Uptake of mannose-terminated glycoproteins in isolated rat liver cells. Evidence for receptor-mediated endocytosis in hepatocytes.

Even though most of the hepatic binding capacity for mannose-terminated glycoproteins has previously been shown to reside in the hepatocytes (not in the non-parenchymal cells), detailed evidence for the specific uptake of mannose-terminated ligands has been lacking. In the present studies, yeast invertase, a large glycoprotein (Mr 270 000) containing about 50% mannose, was shown to be taken up into hepatocytes by receptor-mediated endocytosis. The uptake was saturable and could be specifically inhibited by mannosides or by a Ca2+ chelator. The asialo-glycoprotein receptor was not involved. The low-Mr (13 000) ligand ribonuclease B, which contains a single high-mannose glycan, was not taken up by hepatocytes; however, it was taken up as fast as invertase by non-parenchymal liver cells. After injection of 131I-invertase into a rat in vivo, about one-half of the labelled protein was recovered in the hepatocytes. On a per-cell basis, each endothelial cell contained 3-4 times as much radioactivity as did the hepatocytes. On fractionation of hepatocytes in sucrose gradients, invertase showed a different intracellular distribution from that of asialo-fetuin, in that invertase moved much faster into that region of the gradient where the lysosomes were recovered. This indicates that invertase and asialo-fetuin are not transported intracellularly by identical mechanisms.     

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.     

Mannose-receptor-mediated clearance of lysosomal alpha-mannosidase in scavenger endothelium of cod endocardium.

Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod (Gadus morhua). Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal alpha-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle. Asialo-orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only. In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12 degrees C in a serum free medium. Cod AEC endocytosed mannose-terminated glycoproteins (125I-alpha-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal alpha-mannosidase), whereas 125I-asialo-orosomucoid was not recognised. Uptake of radiolabelled mannose-terminated ligands was inhibited 80-100% in the presence of excess amounts of mannan, invertase, D-mannose, L-fucose or EGTA. Our results suggest that the cod endocardial endothelial cells express a specific Ca(2+)-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells.     

Cholesterol Sulfate Alters Substrate Preference of Matrix Metalloproteinase-7 and Promotes Degradations of Pericellular Laminin-332 and Fibronectin

Localization of secreted matrix metalloproteinases (MMPs) on the cell surface is required not only for processing of cell surface proteins, but also for controlled degradation of the extracellular matrix (ECM). Our previous study demonstrated that binding of MMP-7 (matrilysin) to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of this MMP. In this study, we investigated the role of CS in the MMP-7-catalyzed degradation of protein components of ECM. We found that the degradation of laminin-332 (laminin-5) catalyzed by MMP-7 was accelerated dramatically in the presence of CS, whereas the sulfated lipid inhibited the degradation of casein catalyzed by the protease. The MMP-7-catalyzed degradation of fibronectin was partially inhibited in the presence of low concentrations of CS, whereas it was accelerated significantly at high concentrations of the lipid. Therefore, it is likely that CS alters the substrate preference of MMP-7. We also found that the proteins of which MMP-7-catalyzed degradation were accelerated by CS also had affinities for CS, suggesting that CS facilitates the proteolyses by cross-linking MMP-7 to its substrates. Moreover, MMP-7 tethered to cancer cell surface via CS degraded fibronectin and laminin-332 coated on a culture plate. The degradations of the adhesive proteins led to significant detachment of the cells from the plate. Taken together, our findings provide a novel mechanism in which cell surface CS promotes the proteolytic activities of MMP-7 toward selective substrates in the pericellular ECM, thereby contributing to cancer cell migration and metastasis.     

Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor

The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.     

Purification and characterization of neutral and alkaline invertase from carrot.

Neutral and alkaline invertase were identified in cells of a suspension culture of carrot (Daucus carota L.) and purified to electrophoretic homogeneity. Neutral invertase is an octamer with a molecular mass of 456 kD and subunits of 57 kD, whereas alkaline invertase is a tetramer with a molecular mass of 504 kD and subunits of 126 kD. Both enzymes had sharp pH profiles, with maximal activities at pH 6.8 for neutral invertase and pH 8.0 for alkaline invertase, and both hydrolyzed sucrose with typical hyperbolic kinetics and similar Km values of about 20 mM at pH 7.5. Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a beta-fructofuranosidase. In contrast, alkaline invertase was highly specific for sucrose. Fructose acted as a competitive inhibitor of both enzymes, with Ki values of about 15 mM. Glucose was a noncompetitive inhibitor of both neutral and alkaline invertase, with a Ki of about 30 mM. Neither enzyme was inhibited by HgCl2. Alkaline invertase was markedly inhibited by CaCl2, MgCl2, and MnCl2, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP.     

Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors.

Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial proportion of the ligand could be degraded by incubating mitochondrial fractions prepared 12 min after intravenous injection of the ligand. The results indicate that FSA is very rapidly internalized and transferred through an endosomal compartment to the lysosomes. The endosomes are gradually converted into lysosomes between 9 and 12 min after injection of FSA. The rate-limiting step in the intracellular handling of 125I-TC-FSA is the degradation in the lysosomes.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Regulation and properties of an invertase from Clostridium pasteurianum.

An intracellular invertase was induced in cultures of Clostridium pasteurianum utilizing sucrose as its carbon source for growth. This enzyme synthesis could be repressed by the addition of fructose of a sucrose-growing culture. In contrast, invertase activity was not affected by the addition of glucose to sucrose-growing cells and this enzyme could be induced in a glucose-metabolizing culture by the addition of sucrose. This enzyme was purified 10.5-fold over the induced lese, EC 3.2.1.26) by substrate-specificity studies. Invertase had a pH optimum of 6.5 and an apparent Km of 79.5 mM for sucrose, and required high concentration of potassium phosphate for maximum activity. Invertase was completely inactivated by a 2-min heat treatment at 60 degrees C. This enzyme was strongly inhibited by p-hydroxymercuribenzoate (pCMB) and weakly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), while cysteine could substantially reverse pCMB) inhibition, suggesting that sulfhydryl group(s) were necessary for invertase activity.     

Invertase activity associated with the walls of Solanum tuberosum tubers

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.     

Chondroitin sulfate characterized by the E-disaccharide unit is a potent inhibitor of herpes simplex virus infectivity and provides the virus binding sites on gro2C cells

Although cell surface chondroitin sulfate (CS) is regarded as an auxiliary receptor for binding of herpes simplex virus to cells, and purified CS chain types A, B, and C are known to interfere poorly or not at all with the virus infection of cells, we have found that CS type E (CS-E), derived from squid cartilage, exhibited potent antiviral activity. The IC(50) values ranged from 0.06 to 0.2 mug/ml and substantially exceeded the antiviral potency of heparin, the known inhibitor of virus binding to cells. Furthermore, in mutant gro2C cells that express CS but not heparan sulfate, CS-E showed unusually high anti-herpes virus activity with IC(50) values of <1 ng/ml. Enzymatic degradation of CS-E with chondroitinase ABC abolished its antiviral activity. CS-E inhibited the binding to cells of the purified virus attachment protein gC. A direct interaction of gC with immobilized CS-E and inhibition of this binding by CS-E oligosaccharide fragments greater than octasaccharide were demonstrated. Likewise, the gro2C-specific CS chains interfered with the binding of viral gC to these cells and were found to contain a considerable proportion (13%) of the E-disaccharide unit, suggesting that this unit is an essential component of the CS receptor for herpes simplex virus on gro2C cells and that the antiviral activity of CS-E was due to interference with the binding of viral gC to a CS-E-like receptor on the cell surface. Knowledge of the determinants of antiviral properties of CS-E will help in the development of inhibitors of herpes simplex virus infections in humans.     

Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre- treatment of the cells with either heparitinase, chondroitinase or beta- D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.     

Effect of suramin on growth of androgen-responsive mouse tumor (Shionogi carcinoma 115) and its autonomous subline (Chiba subline 2)

Suramin has been shown to inhibit the binding of various growth factors to their receptors. Shionogi Carcinoma 115 cells (SC 115 cells) and Chiba Subline 2 cells (CS 2 cells) are clones of an androgen-responsive mouse tumor cell and its autonomous subline, respectively. Since the growth of SC 115 and CS 2 cells are assumed to be regulated by their own fibroblast growth factor (FGF)-like growth factors, the present study was undertaken to examine the effect of suramin on these cells. Suramin inhibited the growth of SC 115 and CS 2 cells in a dose dependent manner. The inhibition of suramin was reversible up to 50 micrograms/ml. Suramin reversibly changed the shape of these cells from fibroblast-like to polygonal and epithelial-like ones, and inhibited 3H-thymidine incorporation into these cells which was evoked by acidic and basic FGFs, and conditioned medium obtained from CS 2 cells. The binding of 125I-basic FGF to SC 115 and CS 2 cells was inhibited by suramin. However, suramin had no effect on growth factor production and the hst-1 gene expression on CS 2 cells. In conclusion, suramin inhibited the autocrine and paracrine growth of SC 115 and CS 2 cells by blocking the binding of autocrine growth factors to their receptors.     

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K_m for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective `PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.     

A membrane-associated isozyme of invertase in yeast precursor of the external glycoprotein

An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from Saccharomyces cerevisiae cells which are actively synthesizing external invertase, these membranes show an activity band on sodium dodecyl sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was completely absent. It has a molecular weight of approximately 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a condition of non-synthesizing external invertase to one where the enzyme was synthesized. When the increase in the external enzyme slowed down after some time, the membrane-associated form almost completely disappeared.The addition of tunicamycin to yeast cells synthesizing external invertase inhibited further synthesis of the enzyme by 97%; also the formation of the membrane-associated form was strongly inhibited. The amount of it present before the addition of tunicamycin completely disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those carbohydrate parts which contain N-acetylglucosamine and are transferred via dolichyl phosphate-bound intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.     

Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.     

Effects of chondroitin sulfate and its oligosaccharides on toll-like receptor-mediated IL-6 secretion by macrophage-like J774.1 cells

The effects of two types of chondroitin sulphate (CS), CS-A and CS-C, their oligosaccharides (oligo-CSs), and disaccharides (Di-CSs) on toll-like receptor (TLR)-mediated secretion of interleukin (IL)-6 were compared using macrophage-like cell line J774.1. IL-6 secretion in the J774.1 cells was markedly increased by Pam3CS4, LPS, and CpG, the ligands to TLR1/2, 4, and 9 respectively. Among these three ligands, CpG-induced IL-6 was most clearly suppressed by CSs and their digests. Suppression of IL-6 secretion by smaller sized CS-A was stronger than that by intact CS-A, whereas no such size-dependent suppression was apparent for CS-C. Di-4S, the disaccharide unit of the CS-A digest, also showed much stronger suppression than Di-6S, the disaccharide unit of the CS-C digest, and the non-sulfated disaccharide unit, Di-0S. The suppressing activity of oligo-CSs, particularly Di-CSs, against TLR-mediated inflammation was dependent on the CS structure, including the sulfation site.     

Effects of Chondroitin Sulfate and Its Oligosaccharides on Toll-Like Receptor-Mediated IL-6 Secretion by Macrophage-Like J774.1 Cells

The effects of two types of chondroitin sulphate (CS), CS-A and CS-C, their oligosaccharides (oligo-CSs), and disaccharides (Di-CSs) on toll-like receptor (TLR)-mediated secretion of interleukin (IL)-6 were compared using macrophage-like cell line J774.1. IL-6 secretion in the J774.1 cells was markedly increased by Pam3CS4, LPS, and CpG, the ligands to TLR1/2, 4, and 9 respectively. Among these three ligands, CpG-induced IL-6 was most clearly suppressed by CSs and their digests. Suppression of IL-6 secretion by smaller sized CS-A was stronger than that by intact CS-A, whereas no such size-dependent suppression was apparent for CS-C. Di-4S, the disaccharide unit of the CS-A digest, also showed much stronger suppression than Di-6S, the disaccharide unit of the CS-C digest, and the non-sulfated disaccharide unit, Di-0S. The suppressing activity of oligo-CSs, particularly Di-CSs, against TLR-mediated inflammation was dependent on the CS structure, including the sulfation site.