scholarly journals Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans

1988 ◽  
Vol 256 (1) ◽  
pp. 29-33 ◽  
Author(s):  
K Karlsson ◽  
U Lindahl ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Inhibitory Effect ◽  
Extracellular Superoxide Dismutase ◽  
Net Charge ◽  
Sulphated Glycosaminoglycans ◽  
Sepharose 4B ◽  
Heparin Affinity ◽  
Secretory Enzyme

The secretory enzyme extracellular superoxide dismutase (EC-SOD) occurs in at least three forms, which differ with regard to heparin affinity: A lacks affinity, B has intermediate affinity, and C has relatively strong affinity. The affinity of EC-SOD C for various sulphated glycosaminoglycans (GAGs) was assessed (a) by determining the concentration of NaCl required to release the enzyme from GAG-substituted Sepharose 4B and (b) by determining the relative potencies of the GAGs to release EC-SOD C from heparan sulphate-Sepharose 4B. Both methods indicated the same order of affinity. Heparin bound EC-SOD C about 10 times as avidly as the studied heparan sulphate preparation, which in turn was 10 and 150 times as efficient as dermatan sulphate and chondroitin sulphate respectively. Chondroitin sulphate showed weak interaction with EC-SOD C at physiological ionic strength. Heparin subfractions with high or low affinity for antithrombin III were equally efficient. The binding of EC-SOD C to heparin-Sepharose was essentially independent of pH in the range 6.5-9; below pH 6.5 the affinity increased, and beyond pH 9.5 there was a precipitous fall in affinity. The inhibitory effect of NaCl on the binding of EC-SOD C to GAGs indicates that the interaction is of electrostatic nature. EC-SOD C carries a negative net charge at neutral pH, and it is suggested that the binding occurs between the negative charges of the GAG sulphate groups and a structure in the C-terminal end of the enzyme that has a cluster of positive charges. These results are compatible with the notion that heparan sulphate proteoglycans on cell surfaces or in the intercellular matrix may serve to bind EC-SOD C in tissues.

scholarly journals Proteolytic modification of the heparin-binding affinity of extracellular superoxide dismutase

1993 ◽  
Vol 290 (2) ◽  
pp. 623-626 ◽  
Author(s):  
K Karlsson ◽  
A Edlund ◽  
J Sandström ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Binding Affinity ◽  
Heparan Sulphate ◽  
Limited Proteolysis ◽  
Size Exclusion ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Binding Domains ◽  
Heparin Affinity

The heparin-binding affinity of the tetrameric extracellular superoxide dismutase (EC-SOD) is a result of the cooperative effect of the heparin-binding domains of the subunits, located in the hydrophilic, strongly positively charged C-terminal ends. EC-SOD C, the high-heparin-affinity type, exposed to immobilized trypsin and plasmin was found to rapidly lose its affinity for heparin, without any loss of enzymic activity or major change in molecular mass as judged by size-exclusion chromatography. Heparin and dextran sulphate 5000 inhibited the proteolysis, suggesting that EC-SOD C sequestered by heparan sulphate proteoglycan in vivo is partially protected against proteolysis. The loss of heparin-affinity occurred with the stepwise formation of intermediates, and the pattern upon chromatography on heparin-Sepharose and subsequent immunoblotting was compatible with the notion that the changes are due to sequential truncations of heparin-binding domains from subunits composing the EC-SOD tetramers. A similar pattern with intermediates and apparent truncations has previously been found with EC-SOD of human plasma. The findings show that the unique design of the heparin-binding domain of EC-SOD allows easy modification of the heparin-affinity by means of limited proteolysis, and suggest that such proteolysis is a major contributor to the heterogeneity in heparin-affinity of EC-SOD in mammalian plasma.

scholarly journals Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues

1993 ◽  
Vol 294 (3) ◽  
pp. 853-857 ◽  
Author(s):  
J Sandström ◽  
K Karlsson ◽  
T Edlund ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Umbilical Cord ◽  
Heparan Sulphate ◽  
Complex Mixture ◽  
Distribution Volume ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Intracellular Space ◽  
Heparin Binding Domain ◽  
Heparin Affinity

The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.

scholarly journals Non-enzymic glycation of human extracellular superoxide dismutase

1991 ◽  
Vol 279 (1) ◽  
pp. 263-267 ◽  
Author(s):  
T Adachi ◽  
H Ohta ◽  
K Hirano ◽  
K Hayashi ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Heparan Sulphate ◽  
Normal Subjects ◽  
Enzymic Activity ◽  
Diabetic Patients ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
A Minor ◽  
Heparin Affinity

The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.

scholarly journals The high concentration of Arg213→Gly extracellular superoxide dismutase (EC-SOD) in plasma is caused by a reduction of both heparin and collagen affinities

2005 ◽  
Vol 385 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Steen V. PETERSEN ◽  
Dorte Aa. OLSEN ◽  
John M. KENNEY ◽  
Tim D. OURY ◽  
Zuzana VALNICKOVA ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Superoxide Dismutase ◽  
Type I Collagen ◽  
Heparan Sulphate ◽  
Common Mutation ◽  
Type I ◽  
Extracellular Superoxide Dismutase ◽  
High Concentration ◽  
Homozygous Individual ◽  
Heparin Affinity

The C-terminal region of EC-SOD (extracellular superoxide dismutase) mediates the binding to both heparin/heparan sulphate and type I collagen. A mutation (Arg213→Gly; R213G) within this extracellular matrix-binding region has recently been implicated in the development of heart disease. This relatively common mutation affects the heparin affinity, and the concentration of EC-SOD in the plasma of R213G homozygous individuals is increased 10- to 30-fold. In the present study we confirm, using R213G EC-SOD purified from a homozygous individual, that the heparin affinity is reduced. Significantly, the collagen affinity of the R213G EC-SOD variant was similarly affected and both the heparin and collagen affinities were reduced by 12-fold. Structural analysis of synthetic extracellular matrix-binding regions suggests that the mutation alters the secondary structure. We conclude that the increased concentration of EC-SOD in the plasma of R213G carriers is caused by a reduction in both heparin and collagen affinities.

scholarly journals Human mononuclear cells contain an endoglycosidase specific for heparan sulphate glycosaminoglycan demonstrable with the use of a specific solid-phase metabolically radiolabelled substrate

1989 ◽  
Vol 264 (3) ◽  
pp. 777-783 ◽  
Author(s):  
R F Sewell ◽  
P E C Brenchley ◽  
N P Mallick
Keyword(s):  
Affinity Chromatography ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Human Spleen ◽  
Human Mononuclear Cells ◽  
Normal Human ◽  
Sepharose 4B ◽  
Heparin Affinity

Xyloside-initiated 35SO4(2-)-labelled glycosaminoglycans were isolated from the medium of cultured bovine glomeruli and covalently coupled to Sepharose 4B to construct a solid-phase substrate suitable for the detection of endoglycosidases. The substrate is rendered specific for heparitinase by prior digestion with chondroitin sulphate ABC lyase and is insensitive to proteinase, neuraminidase and hyaluronidase. Normal human mononuclear cells are shown to contain a heparitinase. This enzyme appears to be cell-associated and can be partially purified from human spleen by heparin affinity chromatography.

scholarly journals Heparin-Affinity of Human Extracellular-Superoxide Dismutase in the Brain.

2001 ◽  
Vol 24 (2) ◽  
pp. 191-193 ◽  
Author(s):  
Tetsuo ADACHI ◽  
Masayuki YAMAMOTO ◽  
Hirokazu HARA
Keyword(s):  
Superoxide Dismutase ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Affinity ◽  
The Brain

scholarly journals Sulphated glycosaminoglycans in regenerating rat liver

1980 ◽  
Vol 188 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M Edward ◽  
W F Long ◽  
H H Watson ◽  
F B Williamson
Keyword(s):  
Molecular Mechanisms ◽  
Temporal Trend ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Fold Increase ◽  
Sham Operation ◽  
Dermatan Sulphate ◽  
Weight Percentage ◽  
Biphasic Pattern ◽  
Sulphated Glycosaminoglycans

The total weight percentage glycosaminoglycan content of rat liber was found to increase by 50% in the first 30 h after partial hepatectomy. The content returned to near normal by the third day, but then increased again to a second maximum at 5-6 days, only to gradually decline to normal by the ninth day, when regeneration was nearly complete. This biphasic pattern was most marked in the chondroitin sulphate A/C component, with a 6-fold increase by the sixth day. Dermatan sulphate showed the same temporal trend, whereas heparan sulphate remained relatively unaltered. No such changes were detected in the livers of rats subjected to sham operation. The possible molecular mechanisms underlying the apparent link between cellular glycosaminoglycan content and proliferative tendency are discussed.

scholarly journals Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

1980 ◽  
Vol 192 (2) ◽  
pp. 395-402 ◽  
Author(s):  
Perumana R. Sudhakaran ◽  
Wolfgang Sinn ◽  
Kurt von Figura
Keyword(s):  
Cyclic Amp ◽  
Heparan Sulphate ◽  
Molecular Size ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Dibutyryl Cyclic Amp ◽  
Isolated Rat Hepatocytes ◽  
Intracellular Compartments ◽  
Sulphated Glycosaminoglycans ◽  
Chain Synthesis

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E1, and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of 35SO42− into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of 35SO42− or [14C]glucosamine into heparan sulphate was decreased to 40–50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse–chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by β-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, 35SO42− incorporation was decreased to 5%. Addition of p-nitrophenyl β-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl β-d-xylosides. Incorporation of [3H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.

scholarly journals Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1971 ◽  
Vol 124 (4) ◽  
pp. 677-683 ◽  
Author(s):  
P.-H. Iverius
Keyword(s):  
Cyanogen Bromide ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Direct Analysis ◽  
Dermatan Sulphate ◽  
Peptide Residue ◽  
Agarose Beads ◽  
Freeze Dried ◽  
Coupling Procedure ◽  
Sepharose 4B

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.

Chlorate: a reversible inhibitor of proteoglycan sulphation in Chlamydia trachomatis-infected cells

2004 ◽  
Vol 53 (2) ◽  
pp. 93-95 ◽  
Author(s):  
Sanaa Fadel ◽  
Adrian Eley
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Chlamydial Infection ◽  
Heparan Sulphate ◽  
Sodium Chlorate ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Reversible Inhibitor ◽  
Sulphated Glycosaminoglycans ◽  
Necessary And Sufficient

Sulphated glycosaminoglycans, such as heparan sulphate, have been shown to be essential for the infectivity of many organisms. The aims of this study were to verify the role of sulphated glycosaminoglycans in chlamydial infection and to investigate whether they are present on chlamydia or chlamydial host cells. The effect of undersulphation of host cells and chlamydial elementary bodies was examined using sodium chlorate. Also studied was whether any inhibitory effect was reversible. The results strongly suggest that Chlamydia trachomatis does not produce heparan sulphate and that heparan sulphate of the host cell is necessary and sufficient to mediate chlamydial infection. The essential role played by the sulphate constituents of the host-cell glycosaminoglycan in the infectivity of LGV serovars, and to a lesser extent of serovar E, was also confirmed.

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