Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity

A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins

Antibodies to Lactobacilli and Bifidobacteria in Young Children with Different Propensity to Develop Islet Autoimmunity

Peer reviewe

Glükoomika

BeSt programmi toetusel loodud sisupakett „Glükoomika“ on mõeldud TÜ Arstiteaduskonna ning Loodus-ja tehnoloogiateaduskonna doktorantidele, magistrantidele ja noorteadlastele. Õpiobjekti kasutamine eeldab üldisi teadmisi biokeemiast, valkudest ja sahhariididest. Lühikursus tutvustab glükoomika mõisteid, peamiste glükaanide ehitust ja kasutatavaid uurimismeetodeid

Antigeenide identifitseerimine

BeSt programmi toetusel loodud sisupakett „Antigeenide identifitseerimine" tutvustab antikehadega ja antigeenidega seotud mõisteid ning meetodeid nende uurimiseks. Õpiobjekti kasutamine eeldab üldisi teadmisi immunoloogiast, biokeemiast ja valkudest

Helicobacter pylori cell surface interactions with glycosaminoglycans. Identification and characterisation of proteins binding to heparin/heparan sulphate.

Helicobacter pylori is a gastric pathogen which cause chronic type B gastritis and peptic ulcer disease. H. pylori produces virulence factors such as urease, vacuolating cytotoxin VacA, cag pathogenicity island-associated proteins, flagella and adhesins. These proteins interact with several host cell molecules such as sialylated cell surface glycoproteins, some of glycolipids and mucins. Glycosaminoglycans are widely distributed molecules in human cells and extracellular matrix and interact with H. pylori cell surface proteins. Heparan sulphate shows a pH-dependent binding to all strains, which is inhibited by sulphated glycosaminoglycans and other polysulphated molecules such as dextran sulphate and fucoidan. Using bioinformatics methods, the binding of H. pylori VacA cytotoxin to heparin and heparan sulphate was predicted. Specific peptides were synthesised and their interactions with surface-immobilised heparan sulphate studied in a BIAcore biosensor. Synthetic peptides bound to heparan sulphate with a moderate affinity. It was shown that native toxin interacts with immobilised heparin. Heparan sulphate was proposed as a receptor/co-receptor for VacA cytotoxin binding to host cell surfaces. H. pylori urease binds to surface-immobilised heparin and heparan sulphate. The interaction was characterised using a BIAcore biosensor. The binding was a thousand-fold higher at pH 5.5 compared to pH 6.5. The binding epitopes were identified by SELDI-TOF MS technology. Using a proteomic technology, four immunogenic heparin-binding proteins were identified: cell binding factor 2, urease, one outer membrane protein and one hypothetical protein. The binding of three of these proteins to heparin was demonstrated for the first time. The cell binding factor was identified as a specific and highly immunogenic H. pylori surface protein. 2-DE and 2-D immunoblotting allow efficient separation and identification of immunogenic proteins. Understanding how GAGs interact with pathogens, will allow the design of new experimental putative anti-microbial compounds based on sulphated polysaccharide structures

Separation and surveys of proteins of Helicobacter pylori.

The analysis of Helicobacter pylori proteins is a demanding task for the elucidation of virulence factors, antigens and vaccines, all important for diagnosis, therapy and protection. In the "pre-genomic era" the purification of proteins was mostly performed by using various techniques such as detergent treatment of the bacterial cells, ultra-centrifugation, various chromatographic methods, antibody detection, N-terminal sequence determination and finally cloning and identification of the corresponding gene. In this review, the most representative methods used for purification, separation and identification of H. pylori proteins will be presented as well as some important developments in the "post-genomic era" that have improved the performance of these characterisation techniques

Terapeutilised antikehad

BeSt programmi raames loodud õpiobjekt täiendab arstiteaduskonna immunoloogia õppeainete immunotehnoloogia seminari ja käsitleb üht immunotehnoloogia peamist rakendusvaldkonda, milleks on terapeutiliste antikehade arendamine. Õpiobjekti üldeesmärgiks on anda ülevaade terapeutiliste antikehade saamisviisidest, tüüpidest, ravimiarendusprotsessist ja kasutamisest haiguste ravis. Õpiobjekti mahuks on 0,15 EAP-d ja selle läbimiseks kulub umbes 4 tundi. Õpiväljundite saavutamist toetavad küsimused mõtlemiseks, ülesanded, lisainfo, olulise rõhutamine ja enesetest

Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology.

Cell surface proteins of the human gastric pathogen Helicobacter pylori, reference strain CCUG 17874, were extracted with acid glycine and fractionated by heparin affinity chromatography. The extracts were subsequently analysed using high-resolution two-dimensional gel electrophoresis (2-DE) and immunoblotting. Four proteins of low molecular masses (25-30 kDa) stained by Coomassie R-350, were identified by peptide ESI-MS/MS sequencing after in-gel tryptic digestion. The identified proteins were recognised by sera from H. pylori-infected patients. Two of them are now described for the first time as immunogenic proteins of which one protein was determined to be distinct from all H. pylori proteins previously described. In addition, the specificity of the identified peptides was evaluated using both 1-D and 2-D immunoblotting against a panel of sera from patients with various bacterial infections. The present identification of highly specific antigens of H. pylori will encourage the improvement of serological diagnostic tests to diagnose and monitor H. pylori infection

Genetic variants in humanin nuclear isoform gene regions show no association with coronary artery disease.

ObjectiveCoronary artery disease contributes to noncommunicable disease deaths worldwide. In order to make preventive methods more accurate, we need to know more about the development and progress of this pathology, including the genetic aspects. Humanin is a small peptide known for its cytoprotective and anti-apoptotic properties. Our study looked for genomic associations between humanin-like nuclear isoform genes and coronary artery disease using CARDIoGRAMplusC4D Consortium data.ResultsLookup from meta-analysis datasets gave single nucleotide polymorphisms in all 13 humanin-like nuclear isoform genes with the lowest P value for rs6151662 from the MTRNR2L2 gene including the 50 kb flanking region in both directions (P-value = 0.0037). Within the gene region alone the top variant was rs78083998 from the MTRNR2L13 region (meta-analysis P-value = 0.042). None of the found associations were statistically significant after correction for multiple testing. Lookup for expression trait loci in these gene regions gave no statistically significant variants