Svein Olav Kolset

Selective Effects of Sodium Chlorate Treatment on the Sulfation of Heparan Sulfate

We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3′-phosphoadenosine 5′-phosphosulfate. Metabolically [3H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternatingN-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mm chlorate, overall O-sulfation of HS was inhibited by ∼70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mm) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mm chlorate reduced both 2-O- and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-O-sulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3′-phosphoadenosine 5′-phosphosulfate availability has distinct effects on the N- and O-sulfation of HS and thatO-sulfation is affected in a domain-specific fashion.

Serglycin - Structure and biology

Abstract.Serglycin is a proteoglycan found in hematopoietic cells and endothelial cells. It has important functions related to formation of several types of storage granules. In connective tissue mast cells the covalently attached glycosaminoglycan is heparin, whereas mucosal mast cells and activated macrophages contain oversulfated chondroitin sulfate (type E). In mast cells, serglycin interact with histamine, chymase, tryptase and carboxypeptidase, in neutrophils with elastase, in cytotoxic T cells with granzyme B, in endothelial cells with tissue-type plasminogen activator and in macrophages with tumor necrosis factor-α. Serglycin is important for the retention of key inflammatory mediators inside storage granules and secretory vesicles. Serglycin can further modulate the activities of partner molecules in different ways after secretion from activated immune cells, through protection, transport, activation and interactions with substrates or target cells. Serglycin is a proteoglycan with important roles in inflammatory reactions.

Diabetic Nephropathy and Extracellular Matrix

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typical morphological changes are the result of changes in the extracellular matrix (ECM). Thus, basement membranes are thickened and the glomerular mesangial matrix and the tubulointerstitial space are expanded, due to increased amounts of ECM. One important ECM component, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs in basement membranes are decreased but increased in the mesangium and the tubulointerstitial space. The amounts and structures of heparan sulfate chains are changed, and such changes affect levels of growth factors regulating cell proliferation and ECM synthesis, with cell attachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfate structures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classes also modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serine proteases, such as plasminogen activator, as well as their corresponding inhibitors. The levels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN. Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesis and turnover in DN. Whether ECM components can be used as markers for early kidney changes is an important research topic, whereas at present, the clinical use remains to be established.

Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFA) may reduce cell multiplication in cultures of normal, as well as transformed, white blood cells. We assessed the sensitivity of 14 different leukemia cell lines to PUFA by measuring cell number after 3 days of incubation. Ten of the examined cell lines were sensitive to 30, 60 and/or 120 μM of arachidonic, eicosapentaenoic and docosahexaenoic acid, whereas four cell lines were resistant. The sensitivity to PUFA was not associated with any particular cell lineage, clinical origin or specific mRNA pattern of bcl-2 and c-myc. Effects on cell viability were assessed by studying cell membrane integrity, DNA fragmentation and cell morphology. The sensitive cell lines Raji and Ramos died by necrosis and apoptosis, respectively, during incubation with eicosapentaenoic acid, whereas the viability of the resistant U-698 cell line was unaffected. The effects of EPA on Raji cells, was counteracted by vitamin E, indicating that lipid peroxidation was involved. However, apoptosis induced by eicosa- pentaenoic acid in Ramos cells, was unaffected by vitamin E, as well as eicosanoid synthesis inhibitors. In conclusion, our results indicate that a majority of leukemia cell lines are sensitive to PUFA. This sensitivity may be caused by induction of apoptosis or necrosis by very long-chain polyunsaturated fatty acids.

The effects of colchicine and cytochalasin B on uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes.

Abstract We have studied the effects of colchicine, an inhibitor of microtubular function, and cytochalasin B (CB), an inhibitor of microfilaments, on the uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes. CB inhibited degradation only, while colchicine inhibited uptake as well as degradation. When the two were combined, no additive effect on degradation was found. The inhibition of uptake by colchicine could be accounted for by a reduction in the binding capacity of the plasma membrane for asialo-glycoprotein. The intracellular distribution of endocytosed asialo-glycoprotein was examined by isopycnic centrifugation in sucrose gradients. The results suggest that in cells treated with colchicine or CB, access of the endocytosed protein to the lysosomes is impeded.

Serglycin: A Structural and Functional Chameleon with Wide Impact on Immune Cells

Among the different proteoglycans expressed by mammals, serglycin is in most immune cells the dominating species. A unique property of serglycin is its ability to adopt highly divergent structures, because of glycosylation with variable types of glycosaminoglycans when expressed by different cell types. Recent studies of serglycin-deficient animals have revealed crucial functions for serglycin in a diverse array of immunological processes. However, its exact function varies to a large extent depending on the cellular context of serglycin expression. Based on these findings, serglycin is emerging as a structural and functional chameleon, with radically different properties depending on its exact cellular and immunological context.

Cell surface heparan sulfate proteoglycans and lipoprotein metabolism

Abstract. Cell surface heparan sulfate proteoglycans are involved in several aspects of the lipoprotein metabolism. Most of the biological activities of these proteoglycans are mediated via interactions of their heparan sulfate moieties with various protein ligands, including lipoproteins and lipases. The binding of lipoproteins to heparan sulfate is largely determined by their apoprotein composition, and apoproteins B and E display the highest affinity for heparan sulfate. Interactions of lipoproteins with heparan sulfate are important for the cellular uptake and turnover of lipoproteins, in part by enhancing the accessibility of lipoproteins to lipoprotein receptors and lipases. Apoprotein B may interact with receptors without involving heparan sulfate. Heparan sulfate has been further implicated in presentation and stabilization of lipoprotein lipase and hepatic lipase on cell surfaces and in the transport of lipoprotein lipase from extravascular cells to the luminal surface of the endothelia. In atherosclerosis, heparan sulfate is intimately involved in several events important to the pathophysiology of the disease. Heparan sulfate thus binds and regulates the activity of growth factors, cytokines, superoxide dismutase and antithrombin, which contribute to aberrant cell proliferation, migration and matrix production, scavenging of reactive oxygen radicals and thrombosis. In this review we discuss the various roles of heparan sulfate proteoglycans in vascular biology, with emphasis on interactions of heparan sulfate with lipoproteins and lipases and the molecular basis of such interactions.

Serglycin-binding proteins in activated macrophages and platelets.

The major proteoglycan in macrophages and platelets is the chondroitin sulfate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell‐associated proteins from macrophages and blood platelets was examined. Affinity chromatography with serglycin‐Sepharose and chondroitin sulfate‐Sepharose was used to isolate proteoglycan‐binding proteins from macrophages and platelets. Antibodies against human macrophage inflammatory protein‐1α (MIP‐1α) precipitated a 14‐kDa 35S‐methionine‐labeled protein among the chondroitin sulfate binding proteins secreted from the macrophage‐like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of ~10 and 14 kDa were precipitated by an antiserum against the murine MIP‐1α. Protein sequencing of fragments obtained by trypsin digestion of a 14‐kDa chondroitin sulfate‐binding protein from cell extracts of stimulated U937 cells revealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed high homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6‐sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4‐sulfate. Amino‐terminal sequencing of two proteins from platelet extracts that bound to serglycin‐Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PF4). Chondroitin sulfate‐Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin‐Sepharose, indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparan sulfate in blocking the binding of [3H]chondroitin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine‐labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP‐1α, and lysozyme play important roles in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response.

Serglycin Is the Major Secreted Proteoglycan in Macrophages and Has a Role in the Regulation of Macrophage Tumor Necrosis Factor-α Secretion in Response to Lipopolysaccharide

It has recently been shown that serglycin is essential for maturation of mast cell secretory granules. However, serglycin is expressed also by other cell types, and in this study we addressed the role of serglycin in macrophages. Adherent cells were prepared from murine peritoneal cell populations and from spleens, and analyzed for proteoglycan synthesis by biosynthetic labeling with [35S]sulfate. Conditioned media from serglycin–/– peritoneal macrophages and adherent spleen cells displayed a 65–80% reduction of 35S-labeled proteoglycans, compared with corresponding material from serglycin+/+ cells, indicating that serglycin is the dominant secretory proteoglycan in macrophages of these origins. In contrast, the levels of intracellular proteoglycans were similar in serglycin+/+ and serglycin–/– cells, suggesting that serglycin is not stored intracellularly to a major extent in macrophages. This is in contrast to mast cells, in which serglycin is predominantly stored intracellularly. Transmission electron microscopy revealed that the absence of serglycin did not cause any major morphological effects on peritoneal macrophages, in contrast to dramatic defects in intracellular storage vesicles in peritoneal mast cells. Several secretory products were not found to be affected by the lack of serglycin. However, the secretion of tumor necrosis factor-α in response to lipopolysaccharide stimulation was markedly higher in serglycin–/– cultures than in those of serglycin+/+. The present report thus demonstrates that serglycin is the major proteoglycan secreted by peritoneal macrophages and suggests that the macrophage serglycin may have a role in regulating secretion of tumor necrosis factor-α.

Syndecan-1 plasma levels during coronary artery bypass surgery with and without cardiopulmonary bypass

The glycocalyx covering the endothelium is shed during ischemia and reperfusion. The shedding is accompanied by increased levels of the glycocalyx component syndecan-1 in the circulation. Our aim was to compare plasma levels of syndecan-1 in patients undergoing coronary artery bypass grafting (CABG), with or without the use of cardiopulmonary bypass (CPB). Syndecan-1 plasma concentrations were measured in patients undergoing CABG on-pump (n = 22) or off-pump (n = 22). The syndecan-1 concentration increased significantly from 29.5 ± 4.6 ng/mL at baseline to 98.7 ± 9.8 ng/mL (p < 0.01) after the start of CPB or 30 minutes after the induction of anesthesia in the off-pump group. There were no significant differences in peak syndecan-1 plasma concentrations between on-pump and off-pump patients. Plasma levels of syndecan-1 increased significantly during CABG, with or without the use of CPB. There were no significant differences in syndecan-1 concentrations in the two groups.