosis (3), and asthma (4). The importance of these pathological changes in proteoglycans has driven the need to study and design novel chemical tools which can control proteoglycan biosynthesis. Thus, we have studied the effect of a fluorinated analogue of glucosamine on proteoglycan synthesis in murine airway smooth muscle cells.Mono-, di-, and oligosaccharides that contain fluorine have been developed to study the enzymes involved in carbohydrate metabolism, and some of these have been shown to be inhibitors. The atomic size of fluorine is only slightly smaller (van der Waals'' radius (r′) of 135 pm) than that of oxygen (140 pm), and the C-F bond has a higher energy (485 kJ/mol) compared with that of C-O (370 kJ/mol) (5). The substitution of fluorine for oxygen at the 4-position of N-acetylglucosamine (4-F-GlcNAc)² confers a greater electronegativity on the bond and makes it less likely to be removed from the GlcN carbon ring. It is the properties of fluorine that contribute to the unique characteristics of 4-F-GlcNAc.4-F-GlcNAc used for cell culture experiments has O-acetyl groups at several of its ring positions, which in effect increases its cell permeability compared with that of unmodified forms (6). After hydrolysis to remove the O-acetyl residues, 4-F-GlcNAc, like GlcNAc, must be converted to UDP-4-F-GlcNAc, which in turn can be a substrate (or inhibitor) of enzyme reactions that use UDP-GlcNAc. GlcN is typically used as a control compound for 4-F-GlcNAc in vitro because of its superior cell permeability characteristics when compared with acetylated GlcN derivatives. Although acetylated GlcN derivatives enter the cell via passive diffusion, GlcN can enter by both passive diffusion and through the glucose transporter 4 (7).4-F-GlcNAc and 4-F-N-acetylgalactosamine (4-F-GalNAc) have been specifically studied as potential inhibitors of cell growth for the treatment of leukemia. The IC₅₀ values for 4-F-GlcNAc and 4F-GalNAc inhibition of leukemic cell proliferation are 34 and 35 μm, respectively (8). Moreover, by blocking polylactosamine synthesis necessary for elaboration of selectin ligands, 4-F-GlcNAc exhibits anti-inflammatory effects by reducing leukocyte homing to areas of contact allergic hypersensitivity in mice in vivo (9). Beyond effects on cell membrane glycoproteins, it has been proposed that the 4-fluorinated analogue of glucosamine truncates the GlcNAc-hexuronic acid chains on heparan sulfate (HS) by preventing the formation of the normal 1,4-glycosidic linkage between glucuronate (GlcUA) and on the nonreducing end of the growing chain (10). Thus, 4-F-GlcNAc has been suggested as a therapy for reducing amyloid deposition, which can feature HS accumulation (10, 11). Treatment of cultured hepatocytes in vitro with 4-F-GlcNAc and 4F-GalNAc (10–1000 μm) for 24 h reduced [³H]glucosamine and [³⁵S]sulfate incorporation into cellular glycosaminoglycans (11). However, total protein synthesis was also reduced at 1000 μm (11).Although the effects of 4-F-GlcNAc on HS production have been described (10), its effects on other extracellular matrix glycosaminoglycans, chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA), have not been reported.Airway smooth muscle (ASM) cells produce HS- and CS/DS-containing proteoglycans, including perlecan, versican, and decorin (12). Using these cells, we observed that 4F-GlcNAc inhibits CS/DS synthesis nearly as effectively as it inhibits HS synthesis. Although the 4-F on a nonreducing terminal F-GlcNAc-HS chain would block further HS synthesis by preventing the formation of the GlcUAβ1,4 bond required for elongation, the glycosidic bond in CS/DS is β1,3 between hexuronic acid and GalNAc. Thus, UDP-4-F-GlcNAc could not interfere with CS/DS synthesis via the same mechanism because it cannot be 4-epimerized to UDP-4F-GalNAc. Thus, we hypothesized that UDP-4-F-GlcNAc is a potent inhibitor of the 4-epimerase required to convert UDP-GlcNAc to UDP-GalNAc, thereby depleting the cell of UDP-GalNAc, a necessary substrate for CS/DS synthesis. To explore this putative mechanism, we analyzed the inhibitory effects of 4-F-GlcNAc on intrinsic and xyloside-stimulated CS synthesis in ASM cells (13).     

Iduronic Acid in Chondroitin/Dermatan Sulfate Affects Directional Migration of Aortic Smooth Muscle Cells

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1−/− aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.     

A Monoclonal Antibody to Alpha Tubulin Recognizes Host Cell and Trypanosoma cruzi Tubulins1

ABSTRACT. A mouse monoclonal anti-α-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines–J774 macrophages, BSC-1, and Vero cells–infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.     

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.Chondroitin sulfate (CS) is an unbranched polymer chain composed of alternating glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) units (36, 49). In dermatan sulfate (DS), d-glucuronic acid is converted to its epimer l-iduronic acid (IdoA) (25). The extent of this modification, which varies from a few percent of the glucuronic acid being epimerized to a predominant presence of iduronic acid, depends on the variable epimerase activity in tissues and on the core protein attached to the chain in CS/DS proteoglycans (PGs) (41, 47). The same CS/DS PG has a different iduronic acid content, depending on the cell type and tissue of origin (4, 5). The name CS/DS denotes the hybrid GlcA-IdoA nature of the chain. It has long been known that the distribution of iduronic acids within the chain is not random but follows two patterns: either they are clustered together, forming long iduronic acid blocks, or they are isolated, i.e., interspersed among surrounding glucuronic acids (11). DS epimerase 1 (DS-epi1) and DS-epi2, encoded in mouse by the Dse and Dsel (Dse-like) genes, respectively, are present in organisms ranging from Xenopus tropicalis to humans but not in worms and flies (23, 34). During DS biosynthesis, epimerization is followed by the action of eight C-specific O-sulfotranferases, which transfer a sulfate group to C-2 of both IdoA and GlcA and to C-4, C-6, and C-4/C-6 of GalNAc (18). These modification reactions, individually affecting only part of the available substrate, produce structural variability in the CS/DS chain. Considerable efforts have been made to characterize specific sequences in the CS/DS chains responsible for binding to protein and the subsequent mediation of a biological effect (28). For instance, (IdoA-2OS-GalNAc-4OS)₃- and GalNAc-4/6-diOS-containing structures bind and activate heparin cofactor II, which is the major antithrombotic system in the subendothelial layer (48). IdoA/GlcA-2OS-GalNAc-6OS-containing structures bind to pleiotrophin, mediating neuritogenic activity (3, 44). IdoA-GalNAc-4OS-containing structures bind to basic fibroblast growth factor, and the complex has been shown to be active in wound healing (46).CS/DS PGs are mainly found in the extracellular matrix. They belong to four families: lecticans, e.g., versican, aggrecan, brevican, and neurocan; collagens, e.g., collagen IX; basement membrane PGs, e.g., SMC3, collagen XV, and perlecan, containing both heparan sulfate (HS) and CS/DS; and small leucine-rich repeat PGs. Some PGs of the first three groups are referred to as CS PGs. The actual presence of iduronic acid, depending on the tissue examined and on the developmental stage, has been overlooked in many cases (37, 44). The archetypical small leucine-rich repeat PG family members decorin, biglycan, fibromodulin, and lumican bind fibrillar collagens and affect collagen fibril and scaffold formation in connective tissues (15). Decorin and biglycan are substituted with one and two CS/DS chains, respectively. Decorin is involved in collagen type I fibril formation and matrix assembly in a wide range of connective tissues and binds near the C terminus of collagen monomers, delaying their accretion to the growing fibrils. We have identified an SYIRIADTNIT sequence in decorin as essential for binding to collagen (16). The role of the decorin CS/DS chain in vivo has not been explored, although in vitro studies suggest that IdoA promotes the binding of CS/DS to collagen (31) and is required for self-association of CS/DS chains (6, 10, 22).Here the function of DS-epi1 in mice was disrupted. DS-epi1-deficient mice show CS/DS with a marked deficiency in iduronic acid-containing structures. The deletion of DS-epi1 is likely to affect many types of PGs and to result in a complex phenotype. We focus on skin alterations presumably caused by altered decorin/biglycan CS/DS chains.     

Characterization of Human DSPG3, a Small Dermatan Sulfate Proteoglycan

PG-Lb is a small dermatan sulfate proteoglycan that has been previously characterized in chicken. In the developing limb, chick PG-Lb appears to be exclusively expressed in the zone of flattened chondrocytes. We have cloned and sequenced the human homolog to chick PG-Lb from two human chondrocyte cDNA libraries and a human chondrocyte RNA sample. The human homolog has been named DSPG3, as it is the third member of the small dermatan sulfate proteoglycan family to be identified and characterized along with biglycan (PG-I) and decorin (PG-II). DSPG3 maps to chromosome 12q21 and is composed of 1515 nucleotides of cDNA that code for a 322-amino-acid protein. The protein contains three potential glycosaminoglycan attachment sites, two N-glycosylation sites, a poly- glutamic acid stretch, and six cysteines. By Northern analysis, we have demonstrated that DSPG3 is expressed in cartilage, as well as ligament and placental tissues.     

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary,Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.     

猪链球菌2型次黄嘌呤核苷酸脱氢酶单克隆抗体的制备及其识别表位分析

用纯化的硫氧还蛋白-IMPDH融合蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,对杂交瘤细胞及时筛选,阳性孔经4次有限稀释法克隆,成功获得1A8、1F2、2D2和2D12共4株能稳定传代并分泌抗IMPDH的单克隆抗体(McAb)的杂交瘤细胞株.4株腹水型单克隆抗体间接ELISA效价分别为100x211、100x211、100x210和100x28,经Western-blot分析表明,4株单抗与硫氧还蛋白-IMPDH融合蛋白均具有特异性反应,并且通过4种IMPDH全基因分片段缺失表达的融合蛋白,分析了4株单抗所识别抗原决定簇的差异性,发现1A8、1F2,2D2识别表位的编码基因集中在IMPDH基因片段的627 bp～790 bp之间,2D12识别表位的编码基因则集中在IMPDH基因片段的411 bp～790 bp之间.猪链球菌2型中IMPDH单克隆抗体的获得及相应表位分析为研究IMPDH蛋白的生物学活性及免疫学活性奠定了基础.     

Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.     

The Protective Role of Fucosylated Chondroitin Sulfate,a Distinct Glycosaminoglycan,in a Murine Model of Streptozotocin-Induced Diabetic Nephropathy

Background

Heparanase-1 activation, albuminuria, and a decrease in glomerular heparan sulfate (HS) have been described in diabetic nephropathy (DN). Glycosaminoglycan (GAG)-based drugs have been shown to have renoprotective effects in this setting, although recent trials have questioned their clinical effectiveness. Here, we describe the effects of fucosylated chondroitin sulfate (FCS), a novel GAG extracted from a marine echinoderm, in experimentally induced DN compared to a widely used GAG, enoxaparin (ENX).

Methods

Diabetes mellitus (DM) was induced by streptozotocin in male Wistar rats divided into three groups: DM (without treatment), FCS (8 mg/kg), and ENX (4 mg/kg), administered subcutaneously. After 12 weeks, we measured blood glucose, blood pressure, albuminuria, and renal function. The kidneys were evaluated for mesangial expansion and collagen content. Immunohistochemical quantifications of macrophages, TGF-β, nestin and immunofluorescence analysis of heparanase-1 and glomerular basement membrane (GBM) HS content was also performed. Gene expression of proteoglycan core proteins and enzymes involved in GAG assembly/degradation were analyzed by TaqMan real-time PCR.

Results

Treatment with GAGs prevented albuminuria and did not affect the glucose level or other functional aspects. The DM group exhibited increased mesangial matrix deposition and tubulointerstitial expansion, and prevention was observed in both GAG groups. TGF-β expression and macrophage infiltration were prevented by the GAG treatments, and podocyte damage was halted. The diabetic milieu resulted in the down-regulation of agrin, perlecan and collagen XVIII mRNAs, along with the expression of enzymes involved in GAG biosynthesis. Treatment with FCS and ENX positively modulated such changes. Heparanase-1 expression was significantly reduced after GAG treatment without affecting the GBM HS content, which was uniformly reduced in all of the diabetic animals.

Conclusions

Our results demonstrate that the administration of FCS prevented several pathological features of ND in rats. This finding should stimulate further research on GAG treatment for this complication of diabetes.