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1.
The potential relationship of an intact membrane organization for the synthesis of chondroitin and chondroitin 4-sulfate was examined after modification of a mouse mast cell microsomal system with the nonionic detergent, Triton X-100. The results indicated that Triton X-100 had no effect on the rate of polymerization but had a slight effect on the size of glycosaminoglycan chains. An "all or nothing" pattern of sulfation of newly formed chondroitin was obtained in both the presence and the absence of Triton X-100, and this pattern did not change whether sulfation was initiated concurrent with or subsequent to polymerization. Sulfation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system required Triton X-100 but still produced an all or nothing pattern rather than a random sulfation pattern. When a 100,000 x g supernatant fraction was utilized for sulfation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and a partial sulfation pattern was obtained. However, it was similar to the all or nothing pattern in that it still produced two populations, with some chains nonsulfated and others approximately 50% sulfated. When chondroitin hexasaccharide was used with 3'-phosphoadenylylphospho[35S]sulfate, multiple GalNAc residues of the individual hexasaccharides were found to be sulfated. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulfation of multiple GalNAc residues on the individual hexasaccharide molecules was even greater, so that trisulfated products were found. These results suggest that efficient sulfation of chondroitin is related to enzyme-substrate interaction more than to membrane organization. 相似文献
2.
The transfer of [35S] sulfate from [35S]PAPS, by means of PAPS: chondroitin sulfate sulfotransferase, to various chondroitin sulfates, with different degrees of sulfation and molecular weights is reported. Analyses by digestion with chondroitin AC and specific 4- or 6-sulfatases indicate that the sulfation occurs only in position 6 of the non-sulfated N-acetyl galactosamine moiety. The 50-70% desulfated chondroitin 4/6-sulfates are two times better sulfate acceptors than totally desulfated chondroitin, and the affinity of the sulfotransferase increases markedly from the octa-to the deca-saccharide. These results suggest that sulfation increases sharply only after the growing polysaccharide contains about 10 sugar residues, in the early stages of polymerization, and that the sulfation of chondroitin sulfate may be a process in which the addition of some sulfate groups facilitates further sulfation. 相似文献
3.
Heparin and heparan sulfate biosynthesis 总被引:2,自引:0,他引:2
4.
Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains. 相似文献
5.
Lamanna WC Kalus I Padva M Baldwin RJ Merry CL Dierks T 《Journal of biotechnology》2007,129(2):290-307
Heparan sulfate (HS) is a cell surface carbohydrate polymer modified with sulfate moieties whose highly ordered composition is central to directing specific cell signaling events. The ability of the cell to generate these information rich glycans with such specificity has opened up a new field of "heparanomics" which seeks to understand the systems involved in generating these cell type and developmental stage specific HS sulfation patterns. Unlike other instances where biological information is encrypted as linear sequences in molecules such as DNA, HS sulfation patterns are generated through a non-template driven process. Thus, deciphering the sulfation code and the dynamic nature of its generation has posed a new challenge to system biologists. The recent discovery of two sulfatases, Sulf1 and Sulf2, with the unique ability to edit sulfation patterns at the cell surface, has opened up a new dimension as to how we understand the regulation of HS sulfation patterning and pattern-dependent cell signaling events. This review will focus on the functional relationship between HS sulfation patterning and biological processes. Special attention will be given to Sulf1 and Sulf2 and how these key editing enzymes might act in concert with the HS biosynthetic enzymes to generate and regulate specific HS sulfation patterns in vivo. We will further explore the use of knock out mice as biological models for understanding the dynamic systems involved in generating HS sulfation patterns and their biological relevance. A brief overview of new technologies and innovations summarizes advances in the systems biology field for understanding non-template molecular networks and their influence on the "heparanome". 相似文献
6.
Dipanwita Mitra Mohammad H. Hasan John T. Bates Michael A. Bierdeman Dallas R. Ederer Rinkuben C. Parmar Lauren A. Fassero Quntao Liang Hong Qiu Vaibhav Tiwari Fuming Zhang Robert J. Linhardt Joshua S. Sharp Lianchun Wang Ritesh Tandon 《PLoS pathogens》2021,17(8)
Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity. 相似文献
7.
The replica filter technique has been used to isolate variants of Swiss mouse 3T3 cells which produce heparan sulfates with altered levels of sulfation. These changes in the extent of sulfation correlate with alterations in cell morphology, in the organization of cytoskeletal elements, focal contacts, and the extracellular matrix, and in the growth regulation of cells, as expressed by saturation density. An increase in the extent of heparan sulfate sulfation occurs concomitantly with a decreased saturation density and enhanced focal contact formation. In contrast, graded decreases in sulfation correlate with graded increases in saturation density and losses of cytoskeletal and extracellular matrix organization. These graded responses appear very similar to those which have been reported for the transformation of cells with fusiform mutants of Rous sarcoma virus or the adenovirus type 2 Ela transforming gene and suggest that the morphological changes observed in the transformed cells can be controlled by cellular systems. 相似文献
8.
Chondroitin sulfate (CS) and dermatan sulfate (DS) interact with various extracellular molecules such as growth factors, cytokines/chemokines, neurotrophic factors, morphogens, and viral proteins, thereby playing roles in a variety of biological processes including cell adhesion, proliferation, tissue morphogenesis, neurite outgrowth, infections, and inflammation/leukocyte trafficking. CS/DS are modified with sulfate groups at C-2 of uronic acid residues as well as C-4 and/or C-6 of N-acetyl-D-galactosamine residues, yielding enormous structural diversity, which enables the binding with numerous proteins. We have demonstrated that highly sulfated CS-E from squid cartilage, for example, interacts with heparin-binding proteins including midkine, pleiotrophin, and fibroblast growth factors expressed in brain with high affinity (Kd values in the nM range). Here, we analyzed the binding of CS and DS, which have a relatively low degree of sulfation and have been widely used as a nutraceutical and a drug for osteoarthritis etc., with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the CS/DS chains. SPR showed that relatively low sulfated CS-A, DS, and CS-C also bound with significant affinity to midkine, pleiotrophin, hepatocyte growth factor, monokine-induced by interferon-γ, and stromal cell derived factor-1β, although the binding was less intense than that with highly sulfated CS-D and CS-E. These findings suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition. 相似文献
9.
Shinya Sugimoto Kunitoshi Yamanaka Shingo Nishikori Atsushi Miyagi Toshio Ando Teru Ogura 《The Journal of biological chemistry》2010,285(9):6648-6657
AAA+ chaperone ClpX has been suggested to be a modulator of prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown. In this study, we have extensively investigated the nature of FtsZ polymers and mechanisms of ClpX-regulated FtsZ polymer dynamics. We found that FtsZ polymerization is inhibited by ClpX in an ATP-independent manner and that the N-terminal domain of ClpX plays a crucial role for the inhibition of FtsZ polymerization. Single molecule analysis with high speed atomic force microscopy directly revealed that FtsZ polymer is in a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology with abnormal localization of FtsZ. These data together suggest that ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the N-terminal domain of ClpX and FtsZ monomers or oligomers. 相似文献
10.
The sulfation of glycosaminoglycans by ascorbic acid 2-[35S]sulfate was studied in costal cartilage and chondrocytes in vitro. Negligable (if any) sulfation of glycosaminoglycans was detected with immediately isolated ascorbic acid 2-[35S]sulfate. However, formation of [35S]glycosaminoglycans was readily detected with ascorbic acid 2-[35S]sulfate which had been stored at minus 20 degrees C for several days. The [35S]glycosaminoglycans did not result from the direct transfer of 35S from ascorbic acid 2-sulfate but rather from a decomposition product of ascorbic acid 2-[35S]sulfate. Evidence is presented to show that the sulfation pathway with the decomposition product involves exchange with inorganic sulfate, and strongly suggests that sulfation proceeds via 3'-phosphoadenosine 5'-phosphosulfate. The decomposition product appears similar to inorganic sulfate in several test systems. In view of these observations, it is suggested that previous conclusions implicating as acid 2-sulfate as a biological sulfate donor, based on the use of ascorbic acid 2-[35S]sulfate be re-evaluated. 相似文献
11.
Heparan sulfate (HS) proteoglycans, at the cell surface and extracellular matrix, facilitate ligand-receptor interactions crucial to many physiological processes. The distinct sulfation patterns of HS sugar chains presented by their protein core are key to HS proteoglycan activity. Tight regulation of several Golgi complex enzyme families is crucial to produce complex tissue-specific HS sequences. Several in vivo models deficient in HS biosynthesis enzymes demonstrate that developmental abnormalities result from modified HS structure. This review will discuss the plasticity of sulfation requirements on HS for activating protein ligands, which might reflect a flexible HS biosynthetic mechanism. In addition, the latest discovery of HS acting enzymes, the Sulfs, responsible for extracellular tweaking of HS sulfation levels subsequent to biosynthesis will be considered. 相似文献
12.
Gulberti S Jacquinet JC Chabel M Ramalanjaona N Magdalou J Netter P Coughtrie MW Ouzzine M Fournel-Gigleux S 《Glycobiology》2012,22(4):561-571
Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis. We synthesized a series of sulfated and unsulfated analogs of the linkage oligosaccharide and of the constitutive unit of CS and tested these molecules as potential acceptor substrates for the recombinant human CSGalNAcT-1. We show here that sulfation at C4 or C6 of the Gal residues markedly influences CSGalNAcT-1 initiation activity and catalytic efficiency. Kinetic analysis indicates that CSGalNAcT-1 exhibited 3.6-, 1.6-, and 2.2-fold higher enzymatic efficiency due to lower K(m) values toward monosulfated trisaccharides substituted at C4 or C6 position of Gal1, and at C6 of Gal2, respectively, compared with the unsulfated oligosaccharide. This highlights the critical influence of Gal substitution on both CSGalNAcT-1 activity and specifity. No GalNAcT activity was detected toward sulfated and unsulfated analogs of the CS constitutive disaccharide (GlcA-β1,3-GalNAc), indicating that CSGalNAcT-1 was involved in initiation but not in elongation of CS chains. Our results strongly suggest that sulfation of the linkage region acts as a regulatory signal in CS chain initiation. 相似文献
13.
A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides 总被引:3,自引:0,他引:3
A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure. 相似文献
14.
R V Iozzo 《The Journal of biological chemistry》1989,264(5):2690-2699
We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system. 相似文献
15.
Heparin modulation of laminin polymerization 总被引:4,自引:0,他引:4
Previously, it has been shown that laminin will self-assemble by a two-step calcium-dependent process using end-domain interactions (Yurchenco, P. D., Tsi-library, E. C., Charonis, A. S., and Furthmayr, H. (1985) J. Biol. Chem. 260, 7636-7644). We now find that heparin, at low concentrations, modifies this polymerization by driving the equilibrium further toward aggregation, by producing a denser polymer, and by inducing aggregation in the absence of calcium. This effect on self-assembly is specific in that it is observed with heparin but not with several heparan sulfates or other glycosaminoglycans: it correlates with affinity and depends on the degree of polysaccharide sulfation. Heparin binds to laminin in a calcium-dependent manner with a single class of interaction (KD = 118 +/- 18 nM) and with a binding capacity of one heparin for two laminins. We find the long arm globule (E3) is the only laminin domain which exhibits substantial heparin binding: heparin binds E3 with an affinity (KD = 94 +/- 12 nM) and calcium dependence similar to that for intact laminin. These data strongly suggest that heparin modifies laminin assembly by binding to pairs of long arm globular domains. As a result the polymer may be stabilized at domain E3 and laminin interdomain interactions induced or modified. We further postulate that heparins may act in vivo as specific regulators of the structure and functions of basement membranes by both altering the laminin matrix and by displacing weakly binding heparan sulfates. 相似文献
16.
Kitagawa Hiroshi; Tsutsumi Kae; Ujikawa Miho; Goto Fumitaka; Tamura Jun-ichi; Neumann Klaus W.; Ogawa Tomoya; Sugahara Kazuyuki 《Glycobiology》1997,7(4):531-537
The relationship between sulfation and polymerization in chondroitinsulfate (CS) biosynthesis has been poorly understood. In thisstudy, we investigated the specificity of bovine serum UDP-GalNAc:CS ß-GalNAc transferase responsible for chain elongationusing structurally defined acceptor substrates. They consistedof tetra- and hexasaccharide-serines that were chemically synthesizedand various regular oligosaccharides with a GlcA residue atthe nonreducing terminus, prepared from chondroitin and CS usingtesticular hyaluronidase. The enzyme preparation was obtainedfrom fetal bovine serum by means of heparin-Sepharose affinitychromatography. The preparation did not contain the 相似文献
17.
Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate 总被引:18,自引:0,他引:18
Sugahara K Mikami T Uyama T Mizuguchi S Nomura K Kitagawa H 《Current opinion in structural biology》2003,13(5):612-620
Recent glycobiology studies have suggested fundamental biological functions for chondroitin, chondroitin sulfate and dermatan sulfate, which are widely distributed as glycosaminoglycan sidechains of proteoglycans in the extracellular matrix and at cell surfaces. They have been implicated in the signaling functions of various heparin-binding growth factors and chemokines, and play critical roles in the development of the central nervous system. They also function as receptors for various pathogens. These functions are closely associated with the sulfation patterns of the glycosaminoglycan chains. Surprisingly, nonsulfated chondroitin is indispensable in the morphogenesis and cell division of Caenorhabditis elegans, as revealed by RNA interference experiments of the recently cloned chondroitin synthase gene and by the analysis of mutants of squashed vulva genes. 相似文献
18.
Selenate, a sulfation inhibitor, blocks the synthesis of heparan sulfate and chondroitin sulfate by cultured endothelial cells. In contrast, selenate does not affect the production of hyaluronic acid, a nonsulfated glycosaminoglycan. No differences in molecular weight, [3H]glucosamine/[35S]sulfuric acid ratios, or disaccharide composition were observed when the heparan sulfate synthesized by selenate-treated cells was compared with that of control cells. The absence of undersulfated chains in preparations from cultures exposed to selenate supports the concept that, in the intact cell, the polymerization of heparan sulfate might be dependent on the sulfation of the saccharide units added to the growing glycosaminoglycan chain. 相似文献
19.
Anthony J Hayes Debbie Tudor Mari A Nowell Bruce Caterson Clare E Hughes 《The journal of histochemistry and cytochemistry》2008,56(2):125-138
Osteoarthritis is a chronic, debilitating joint disease characterized by progressive destruction of articular cartilage. Recently, a number of studies have identified a chondroprogenitor cell population within articular cartilage with significant potential for repair/regeneration. As yet, there are few robust biomarkers of these cells. In this study, we show that monoclonal antibodies recognizing novel chondroitin sulfate sulfation motif epitopes in glycosaminoglycans on proteoglycans can be used to identify metabolically distinct subpopulations of cells specifically within the superficial zone of the tissue and that flow cytometric analysis can recognize these cell subpopulations. Fluorochrome co-localization analysis suggests that the chondroitin sulfate sulphation motifs are associated with a range of cell and extracellular matrix proteoglycans within the stem cell niche that include perlecan and aggrecan but not versican. The unique distributions of these sulphation motifs within the microenvironment of superficial zone chondrocytes, seems to designate early stages of stem/progenitor cell differentiation and is consistent with these molecules playing a functional role in regulating aspects of chondrogenesis. The isolation and further characterization of these cells will lead to an improved understanding of the role novel chondroitin sulfate sulfation plays in articular cartilage development and may contribute significantly to the field of articular cartilage repair. 相似文献
20.
Distinct effects on heparan sulfate structure by different active site mutations in NDST-1 总被引:2,自引:0,他引:2
Heparan sulfate polymerization and modification take place in the Golgi compartment. The modification reactions are initiated by glucosaminyl N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme that removes N-acetyl groups from selected N-acetyl-d-glucosamine units followed by N-sulfation of the generated free amino groups. Four isoforms of NDST have been identified. NDST-1 and -2 have a wide and largely overlapping tissue distribution, but it is not known if they can act on the same heparan sulfate chain. We have introduced point mutations into NDST-1 cDNA, which selectively destroy the N-deacetylase or N-sulfotransferase activity of the enzyme [Wei, Z., and Swiedler, S. J. (1999) J. Biol. Chem. 274, 1966-70 and Sueyoshi, T., et al. (1998) FEBS Lett. 433, 211-4]. Stable 293 cell lines expressing the NDST-1 mutants were then generated. Structural analyses of heparan sulfate synthesized by these cells and by cells overexpressing wild-type NDST-1 demonstrate that the N-deacetylation step is not only prerequisite but also rate-limiting, determining the degree of N-sulfation. Transfection of mutant NDST-1 lacking N-deacetylase activity had no effect on heparan sulfate sulfation, while cells expressing wild-type enzyme or NDST-1 lacking N-sulfotransferase activity both resulted in the production of oversulfated heparan sulfate. Since no increase in the amount of N-unsubstituted glucosamine residues was seen after transfection of the mutant lacking N-sulfotransferase activity, the results also suggest that two different enzyme molecules can act on the same glucosamine unit. In addition, we show that oversulfation of heparan sulfate produced by cells tranfected with wild-type NDST-1 or the mutant lacking N-sulfotranferase activity results in decreased sulfation of chondroitin sulfate. 相似文献