Analysis of Oversulfation in a Chondroitin Sulfate Oligosaccharide Fraction from Bovine Aorta by Nanoelectrospray Ionization Quadrupole Time-of-Flight and Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions

Markedly enhanced intratumoral spread and antitumor effect of oncolytic adenovirus expressing decorin

With the aim of improving viral distribution and tumor penetration, we have engineered decorin expressing replication-incompetent (dl-LacZ-DCNG) and -competent (Ad-[DELTA]E1B-DCNG) adenoviruses. In both tumor spheroids and established solid tumors in vivo, administration of dl-LacZ-DCNG resulted in greater transduction efficiency and viral spread throughout the tumor mass. Ad-[DELTA]E1B-DCNG also enhanced viral distribution and tumor spread, leading to an increased anti-tumor effect and survival advantage. Upon histological analysis, Ad-[DELTA]E1B-DCNG also elicited greater percentage of apoptotic cells and extensive necrosis compared to those from untreated or control virus-treated tumors. Furthermore, Ad-[DELTA]E1B-DCNG substantially decreased extracellular matrix components within the tumor tissue, while normal tissue adjacent to the tumor was not affected. Finally, intratumoral administration of Ad-[DELTA]E1B-DCNG did not enhance but inhibited the formation of pulmonary metastases of B16BL6 melanoma cells in mice. Taken together, these data demonstrate the utility of decorin as a dispersion agent and suggest its utility and potential in improving the efficacy of replicating adenovirus-mediated cancer gene therapy

High performance mass spectrometry for advanced interactomics studies

Fibroblast growth factor-2 (FGF-2) is a glycosaminoglycan (GAG) binding protein, involved in different biological processes, such as angiogenesis, bone signaling, embryonic development, morphogenesis or cartilage metabolism. GAGs, one of its binding partners, are long-unbranched polysaccharides exhibiting a repeating disaccharide unit. Moreover, preceding studies have shown that GAGs play an important role in tissue development, cellular behavior or extracellular matrix (ECM) organization. The FGF-GAG noncovalent interactions are of high importance in the biological and biomedical fields of research, as a result of their influence in the tissue regeneration and cell proliferation processes. Here, we have employed one of the most advanced mass spectrometric (MS) techniques consisting of fully automated chip-nanoelectrospray (nanoESI), coupled to a quadrupole time-of-flight (QTOF) MS for studying the FGF-GAG noncovalent complexes. The experiments were conducted in 10 mM ammonium acetate/formic acid, pH 6.8, by incubating FGF-2 and CS disaccharides dissolved in buffer; aliquots were collected after 5, 10, 30, 60 and 90 minutes and further submitted to chip-based MS analysis. For the first time, a CS disaccharide was involved in a binding assay with FGF-2. The detected complexes in the screening experiments were further characterized by top-down fragmentation in tandem MS (MS/MS) using collision induced-dissociation (CID) at low ion acceleration energies. CID MS/MS provided data showing for the first time that the binding process occurs via SO3 located at C4 in the GalNAc moiety

The Dermatan Sulfate Proteoglycan Decorin Modulates α2β1 Integrin and the Vimentin Intermediate Filament System during Collagen Synthesis.

Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn(-/-)) mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn(-/-) mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn(-/-) fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn(-/-) fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2β1 integrin at day 6 in Dcn(-/-) fibroblasts, whereas the protein core had no effect on β1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less β1 integrin compared to Dcn(-/-). Furthermore, the α2β1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2β1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn(-/-) phenotype

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

The Angiotensin II Type 1 Receptor Antagonist Losartan Affects NHE1-Dependent Melanoma Cell Behavior

Background/Aims: The peptide hormone angiotensin II (ATII) plays a prominent role in regulating vasoconstriction and blood pressure. Its primary target is the angiotensin II receptor type 1 (AT1), the stimulation of which induces an increase in cytosolic [Ca2+] and calmodulin activation. Ca2+-bound (activated) calmodulin stimulates the activity of the Na+/ H+ exchanger isoform 1 (NHE1); and increased NHE1 activity is known to promote melanoma cell motility. The competitive AT1 receptor inhibitor losartan is often used to lower blood pressure in hypertensive patients. Since AT1 mediates ATII-stimulated NHE1 activity, we set out to investigate whether ATII and losartan have an impact on NHE1-dependent behavior of human melanoma (MV3) cells. Methods: ATII receptor expression was verified by PCR, F-actin was visualized using fluorescently labeled phalloidin, and cytosolic [Ca2+] and pH were determined ratiometrically using Fura-2 and BCECF, respectively. MV3 cell behavior was analyzed using migration, adhesion, invasion and proliferation assays. Results: MV3 cells express both AT1 and the angiotensin II receptor type 2 (AT2). Stimulation of MV3 cells with ATII increased NHE1 activity which could be counteracted by both losartan and the Ca2+/ calmodulin inhibitor ophiobolin-A. ATII stimulation induced a decrease in MV3 cell migration and a more spherical cell morphology accompanied by an increase in the density of F-actin. Independently of the presence of ATII, both NHE1 and migratory activity were reduced when AT1 was blocked by losartan. On the other hand, losartan clearly increased cell adhesion to, and the invasion of, a collagen type I substrate. The AT2 inhibitor PD123319 did not affect NHE1 activity, proliferation and migration, but increased adhesion and invasion. Conclusion: Losartan inhibits NHE1 activity and the migration of human melanoma cells. At the same time, losartan promotes MV3 cell adhesion and invasion. The therapeutic use of AT1 antagonists (sartans) in hypertensive cancer patients should therefore be given critical consideration

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS2 MS3) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS2 MS3, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS2 MS3, which provided sequence patterns consistent with a rare GlcA-3-sulfate GalNAc-6-sulfate structural motif