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 Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase

1996 ◽  
Vol 317 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Tim D. OURY ◽  
James D. CRAPO ◽  
Zuzana VALNICKOVA ◽  
Jan J. ENGHILD
Keyword(s):  
Superoxide Dismutase ◽  
Limited Proteolysis ◽  
Exchange Chromatography ◽  
Disulphide Bond ◽  
High Yield ◽  
Human Aorta ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Sod Activity ◽  
Native Page

Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.

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 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 Mammalian and Drosophila cells adhere to the laminin α4 LG4 domain through syndecans, but not glypicans

2004 ◽  
Vol 382 (3) ◽  
pp. 933-943 ◽  
Author(s):  
Hironobu YAMASHITA ◽  
Akira GOTO ◽  
Tatsuhiko KADOWAKI ◽  
Yasuo KITAGAWA
Keyword(s):  
Cell Adhesion ◽  
Umbilical Vein ◽  
Heparan Sulphate ◽  
Direct Interaction ◽  
Binding Activity ◽  
Main Body ◽  
Heparin Binding ◽  
Adhesion Activity ◽  
Umbilical Vein Endothelial Cells

We have previously shown that the LG4 (laminin G-like) domain of the laminin α4 chain is responsible for the significantly higher affinity of the α4 chain to heparin than found for other α chains [Yamaguchi, Yamashita, Mori, Okazaki, Nomizu, Beck and Kitagawa (2000) J. Biol. Chem. 275, 29458–29465]; four basic residues were identified to be essential for this activity [Yamashita, Beck and Kitagawa (2004) J. Mol. Biol. 335, 1145–1149]. By creating GST (glutathione S-transferase)-fused LG1, LG2, LG4 and LG5 proteins, we found that only LG4 is active for the adhesion of human HT1080 cells, human umbilical vein endothelial cells and Drosophila haemocytes Kc167 with a half-saturating concentration of 20 μg/ml. Adhesion was counteracted by treatment of the cells with heparin, heparan sulphate and heparitinase I. Upon mutating the four basic residues essential for heparin binding within LG4, the adhesion activity was abolished. Pull-down experiments using glutathione beads/GST-fusion proteins indicate a direct interaction of LG4 with syndecan-4, which might be the major receptor for cell adhesion. Neither the release of glypican-1 by treating human cells with phosphatidylinositol-specific phospholipase C nor targeted knockdown of dally or dally-like protein impaired the cell-adhesion activity. As the LG4–LG5 domain of the α4 chain is cleaved in vivo from the main body of laminin-8 (α4β1γ1), we suggest that the heparan sulphate proteoglycan-binding activity of LG4 is significant in modulating the signalling of Wnt, Decapentaplegic and fibroblast growth factors.

Heparin stimulates the proliferation of intestinal epithelial cells in primary culture

1994 ◽  
Vol 107 (2) ◽  
pp. 401-411
Author(s):  
N. Flint ◽  
F.L. Cove ◽  
G.S. Evans
Keyword(s):  
Growth Factors ◽  
Heparan Sulphate ◽  
Primary Cultures ◽  
Cell Types ◽  
Heparin Binding ◽  
Epithelial Proliferation ◽  
Inhibition Of Proliferation ◽  
Trophic Effect ◽  
Heparin Binding Growth Factors

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

scholarly journals Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

2005 ◽  
Vol 390 (2) ◽  
pp. 407-418 ◽  
Author(s):  
Catherine de Coupade ◽  
Antonio Fittipaldi ◽  
Vanessa Chagnas ◽  
Matthieu Michel ◽  
Sophie Carlier ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Heparan Sulphate ◽  
Intracellular Delivery ◽  
Membrane Lipid ◽  
Cell Penetrating Peptides ◽  
Heparin Binding ◽  
Independent Manner ◽  
Cell Penetrating

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell® peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell® peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

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