肝素对人类神经tau蛋白分子聚集及磷酸化的影响

在老年性痴呆患者的脑中,肝素与超磷酸化的tau蛋白共存[7].采用NCLK(neuronalcdc2-likekinase)及PP2B(phosphoproteinphosphatase2B)在含肝素的体系中对人类神经tau蛋白进行磷酸化和脱磷酸化,结果表明,肝素具有促进tau蛋白被磷酸化的功能,并促进该蛋白磷酸化分子二聚体的形成和单体的减少,其一级动力学常数分别为2.88×10-3s-1和1.74×10-3s-1.PP2B可使磷酸化的tau蛋白脱磷酸化,并且脱磷酸化作用随肝素浓度的增加而增强,提示肝素可能具有调节tau蛋白磷酸化状态的作用     

Interaction of neuronal Cdc2-like protein kinase with microtubule-associated protein tau

Neuronal Cdc2-like protein kinase (NCLK), a approximately 58-kDa heterodimer, was isolated from neuronal microtubules (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901). The biochemical nature of NCLK-microtubule association is not known. In this study we found that NCLK is released from microtubules upon microtubule disassembly as a 450-kDa species. The 450-kDa species is an NCLK.tau complex, and NCLK-bound tau is in a nonphosphorylated state. Tau phosphorylation causes NCLK.tau complex dissociation, and phosphorylated tau does not bind to NCLK. In vitro, the Cdk5 subunit of NCLK binds to the microtubule-binding region of tau and NCLK associates with microtubules only in the presence of tau. Our data indicate that in brain extract NCLK is complexed with tau in a tau phosphorylation-dependent manner and that tau anchors NCLK to microtubules. Recently NCLK has been suggested to be aberrantly activated and to hyperphosphorylate tau in Alzheimer's disease brain (Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P, and Tsai, L.-H. (1999) Nature 402, 615-622). Our findings may explain why in Alzheimer's disease NCLK specifically hyperphosphorylates tau, although this kinase has a number of protein substrates in the brain.     

Pseudophosphorylation of tau protein alters its ability for self-aggregation

Filamentous tau protein deposits are a pathological hallmark of a group of neurodegenerative disorders (tauopathies). Tau protein in these aggregates is highly phosphorylated at different phosphorylation sites. Although tau filaments can be formed by heparin-induced aggregation of unphosphorylated recombinant tau, it is not known how tau phosphorylation modulates aggregation behaviour. Analysis of the effect of tau phosphorylation at defined single or multiple sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. To overcome this problem we employed site-directed mutagenesis to convert serine and threonine to aspartic acid or glutamic acid, which introduce a negative charge and conformational change that mimic phosphorylation. We tested 14 different mutated tau proteins for their propensity for self-aggregation and formation of tau filaments. Tau aggregation was monitored with thioflavin S fluorescence in the presence of different inducers such as heparin, Al3+, Fe2+ and Fe3+. We found that mutations in the N-terminal portion up to amino acid 208 mainly suppress tau aggregation, whereas mutations in the C-terminal region mainly lead to an enhanced aggregation. Mutations in the middle portion of tau showed a mixed picture of suppression and enhancement of aggregation. A single amino acid change Ser422Glu has aggregation-favouring properties with all four inducers.     

Regulation of cell motility, morphology, and growth by sulfated glycosaminoglycans

Due to the recent observation that heparin binds to several growth factors and cell adhesion molecules, the effect of heparin on biological processes governed by growth factors and cell adhesion molecules was investigated. Pharmacological doses of heparin were found to alter cell growth rate, cellular morphology, and cell motility. Concentrations (microgram/ml) of heparin or dextran sulfate decreased cell growth rate, but not the final cell density attained in plateau phase. The effect of heparin on cell growth rate was most pronounced when cells were cultured in low concentrations of serum. A heparin-induced decrease in cell growth rate could be reversed by addition of platelet-derived growth factor (PDGF), a heparin-binding growth factor. Heparin altered the morphology of all cell lines studied to various degrees. The effect of heparin on cell morphology was quantitated by measuring the heparin-induced change in cell surface area. HT-1080 and HeLa cells nearly doubled in surface area upon exposure to 10 micrograms/ml heparin. Since several heparin-binding cell adhesion proteins mediate both cell spreading and cell migration, the influence of heparin on cell migration was investigated with an improved version of the phagokinetic track technique. Low concentrations of heparin and dextran sulfate were found to increase the rate of cell migration in a dose-dependent fashion. Since the quantitative effect of heparin on cell growth rate, morphology, and migration depends on the cell line studied, it is suggested that three separate phenomena may be involved. The results presented indicate a central role for sulfated glycosaminoglycans in the control of both cell growth and cell-cell interactions.     

Binding between the integrin alphaXbeta2 (CD11c/CD18) and heparin

The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the alphaXbeta2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.     

14-3-3zeta is an effector of tau protein phosphorylation

Neurofibrillary tangles associated with Alzheimer's disease are composed mainly of paired helical filaments that are formed by the aggregation of abnormally phosphorylated microtubule-associated protein tau. 14-3-3, a highly conserved protein family that exists as seven isoforms and regulates diverse cellular processes is present in neurofibrillary tangles (Layfield, R., Fergusson, J., Aitken, A., Lowe, J., Landon, M., Mayer, R. J. (1996) Neurosci. Lett. 209, 57-60). The role of 14-3-3 in Alzheimer's disease pathogenesis is not known. In this study, we found that the 14-3-3zeta isoform is associated with tau in brain extract and profoundly stimulates cAMP-dependent protein kinase catalyzed in vitro phosphorylation on Ser(262)/Ser(356) located within the microtubule-binding region of tau. 14-3-3zeta binds to both phosphorylated and nonphosphorylated tau, and the binding site is located within the microtubule-binding region of tau. From brain extract, 14-3-3zeta co-purifies with microtubules, and tubulin blocks 14-3-3zeta-tau binding. Among four 14-3-3 isoforms tested, beta and zeta but not gamma and epsilon associate with tau. Our data suggest that 14-3-3zeta is a tau protein effector and may be involved in the abnormal tau phosphorylation occurring during Alzheimer's disease ontogeny.     

Heparin amplifies platelet-derived growth factor (PDGF)- BB-induced PDGF alpha -receptor but not PDGF beta -receptor tyrosine phosphorylation in heparan sulfate-deficient cells. Effects on signal transduction and biological responses

Platelet-derived growth factor (PDGF) induces mitogenic and migratory responses in a wide variety of cells, by activating specific receptor tyrosine kinases denoted the PDGF alpha- and beta-receptors. Different PDGF isoforms bind in a distinct manner to glycosaminoglycans, particularly heparan sulfate. In the present study, we show potentiation by exogenous heparin of PDGF-BB-induced PDGF alpha-receptor tyrosine phosphorylation in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells. This effect was not seen for PDGF-AA treatment, and heparin lacked a potentiating effect on PDGF-BB stimulation of the PDGF beta-receptor. Heparin did not affect the affinity of PDGF-BB binding for the PDGF receptors on CHO 677 cells. The PDGF-BB-stimulated PDGF alpha-receptor phosphorylation was enhanced in a dose-dependent fashion by heparin at low concentration. The effect was modulated by 2-O- and 6-O-desulfation of the polysaccharide. Maximal induction of PDGF alpha-receptor tyrosine phosphorylation (6-fold) in CHO 677 cells was achieved by treatment with a heparin decasaccharide, but shorter oligosaccharides consisting of four or more monosaccharide units were also able to augment PDGF alpha-receptor phosphorylation, albeit at higher concentrations. Heparin potentiated PDGF-BB-induced activation of mitogen-activated protein kinase and protein kinase B (Akt) and allowed increased chemotaxis of the CHO 677 cells toward PDGF-BB. In conclusion, heparin modulates PDGF-BB-induced PDGF alpha-receptor phosphorylation and downstream signaling, with consequences for cellular responsiveness to the growth factor.     

Heparin reduces the alpha-helical content of cobra basic phospholipase A(2) and promotes its complex formation

The interaction of phospholipase A(2) (PLA(2)) with glycosaminoglycans (GAGs) has recently attracted attention in view of its implication on inflammation and cell proliferation. By using Fourier Transformed Infrared (FTIR) spectroscopic measurements, we demonstrate here that binding of cobra basic phospholipase A(2) from Naja nigricollis (N-PLA(2)) to heparin may induce a significant conformational change observed in the amide I region of the enzyme's alpha-helical and beta-sheet structure. It is observed that notable conformational change of N-PLA(2) due to heparin binding occurs only when heparin's chain length is at least an octasaccharide as evidenced by circular dichroism and optical density measurements. This correlation may be an important factor in the aggregation of N-PLA(2) and N-PLA(2)-heparin complexes. Heparin induced change in conformation of PLA(2) is suggested to be a notable link in understanding the diversity in PLA(2) activity when rendered to the extracellular matrix of cell membranes that is full of GAG molecules.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Impact of various glycosaminoglycans upon cAMP-dependent protein kinase

The physiological effects of the second messenger cAMP are displayed by cAMP-dependent protein kinase-medicated phosphorylation of specific target proteins which in turn control diverse cellular functions. We have determined this enzyme substrate phosphorylation in the presence of various glycosaminoglycans using a cAMP-dependent protein kinase isolated from rat liver. The results indicate that sulfated and unsulfated polysaccharides are able to inhibit phosphorylation of histone type IIa catalysed by cAMP-dependent protein kinase. Based on their impact upon substrate phosphorylation, glycosaminoglycans can be divided into three groups: group I with the highest inhibitory effect: dermatan sulfate and heparan sulfate; group II: chondroitin 4-sulfate and group III with the lowest inhibitory effect: chondroitin 6-sulfate, keratan sulfate and hyaluronic acid.     

Measurement of the affinities of heparins, naturally occurring glycosaminoglycans, and other sulfated polymers for antithrombin III and thrombin

Heparin, other glycosaminoglycans, and synthetic sulfated polymers have antithrombotic and anticoagulant activities, which may be mediated through a range of interactions with different proteins. A simple, quantitative method has been developed for assessing the affinity of interaction between sulfated polymers and proteins in the liquid phase. This has been used to compare the binding of a range of glycosaminoglycans and other sulfated polymers to antithrombin III and thrombin, a major inhibitor of and a central protease in the coagulation system, respectively. The results are consistent with the binding of naturally occurring glycosaminoglycans to antithrombin III solely through the well-defined antithrombin III-binding pentasaccharide found in heparin, the apparent affinity of a preparation depending upon its content of this pentasaccharide. Highly sulfated synthetic polymers will, however, bind antithrombin III by a second mechanism. The affinity of heparin for thrombin decreased with decreasing molecular weight. However, results obtained with heparan sulfate preparations did not indicate any clear relationship between either molecular weight or sulfate content and thrombin binding, but suggested that there may be an oligosaccharide sequence containing N-sulfate residues which confers high affinity for thrombin. In addition, some of the synthetic sulfated polymers bound thrombin with very high affinity.     

Interaction of heparin with multimolecular aggregates of acetylcholinesterase

It has been reported previously that heparin, a sulfated glycosaminoglycan, releases the asymmetric 16 S form of acetylcholinesterase (AChE) from cholinergic synapses. Here it is shown that heparin releases the synaptic AChE not as individual 16 S species but as multimolecular aggregates (30 S) of such molecules. Heparin is able to convert low-ionic strength AChE aggregates into a heparin type of AChE aggregates. Our results suggest that the AChE aggregates detached by heparin are likely to be the physiologically important state of aggregation of the 16 S AChE form in the synaptic basal lamina.     

Binding of heparin to basic fibroblast growth factor induces a conformational change.

The binding of heparin to basic fibroblast growth factor (bFGF) induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum. The observed spectral changes suggest that the conformational change is highly localized most likely in the beta-turn regions of the bFGF molecule. Heparan sulfate, a component of the endothelial extracellular matrix, was also observed to bind to bFGF and induce a similar conformational change to that observed for heparin. Further, sucrose octasulfate, a compound which mimics the effects of heparin biologically, was also observed to induce this same conformational change. This spectroscopically observable change has allowed us to probe the functional determinants necessary for heparin to bind the bFGF and to induce the observed conformational change. We have determined the effects of binding of various monomeric and polymeric, sulfated and nonsulfated glycosaminoglycans and carbohydrate compounds. The results indicate that the binding of heparin involves highly specific interactions. Further, heparin was observed to greatly increase the thermal stability of bFGF, raising the Tm by 25 degrees C. Sucrose octasulfate was also able to enhance the thermal stability of bFGF, but not to the same extent as heparin.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

Regulation of glycosaminoglycan function by osmotic potentials. Measurement of water transfer during antithrombin activation by heparin.

The sulfated glycosaminoglycan heparin is an important anticoagulant, widely used to treat and to prevent arterial thrombosis. Heparin triggers conformational changes in, and the functional activation of, the serine proteinase inhibitor antithrombin. We investigated water-transfer reactions during the activation process to explore the possibility that functional interaction between antithrombin and sulfated glycosaminoglycans can be regulated by osmotic potentials. Volume of water transferred upon heparin binding was measured from differences in free energy change, Delta(Delta G), with osmotic stress, pi. Osmotic stress was induced with chemically inert probes that are geometrically excluded from the water-permeable spaces of antithrombin and from intermolecular spaces formed during the association reaction. The free energy change, Delta G, for the antithrombin/heparin interaction was calculated from the dissociation constant, determined by functional titrations of heparin with antithrombin at fixed concentrations of the coagulation protease factor Xa. The effect of osmotic stress was independent of the chemical nature of osmotic probes but correlated with their radius up to radius >17 A. In mixtures including a large and a small probe, the effect of the large probe was not modified by the small probe added at a large molar excess. With an osmotic probe of 4-A radius, the Delta(Delta G)/pi slope corresponds to a transfer of 119 +/- 25 water molecules to bulk solution on formation of the complex. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures revealed complex surfaces with smaller hydration volumes in the bound relative to the free conformation. The residue distribution in, and atomic composition of, the pockets containing atoms from residues implicated in heparin binding were distinct in the bound versus free conformer. The results demonstrate that the heparin/antithrombin interaction is linked to net water transfer and, therefore, can be regulated in biological gels by osmotic potentials.     

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.     

The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.     

Disaccharide analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose-gel electrophoresis.

The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 microg, with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined.