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Johan Hoebeke

Publications and source records attributed to Johan Hoebeke.

At least 37 records · Page 2Linked to original sources
PubMed2005

Type-specific reactivity of anti-glycoprotein G antibodies from herpes simplex virus-infected individuals is maintained by single or dual type-specific residues.

Glycoprotein G-1 (gG-1) of herpes simplex virus type 1 (HSV-1) and gG-2 of HSV-2 are the only known HSV proteins that induce type-specific human antibody responses. Recently, it was shown that purified human anti-gG-1 and anti-gG-2 antibodies presented a type-specific reactivity to immunogenic stretches with high similarity between gG-1 and gG-2. In this study, the molecular basis for this type-specific recognition was investigated employing synthetic peptides covering the indicated regions, including substitutions of the type-specific residues. The results revealed that single or dual type-specific residues localized within regions of high similarity could induce significant structural differences, explaining the type-specific recognition of the human antibody response to the gG proteins.

Amino Acid Substitution↗
PubMed2005

Carbon nanotubes: on the road to deliver.

Over the last few years, considerable advances have been made in the field of nanotechnology. The advent of carbon nanotube functionalization has paved the way for their potential application as a delivery system of diverse molecules such as peptides, proteins, plasmid DNA, and synthetic oligodeoxynucleotides. This opens new therapeutic and preventive opportunities to combat diseases. The scope of this review is to summarize our recent work in this rapidly growing field.

Carbon↗
PubMed2005

Expression of fibrinogen E-fragment and fibrin E-fragment is inhibited in the human infiltrating ductal carcinoma of the breast: the two-dimensional electrophoresis and MALDI-TOF-mass spectrometry analyses.

In the present study, total proteins from a tissue of an infiltrating ductal carcinoma of the breast (IDCA) were compared by the two-dimensional electrophoresis (2D-PAGE) to proteins from an adjacent non-neoplastic breast tissue. Analysis of multiple gels for each sample identified nine proteins present in the tumor sample that were less present in the matched normal adjacent breast tissue and four proteins present at higher levels in the normal tissue. The altered proteins were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and search in protein databases. Protein disulfide isomerase, BiP protein, calreticulin, cathepsin D, inorganic pyrophosphatase, vimentin, apolipoprotein A1 precursor, tropomyosin 4 and beta5-tubulin were identified as being significantly over-expressed in the IDCA with regard to the normal tissue. The expression of fibrinogen E-fragment (known as anti-angiogenic factor) as well as of fibrin E, Pro2619 and actinG1 was found to be inhibited in the tumor sample. The identified proteins might play an important role during malignant transformation, breast cancer progression, and angiogenesis as well as in cellular signaling. This study demonstrates quantitative and qualitative changes in protein abundance between IDCA and normal tissue. The identification of these differentially expressed proteins could lead to a better understanding of the molecular events linked to breast cancer progression.

Breast↗
PubMed2004

Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments.

Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.

Allosteric Site↗
PubMed2004

A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain.

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.

Actins↗
PubMed2004

Variant surface glycoprotein from Trypanosoma evansi is partially responsible for the cross-reaction between Trypanosoma evansi and Trypanosoma vivax.

Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

Amino Acid Sequence↗
PubMed2004

Direct evidence that toll-like receptor 9 (TLR9) functionally binds plasmid DNA by specific cytosine-phosphate-guanine motif recognition.

Cytosine-phosphate-guanine (CpG) motifs in bacterial DNA are known to activate the mammalian immune system, and this activation is thought to depend on the Toll-like receptor 9 (TLR9) signaling pathway. Previous studies strongly suggested that TLR9 is involved as the specific receptor for CpG motifs but did not provide direct evidence of their interaction. In this study, we demonstrate for the first time that murine TLR9 binds an unmethylated CpG-containing plasmid. This interaction is sequence-specific and is influenced by the methylation status of the plasmid. Furthermore, we demonstrate that this interaction leads to the activation of the NF-kappaB pathway in mTLR9-expressing cells. Our results provide a molecular basis for the interaction between CpG-DNA and TLR9.

Amino Acid Motifs↗
PubMed2004

Characterisation and application of antibodies specific for the long platelet-derived growth factor A and B chains.

The platelet-derived growth factor (PDGF) family comprises important mitogens for mesenchymal cells. The active dimeric form of PDGF consists of four structurally related A, B, C, and D chains. All PDGF-variants bind to PDGF-receptors. The A and B chains occur with and without basic C-terminal amino acid extensions as long (A(L) and B(L)) and short (A(S) and B(S)) isoforms. PDGF-A and -B form homo- or heterodimers. The biological relevance of short and long isoforms is unknown, although it may relate to different affinities for glycosaminoglycans of the cell glycocalix and intercellular matrix. Commercially available anti-PDGF-A and anti-PDGF-B antibodies cannot discriminate between the short and the long isoforms. Thus, to investigate the function of the long and short isoforms, we raised antibodies specific for the long A and B chain isoforms. The antibodies were affinity-purified and their properties analysed by surface plasmon resonance. Inhibition studies with different PDGF homodimers and dot-blot studies proved their high specificity for the respective isoforms. Both antibodies recognised the target PDGF homodimers complexed to the glycocalix of human arterial smooth muscle cells and human monocyte-derived macrophages. By using these specific antibodies, we were able to confirm at the protein level the synthesis of PDGF-A and -B during differentiation of human monocyte-derived macrophages and to demonstrate the presence of the PDGF-A(L) and PDGF-B(L) isoforms in human arterial tissue.

Animals↗
PubMed2004

Generation and characterization of neutralizing human monoclonal antibodies against human immunodeficiency virus type 1 Tat antigen.

The human immunodeficiency virus Tat regulatory protein is essential for virus replication and pathogenesis. From human peripheral blood mononuclear cells of three Tat toxoid-immunized volunteers, we isolated five Tat-specific human monoclonal antibodies (HMAbs): two full-length immunoglobulin G (IgG) antibodies and three single-chain fragment-variable (scFv) antibodies. The two IgGs were mapped to distinct epitopes within the basic region of Tat, and the three scFvs were mapped to the N-terminal domain of Tat. The three scFvs were highly reactive with recombinant Tat in Western blotting or immunoprecipitation, but results were in contrast to those for the two IgGs, which are sensitive to a particular folding of the protein. In transactivation assays, scFvs were able to inhibit both active recombinant Tat and native Tat secreted by a transfected CEM cell line while IgGs neutralized only native Tat. These HMAbs were able to reduce viral p24 production in human immunodeficiency virus type 1 strain IIIB chronically infected cell lines in a dose-dependent manner.

Antibodies, Monoclonal↗
PubMed2003

Solid-phase synthesis and characterization of a novel fullerene-peptide derived from histone H3.

A peptide analogue from a histone H3 protein containing the L-fulleropyrrolidino-glutamic acid has been prepared by a solid-phase approach and has been fully characterized. By molecular modelling it was verified that this peptide derivative is able to retain a binding capacity to the MHC (major histocompatibility complex) molecule similar to that of the cognate epitope.

Chromatography, High Pressure Liquid↗
PubMed2003

Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans.

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (A(L) and B(L)) or short (A(S) and B(S)) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AA(L) and PDGF-BB(S) isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.

Animals↗
PubMed2003

Immunological similarity between Schistosoma and bovine cathepsin D.

IgG antibodies from sera of rabbits immunized with a mixture of three synthetic peptides of highly conserved surface-exposed sequences between Schistosoma japonicum and S. mansoni cathepsin D, and a rabbit anti-bovine cathepsin D serum strongly recognized a 45 kDa molecule on immunoblots of adult S. mansoni worm saline extracts (AWSE). This recognition was abolished by immunoadsorption with two of the three selected peptides. The anti-peptide antibodies fixed onto Protein A-Sepharose specifically immunoprecipitated a S. mansoni AWSE component that was able to degrade bovine hemoglobin at pH 3.8. This reaction was inhibited by 7 microM pepstatin A, a classical aspartyl protease inhibitor, suggesting that the parasite cathepsin D was immunoprecipitated. The anti-peptide antibodies also recognized on a dot-blot assay a purified, commercially obtained bovine cathepsin D preparation but not the purified human counterpart. On the other hand, the anti-bovine cathepsin D serum recognized the two above-mentioned schistosome peptides. In addition, S. mansoni-infected patient sera recognized on immunoblots the bovine but not the human cathepsin D. These results, together with a comparative analysis of the selected peptide sequence regions between the schistosome and the two mammal enzymes, allowed us to pinpoint to one amino acid the cross-reactivity between parasite and bovine cathepsin D and the lack of it with human cathepsin D. This difference might be of relevance for immunodiagnosis.

Adult↗
PubMed2003

scFv single chain antibody variable fragment as inverse agonist of the beta2-adrenergic receptor.

Antibodies directed against the second extracellular loop of G protein-coupled receptors were shown to possess functional activities. Using a functional monoclonal antibody against the human beta2-adrenergic receptor, a scFv fragment with high affinity for the target epitope was constructed and produced. The fragment recognized the beta2-adrenergic receptors on A431 cells, blocked cAMP accumulation induced by the beta2-agonist salbutamol, and decreased basal cAMP accumulation in the same cells. Their in vitro activity was tested on neonatal rat cardiomyocytes. The antibody fragments blocked the chronotropic activity induced by the beta2-agonist clenbuterol. They also decreased the in vivo heart beating frequency of mice pretreated with bisoprolol (a beta1-adrenergic receptor antagonist) for 4 min after injection. The immunological approach presented here may serve as a strategy for the synthesis of a new class of allosteric modulators for G protein-coupled receptors.

Albuterol↗
PubMed2003

Synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides.

Carbon nanotubes (NTs) are becoming highly attractive molecules for applications in medicinal chemistry. The main problem of insolubility in aqueous media has been solved by developing a synthetic protocol that allows highly water-soluble carbon NTs to be obtained. As a result, biologically active peptides can be easily linked through a stable covalent bond to carbon NTs. We have demonstrated that a bound peptide from the foot-and-mouth disease virus, corresponding to the 141-159 region of the viral envelope protein VP1, retained the structural integrity and was recognized by monoclonal and polyclonal antibodies. In addition, this peptide-NT conjugate is immunogenic, eliciting antibody responses of the right specificity. Such a system could be greatly advantageous for diagnostic purposes and could find future applications in vaccine delivery.

Capsid Proteins↗
PubMed2003

How biotinylation can interfere with recognition: a surface plasmon resonance study of peptide-antibody interactions.

Biotinylation is one of the most frequently used labelling procedures in biochemistry and molecular biology. To study the influence of biotinylation on peptide antigenicity, we selected a peptide derived from the second extracellular loop of the beta(2)-adrenergic receptor. Interactions between different biotinylated and nonbiotinylated analogs and a monoclonal antibody directed against an epitope present within the N-terminal end of this peptide were studied in detail. Taking advantage of the BIACORE 3000 surface plasmon resonance equipment, we were able to compare antibody interactions with the immobilised peptides and with the same peptides in solution. While the nonbiotinylated peptide, immobilised by its N-terminus, was not recognised by the antibody, it was recognised either after immobilisation by means of the thiol group of the C-terminal cysteine residue or as a free peptide tested as analyte with the monoclonal antibody immobilised on the chip. The N-terminal biotinylated forms were well recognised when immobilised on streptavidin but poorly (for the aminocaproyl-biotin derivative) or not at all (for the biotinylated derivative) when they were allowed to react with immobilised monoclonal antibody. These results indicate that the biotinyl moiety interacts with residues that are important for antibody recognition in solution but such interactions are abrogated when it is bound to the streptavidin. Molecular modeling confirmed that the N-terminus of the peptide mimicked to some extent the streptavidin binding site.

Antibodies, Monoclonal↗
PubMed2003

Sequence analysis, expression, and paratope characterization of a single-chain Fv fragment for the eukaryote ribosomal P proteins.

The variable genes of monoclonal antibody (mAb) B10, specific for the C-terminal region of the eukaryotic ribosomal P protein, have been cloned as a single-chain Fv fragment (scFv) and expressed in Escherichia coli. The primary sequence of the variable regions of the B10 antibody, together with a detailed characterization of the reactive residues of the antigen, allowed the construction of a model of the paratope-epitope interaction, giving a first insight into the binding mechanisms of anti-P autoantibodies to their target peptides. The mAb and scFv could be useful for extensive P protein detection since both recognize the highly conserved motif DDxGF.

Amino Acid Motifs↗
PubMed2003

Autoantibodies to factor VIII with catalytic activity.

Hemophilia A is an X-linked, recessive, bleeding disorder caused by defective or deficient factor VIII (FVIII) molecules. Infusion of purified FVIII to patients with severe hemophilia A results in approximately 25% of the cases, in the emergence of anti-FVIII antibodies (inhibitors) that are known to neutralize the pro-coagulant activity of FVIII by steric hindrance. We recently reported on the proteolysis of FVIII by allo-antibodies in the plasma of high responder patients with severe hemophilia A, demonstrating a new mechanism by which FVIII inhibitors may prevent the pro-coagulant function of FVIII. Hemophilia is the first model where a direct link between the hydrolysis of the target molecule and the occurrence of the clinical manifestations may be established. It also represents the first example in humans, of the induction of catalytic antibodies following the exogenous administration of an antigen. The characterization of FVIII inhibitors as site-specific proteases may provide new approaches to the treatment of inhibitors.

Antibodies, Catalytic↗