MAB2040 Sigma-AldrichAnti-Heparin/Heparan Sulfate Antibody, clone T320.11

Prolonged treatment with a large dose of propofol may cause diffuse cellular cytotoxicity; however, the detailed underlying mechanism remains unclear, particularly in vascular endothelial cells. Previous studies showed that a propofol overdose induces endothelial injury and vascular barrier dysfunction. Regarding the important role of endothelial glycocalyx on the maintenance of vascular barrier integrity, we therefore hypothesized that a propofol overdose-induced endothelial barrier dysfunction is caused by impaired endothelial glycocalyx. In vivo, we intraperitoneally injected ICR mice with overdosed propofol, and the results showed that a propofol overdose significantly induced systemic vascular hyperpermeability and reduced the expression of endothelial glycocalyx, syndecan-1, syndecan-4, perlecan mRNA and heparan sulfate (HS) in the vessels of multiple organs. In vitro, a propofol overdose reduced the expression of syndecan-1, syndecan-4, perlecan, glypican-1 mRNA and HS and induced significant decreases in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio and ATP concentrations in human microvascular endothelial cells (HMEC-1). Oligomycin treatment also induced significant decreases in the NAD+/NADH ratio, in ATP concentrations and in syndecan-4, perlecan and glypican-1 mRNA expression in HMEC-1 cells. These results demonstrate that a propofol overdose induces a partially ATP-dependent reduction of endothelial glycocalyx expression and consequently leads to vascular hyperpermeability due to the loss of endothelial barrier functions.

The chromogranin-A peptide catestatin modulates a wide range of processes, such as cardiovascular functions, innate immunity, inflammation, and metabolism. We recently found that the cardiac antiadrenergic action of catestatin requires a PI3K-dependent NO release from endothelial cells, although the receptor involved is yet to be identified. In the present work, based on the cationic properties of catestatin, we tested the hypothesis of its interaction with membrane heparan sulphate proteoglycans, resulting in the activation of a caveolae-dependent endocytosis. Experiments were performed on bovine aortic endothelial cells. Endocytotic vesicles trafficking was quantified by confocal microscopy using a water-soluble membrane dye; catestatin colocalization with heparan sulphate proteoglycans and caveolin 1 internalization were studied by fluorimetric measurements in live cells. Modulation of the catestatin-dependent eNOS activation was assessed by immunofluorescence and immunoblot analysis. Our results demonstrate that catestatin (5 nM) colocalizes with heparan sulphate proteoglycans and induces a remarkable increase in the caveolae-dependent endocytosis and caveolin 1 internalization, which were significantly reduced by both heparinase and wortmannin. Moreover, catestatin was unable to induce Ser(1179) eNOS phosphorylation after pretreatments with heparinase and methyl-β-cyclodextrin. Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.

Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply β-1,3-N-acetylglucosaminyl-transferase 7 (β3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

Extracellular matrices regulate biological processes at the level of cells, tissues, and in some cases, entire multicellular organisms. The subphylum Urochordata exemplifies the latter case, where animals are partially or completely enclosed in "houses" or "tunics". Despite this common strategy, we show that the house proteome of the appendicularian, Oikopleura, has very little in common with the proteome of the sister class, ascidian, Ciona. Of 80 identified house proteins (oikosins), ∼half lack domain modules or similarity to known proteins, suggesting de novo appearance in appendicularians. Gene duplication has been important in generating almost 1/3 of the current oikosin complement, with serial duplications up to 8 paralogs in one family. Expression pattern analyses revealed that individual oikosins are produced from specific fields of cells within the secretory epithelium, but in some cases, migrate up to at least 20 cell diameters in extracellular space to combine in defined house structures. Interestingly, peroxidasin and secretory phospholipase A(2) domains, implicated in innate immune defence are secreted from the anlage associated with the food-concentrating filter, suggesting that this extra-organismal structure may play, in part, such a role in Oikopleura. We also show that sulfation of proteoglycans is required for the hydration and inflation of pre-house rudiments into functional houses. Though correct proportioning in the production of oikosins would seem important in repetitive assembly of the complex house structure, the genomic organization of oikosin loci appears incompatible with common enhancers or locus control regions exerting such a coordinate regulatory role. Thus, though all tunicates employ extracellular matrices based on a cellulose scaffold as a defining feature of the subphylum, they have evolved radically different protein compositions associated with this common underlying structural theme.

Extracellular superoxide dismutase (ECSOD or SOD3) is highly expressed in lungs and functions as a scavenger of O(2)(* horizontal line ). ECM fragmentation, which can be triggered by oxidative stress, participates in the pathogenesis of chronic obstructive pulmonary disease (COPD) through attracting inflammatory cells into the lungs. The level of SOD3 is significantly decreased in lungs of patients with COPD. However, the role of endogenous SOD3 in the development/progression of emphysema is unknown. We hypothesized that SOD3 protects against emphysema by attenuating oxidative fragmentation of ECM in mice. To test this hypothesis, SOD3-deficient, SOD3-transgenic, and WT C57BL/6J mice were exposed to cigarette smoke (CS) for 3 d (300 mg total particulate matter/m(3)) to 6 mo (100 mg/m(3) total particulate matter) or by intratracheal elastase injection. Airspace enlargement, lung inflammation, lung mechanical properties, and exercise tolerance were determined at different time points during CS exposure or after elastase administration. CS exposure and elastase administration caused airspace enlargement as well as impaired lung function and exercise capacity in SOD3-null mice, which were improved in mice overexpressing SOD3 and by pharmacological SOD mimetic. These phenomena were associated with SOD3-mediated protection against oxidative fragmentation of ECM, such as heparin sulfate and elastin, thereby attenuating lung inflammatory response. In conclusion, SOD3 attenuates emphysema and reduces oxidative fragmentation of ECM in mouse lung. Thus, pharmacological augmentation of SOD3 in the lung may have a therapeutic potential in the intervention of COPD/emphysema.

Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs) have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR) spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

Asbestosis is a form of interstitial lung disease caused by the inhalation of asbestos fibers, leading to inflammation and pulmonary fibrosis. Inflammation and oxidant/antioxidant imbalances are known to contribute to the disease pathogenesis. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. Extracellular matrix (ECM) components, such as collagen and glycosaminoglycans, are known to be sensitive to oxidative fragmentation. Heparan sulfate, a glycosaminoglycan, is highly abundant in the ECM and tightly binds EC-SOD. We investigated the protective role of EC-SOD by evaluating the interaction of EC-SOD with heparan sulfate in the presence of reactive oxygen species (ROS). We found that ROS-induced heparin and heparan sulfate fragments induced neutrophil chemotaxis across a modified Boyden chamber, which was inhibited by the presence of EC-SOD by scavenging oxygen radicals. Chemotaxis in response to oxidatively fragmented heparin was mediated by Toll-like receptor-4. In vivo, bronchoalveolar lavage fluid from EC-SOD knockout mice at 1, 14, and 28 days after asbestos exposure showed increased heparan sulfate shedding from the lung parenchyma. We demonstrate that one mechanism through which EC-SOD inhibits lung inflammation and fibrosis in asbestosis is by protecting heparin/heparan sulfate from oxidative fragmentation.

Using multicolor live cell imaging in combination with biochemical assays, we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan-binding ligands including cationic polymers, lipids and polypeptides. Following clathrin- and caveolin-independent, but flotillin- and dynamin-dependent internalization, proteoglycan-bound ligands associate with flotillin-1-positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin-mediated endocytosis. The proteoglycan-dependent pathway to late endosomes does not require microtubule-dependent transport or phosphatidyl-inositol-3-OH kinase-dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting that this mechanism may be used generally by cell surface proteoglycans and proteoglycan-binding ligands that lack secondary receptors.

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.