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1.
Matrix Biol ; 67: 63-74, 2018 04.
Article in English | MEDLINE | ID: mdl-29331336

ABSTRACT

Increased uptake of glucose, a general hallmark of malignant tumors, leads to an accumulation of intermediate metabolites of glycolysis. We investigated whether the high supply of these intermediates promotes their flow into UDP-sugars, and consequently into hyaluronan, a tumor-promoting matrix molecule. We quantified UDP-N-Acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA) in human breast cancer biopsies, the levels of enzymes contributing to their synthesis, and their association with the hyaluronan accumulation in the tumor. The content of UDP-GlcUA was 4 times, and that of UDP-GlcNAc 12 times higher in the tumors as compared to normal glandular tissue obtained from breast reductions. The surge of UDP-GlcNAc correlated with an elevated mRNA expression of glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), one of the key enzymes in the biosynthesis of UDP-GlcNAc, and the expression of GFAT1 was also elevated. The contents of both UDP-sugars strongly correlated with tumor hyaluronan levels. Interestingly, hyaluronan content did not correlate with the mRNA levels of the hyaluronan synthases (HAS1-3), thus emphasizing the role of the UDP-sugar substrates of these enzymes. The UDP-sugars showed a trend to higher levels in ductal vs. lobular cancer subtypes. The results reveal for the first time a dramatic increase of UDP-sugars in breast cancer, and suggest that their high supply drives the accumulation of hyaluronan, a known promoter of breast cancer and other malignancies. In general, the study shows how the disturbed glucose metabolism typical for malignant tumors can influence cancer microenvironment through UDP-sugars and hyaluronan.


Subject(s)
Breast Neoplasms/metabolism , Hyaluronic Acid/metabolism , Uridine Diphosphate Glucuronic Acid/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolism , Adult , Aged , Aged, 80 and over , Breast Neoplasms/genetics , Case-Control Studies , Female , Gene Expression Regulation, Neoplastic , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Humans , Hyaluronan Synthases/genetics , Middle Aged , Up-Regulation , Young Adult
2.
Proteomics ; 15(17): 2953-65, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25951301

ABSTRACT

The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho-kinase inhibitor Y-27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short-term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 µM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell-IQ time-lapse imaging analysis. Rho-kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin-associated focal adhesions were present in the ROCKi-treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte-specific genes, such as procollagen α1 (II) and aggrecan.


Subject(s)
Amides/pharmacology , Fibroblasts/cytology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Actins/metabolism , Actins/ultrastructure , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cells, Cultured , Fibroblasts/drug effects , Fibroblasts/metabolism , Fluorescence , Focal Adhesions/drug effects , Foreskin/cytology , Gene Expression Regulation/drug effects , Humans , Male , Proteins/analysis , Proteins/genetics , Proteins/metabolism , Proteomics/methods , rho-Associated Kinases/metabolism
3.
Allergy Asthma Immunol Res ; 7(4): 384-92, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25749774

ABSTRACT

PURPOSE: Divergent results on the IgE reactivity of dog-allergic subjects to Can f 4 have been reported. The aim of this study was to evaluate the significance of Can f 4 in dog allergy and to develop an immunochemical method for measuring Can f 4 content in environmental samples. METHODS: We purified the natural dog allergen Can f 4 from a dog dander extract by monoclonal antibody-based affinity chromatography and generated its variant in a recombinant form. Sixty-three dog-allergic patients and 12 nonallergic control subjects were recruited in the study. The IgE-binding capacity of natural Can f 4 and its recombinant variant was assessed by ELISA, immunoblotting, and skin prick tests (SPT). RESULTS: Eighty-one percent of the dog-allergic patients showed a positive result to the immunoaffinity-purified natural Can f 4 in IgE ELISA, but only 46% in IgE immunoblotting. Respective results with the recombinant Can f 4 variant were 54% and 49%. SPT results reflected those obtained in ELISA and immunoblotting. The overall IgE reactivity of the immunoaffinity-purified natural Can f 4 was found to depend strongly on the integrity of the allergen's conformation. A sandwich ELISA based on monoclonal antibodies was found to be functional for measuring Can f 4 in environmental samples. CONCLUSIONS: Can f 4 is a major allergen of dog together with Can f 1 and Can f 5. In combination with other dog allergens, it improves the reliability of allergy tests in dog allergy.

4.
Front Cell Neurosci ; 8: 214, 2014.
Article in English | MEDLINE | ID: mdl-25147500

ABSTRACT

Chronic inflammation and oxidative stress (OS) are present in Alzheimer's disease (AD) brains in addition to neuronal loss, Amyloid-ß (Aß) plaques and hyperphosphorylated tau-protein neurofibrillary tangles (NFTs). Previously we showed that levels of the pro-inflammatory cytokine, interleukin-18 (IL-18), are elevated in post-mortem AD brains. IL-18 can modulate the tau kinases, Cdk5 and GSK3ß, as well as Aß-production. IL-18 levels are also increased in AD risk diseases, including type-2 diabetes and obesity. Here, we explored other IL-18 regulated proteins in neuron-like SH-SY5Y cells. Differentiated SH-SY5Y cells, incubated with IL-18 for 24, 48, or 72 h, were analyzed by two-dimensional gel electrophoresis (2D-DIGE). Specific altered protein spots were chosen and identified with mass spectrometry (MS) and verified by western immunoblotting (WIB). IL-18 had time-dependent effects on the SH-SY5Y proteome, modulating numerous protein levels/modifications. We concentrated on those related to OS (DDAH2, peroxiredoxins 2, 3, and 6, DJ-1, BLVRA), Aß-degradation (MMP14, TIMP2), Aß-aggregation (Septin-2), and modifications of axon growth and guidance associated, collapsin response mediator protein 2 (CRMP2). IL-18 significantly increased antioxidative enzymes, indicative of OS, and altered levels of glycolytic α- and γ-enolase and multifunctional 14-3-3γ and -ε, commonly affected in neurodegenerative diseases. MMP14, TIMP2, α-enolase and 14-3-3ε, indirectly involved in Aß metabolism, as well as Septin-2 showed changes that increase Aß levels. Increased 14-3-3γ may contribute to GSK3ß driven tau hyperphosphorylation and CRMP2 Thr514 and Ser522 phosphorylation with the Thr555-site, a target for Rho kinase, showing time-dependent changes. IL-18 also increased caspase-1 levels and vacuolization of the cells. Although our SH-SY5Y cells were not aged, as neurons in AD, our work suggests that heightened or prolonged IL-18 levels can drive protein changes of known relevance to AD pathogenesis.

5.
J Biol Chem ; 289(12): 8375-89, 2014 Mar 21.
Article in English | MEDLINE | ID: mdl-24509846

ABSTRACT

Hyaluronan synthases (HAS1-3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth.


Subject(s)
Collagen Type I/metabolism , Endocytosis , Glucuronosyltransferase/metabolism , Hyaluronic Acid/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Cell Adhesion , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Dogs , Glucuronosyltransferase/analysis , Humans , Hyaluronan Synthases , Protein Transport , RNA Interference , Up-Regulation , rab GTP-Binding Proteins/analysis , rab GTP-Binding Proteins/genetics
6.
Chem Biol ; 20(3): 379-90, 2013 Mar 21.
Article in English | MEDLINE | ID: mdl-23521796

ABSTRACT

Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, 2-arachidonoylglycerol (2-AG). During 2-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.


Subject(s)
Benzodioxoles/chemistry , Monoacylglycerol Lipases/antagonists & inhibitors , Piperazines/pharmacology , Piperidines/chemistry , Triazoles/chemistry , Urea/chemistry , Urea/pharmacology , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Mice , Piperazine , Rats , Substrate Specificity
7.
Toxicol Lett ; 207(1): 34-41, 2011 Nov 10.
Article in English | MEDLINE | ID: mdl-21907773

ABSTRACT

Drug metabolism can result in the production of highly reactive metabolites that may form adducts with cellular macromolecules, and thus initiate adverse drug reactions, cause toxicity, and even require the withdrawal of drug from the market. In this study, a 2'-deoxyguanosine (dG)-based chemical trapping test system was developed for use as a fast screening tool for DNA adducting metabolites of new drug candidates. Reactive metabolites were generated from parent compounds in in vitro incubations with phenobarbital-induced mouse liver microsomes, human liver microsomes and different recombinant human CYP enzymes in the presence of dG. The formed dG-adducts were separated, characterized and their stability was studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was evaluated with six test compounds, aflatoxin B1, estrone, clozapine, tolcapone, ticlopidine and imipramine. Estrone and aflatoxin B1 formed dG adducts with phenobarbital-induced mouse liver microsomes, human liver microsomes and human recombinant CYP enzymes. Adduct formation was also observed with tolcapone when phenobarbital-induced mouse liver microsomes were used as the enzyme source. The stability of each formed adduct was independent of the different enzyme sources. No dG-adducts were identified with ticlopidine, clozapine and imipramine. Compared to other classical DNA reactivity tests, e.g. Ames test, the present surrogate endpoint, the dG adduct, is faster, enables the characterization of the formed compounds, and also permits the investigation of more unstable adducts.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , DNA Adducts/analysis , Deoxyguanosine/metabolism , Liver/metabolism , Microsomes, Liver/metabolism , Pharmaceutical Preparations/metabolism , Animals , Chromatography, Liquid , Female , Humans , Mice , Mice, Inbred DBA , Microsomes, Liver/enzymology , Tandem Mass Spectrometry
8.
J Exp Bot ; 61(4): 1075-87, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20048332

ABSTRACT

Metal hyperaccumulator plants have previously been characterized by transcriptomics, but reports on other profiling techniques are scarce. Protein profiles of Thlaspi caerulescens accessions La Calamine (LC) and Lellingen (LE) and lines derived from an LCxLE cross were examined here to determine the co-segregation of protein expression with the level of zinc (Zn) hyperaccumulation. Although hydrophobic proteins such as membrane transporters are not disclosed, this approach has the potential to reveal other proteins important for the Zn hyperaccumulation trait. Plants were exposed to metals. Proteins were separated using two-dimensional electrophoresis and those showing differences among accessions, lines or metal exposures were subjected to mass-spectrometric analysis for identification. Crossing decreased the number of different proteins in the lines compared with the parents, more so in the shoots than in the roots, but the frequencies of Zn-responsive proteins were about the same in the accessions and the selection lines. This supports the finding that the Zn accumulation traits are mainly determined by the root and that Zn accumulation itself is not the reason for the co-segregation. This study demonstrates that crossing accessions with contrasting Zn accumulation traits is a potent tool to investigate the mechanisms behind metal hyperaccumulation. Four tentatively identified root proteins showed co-segregation with high or low Zn accumulation: manganese superoxide dismutase, glutathione S-transferase, S-formyl glutathione hydrolase, and translation elongation factor 5A-2. However, these proteins may not be the direct determinants of Zn accumulation. The role of these and other tentatively identified proteins in Zn accumulation and tolerance is discussed.


Subject(s)
Proteomics , Thlaspi/chemistry , Zinc/metabolism , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolism , Thlaspi/genetics , Thlaspi/metabolism
9.
Amino Acids ; 38(2): 613-22, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20012117

ABSTRACT

We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues. To study how the activation of polyamine catabolism affects other metabolic pathways, protein expression pattern of the livers of transgenic animals was analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. A total of 23 proteins were shown to be differentially expressed in the transgenic from the wild-type animals. Many of the identified proteins showed expression patterns associated with polyamine catabolism activation. However, the expression pattern of other proteins, such as repression of GST pi and selenium-binding protein 2 and 60 kDa heat-shock protein, could be explained by the overexpression of peroxisome proliferator-activated receptor gamma co-activator 1alpha in response to depleted ATP pools. The activation of the latter proteins is thought to lead to the improved insulin sensitivity seen in the MT-SSAT animals.


Subject(s)
Liver/chemistry , Polyamines/metabolism , Proteomics , Acetyltransferases/chemistry , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Electrophoresis, Gel, Two-Dimensional , Female , Gene Expression , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Transgenic , Molecular Sequence Data
10.
Mol Plant Pathol ; 9(6): 799-808, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19019008

ABSTRACT

Benzothiadiazole (BTH) induces resistance to the downy mildew pathogen, Peronospora sparsa, in arctic bramble, but the basis for the BTH-induced resistance is unknown. Arctic bramble cv. Mespi was treated with BTH to study the changes in leaf proteome and to identify proteins with a putative role in disease resistance. First, BTH induced strong expression of one PR-1 protein isoform, which was also induced by salicylic acid (SA). The PR-1 was responsive to BTH and exogenous SA despite a high endogenous SA content (20-25 microg/g fresh weight), which increased to an even higher level after treatment with BTH. Secondly, a total of 792 protein spots were detected in two-dimensional gel electrophoresis, eight proteins being detected solely in the BTH-treated plants. BTH caused up- or down-regulation of 72 and 31 proteins, respectively, of which 18 were tentatively identified by mass spectrometry. The up-regulation of flavanone-3-hydroxylase, alanine aminotransferase, 1-aminocyclopropane-1-carboxylate oxidase, PR-1 and PR-10 proteins may partly explain the BTH-induced resistance against P. sparsa. Other proteins with changes in intensity appear to be involved in, for example, energy metabolism and protein processing. The decline in ATP synthase, triosephosphate isomerase, fructose bisphosphate aldolase and glutamine synthetase suggests that BTH causes significant changes in primary metabolism, which provides one possible explanation for the decreased vegetative growth of foliage and rhizome observed in BTH-treated plants.


Subject(s)
Plant Leaves/drug effects , Plant Leaves/metabolism , Rosaceae/drug effects , Rosaceae/metabolism , Thiadiazoles/pharmacology , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Plant/drug effects , Plant Proteins/metabolism , Proteome/drug effects , Salicylic Acid/metabolism
11.
Biorheology ; 45(3-4): 323-35, 2008.
Article in English | MEDLINE | ID: mdl-18836233

ABSTRACT

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.


Subject(s)
Chondrocytes/metabolism , Chondrosarcoma/metabolism , Chondrosarcoma/pathology , Mitogen-Activated Protein Kinases/metabolism , Peptide Elongation Factor 2/metabolism , Phosphoproteins/metabolism , Proteomics , Cell Line, Tumor , Humans , Microfilament Proteins/metabolism , Peptide Elongation Factor 1/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Phosphoproteins/analysis , Phosphoproteins/chemistry , Phosphorylation , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Stress, Mechanical
12.
Electrophoresis ; 27(13): 2687-701, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16739228

ABSTRACT

The skeleton of the human body is built of cartilage and bone, which are tissues that contain extensive amounts of extracellular matrix (ECM). In bone, inorganic mineral hydroxyapatite forms 50-70% of the whole weight of the tissue. Although the organic matrix of bone consists of numerous proteins, 90% of it is composed of type I collagen. In cartilage, ECM forms a major fraction of the tissue, type II collagen and aggrecans being the most abundant macromolecules. It is obvious that the high content of ECM components causes analytical problems in the proteomic analysis of cartilage and bone, analogous to those in the analysis of low-abundance proteins present in serum. The massive contents of carbohydrates present in cartilage proteoglycans, and hydroxyapatite in bone, further complicate the situation. However, the development of proteomic tools makes them more and more tempting also for research of musculoskeletal tissues. Application of proteomic techniques to the research of chondrocytes, osteoblasts, osteocytes, and osteoclasts in cell cultures can immediately benefit from the present knowledge. Here we make an overview to previous proteomic research of cartilage- and bone-associated samples and evaluate the future prospects of applying proteomic techniques to investigate key events, such as cellular signal transduction, in cartilage- and bone-derived cells.


Subject(s)
Bone and Bones/chemistry , Cartilage/chemistry , Extracellular Matrix/chemistry , Proteins/analysis , Proteomics/methods , Amino Acid Sequence , Biomarkers/analysis , Bone and Bones/ultrastructure , Cartilage/ultrastructure , Humans , Molecular Sequence Data , Protein Processing, Post-Translational
13.
Phytochemistry ; 66(21): 2524-33, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16246382

ABSTRACT

PR-10c is a unique member of PR-10 proteins in birch, since it is the only one known to be post-translationally modified by glutathione and is not constitutively expressed in pollen. Both reduced and S-glutathiolated forms of PR-10c show low ribonuclease activity. However, the major function of the protein is apparently not yet resolved. Our protein-ligand interaction studies with saturation transfer difference (STD) NMR revealed that PR-10c interacts with several biologically important molecules, including cytokinin, flavonoid glycosides, sterols and emodin. Competition study with deoxycholate and kinetin revealed no statistically significant binding interference, indicating that these ligands have different binding sites in PR-10c. Ligand docking studies with a molecular model of PR-10c support the STD NMR results of ligand binding and binding epitopes, suggesting that there are three potential binding sites in PR-10c: two in the hydrophobic cavity and one in the glycine-rich loop. Our docking calculations suggested that only kinetin interacts with the glycine-rich loop, the binding occurring through its adenine moiety. Clear ligand specificity could be observed in the binding of nucleotide derivatives. S-glutathiolation of PR-10c did not affect kinetin binding. The present results suggest that birch PR-10c is a multifunctional protein, which has diverse roles in plant stress responses.


Subject(s)
Betula , Plant Proteins/metabolism , Deoxycholic Acid/metabolism , Emodin/metabolism , Kinetin/metabolism , Ligands , Magnetic Resonance Spectroscopy , Protein Binding , Protein Conformation , Quercetin/analogs & derivatives , Quercetin/metabolism , Rutin/metabolism
14.
Mol Immunol ; 39(7-8): 399-411, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12413691

ABSTRACT

Long-chain polysialic acid (PSA) is expressed on the vertebrate neural cell adhesion molecule (NCAM) during neuronal plasticity. Its structural similarity to the capsular PSAs of some pathogenic bacteria has hampered the development of polysaccharide vaccines against meningitis. The antibodies formed during immunization require a long epitope for binding, and cross-react with host tissue PSA. The nature of the epitope and possible external effectors involved are still unclear. We have evaluated the interaction of PSA with its antibody mAb735 by surface plasmon resonance. The influences of PSA chain length, pH, temperature, ionic environment, and polyamines were also determined. The antibody binding affinity was found to dramatically increase with PSA chain length. A sub-nanomolar dissociation constant (K(D)=8.5 x 10(-10)M) was obtained for the binding of very long chain native MenB polysaccharides (approximately 200 Neu5Ac-residues). Colominic acid from Escherichia coli K1 (approximately 100 residues) and shorter polymers exhibited progressively weaker affinities. The antibody also bound tightly (K(D) approximately 5 x 10(-9)M) to polysialylated glycopeptides from human embryonal brain. The effects of pH and ionic strength suggested that the interaction is largely electrostatic. Ca2+ and Mn2+ ions promoted the observed surface plasmon resonance response in a concentration dependent fashion. Spermine increased the response in a similar way. Our results suggest that divalent cations and polyamines may play significant role in the regulation of the PSA epitope presentation in vivo.


Subject(s)
Antibodies, Monoclonal/metabolism , Polysaccharides, Bacterial/immunology , Sialic Acids/metabolism , Animals , Antibody Affinity , Bacterial Capsules , Calcium/pharmacology , Humans , Hydrogen-Ion Concentration , Imidazoles/pharmacology , Magnesium/pharmacology , Magnetic Resonance Spectroscopy , Mice , Osmolar Concentration , Polyamines/pharmacology , Sialic Acids/chemistry , Surface Plasmon Resonance , Temperature
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