Serge Muyldermans

Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake

The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c. A CcmC‐deficient mutant is also compromised in the production and utilization of pyoverdine, the high‐affinity fluorescent siderophore. A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane‐spanning regions with three periplasmic loops. Site‐directed mutagenesis was used in order to assess the importance of some periplasm‐exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization. Despite the conservation of the residues His‐61, Val‐62 and Pro‐63 in the first periplasmic loop, and Leu‐184, His‐185 and Gln‐186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization. Simultaneous replacements of residues Trp‐115 and Gly‐116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production. An Ala substitution of Asp‐127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production. On the other hand, a mutation in Trp‐126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine. These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.

Influence of histones H1/H5 on the DNA coiling in the nucleosome — electric dichroism and birefringence study

Abstract Removal of histones H1 and H5 from chicken erythrocyte mononucleosomes results in a large increase of the negative electric birefringence and dichroism, and of the relaxation times, towards the values observed for mononucleosomal DNA. Cross-linking with dimethylsuberimidate does not yield important changes in the electro-optical properties of mononucleosomes, provided that the reaction is performed at low ionic strength. We suggest that in the absence of H1/H5 the linker DNA is flexible, and that this DNA tail is unwound at low ionic strength and responsible for most of the negative anisotropy of these particles. Bipolar pulse experiments revealed that the orientation mechanism of chromatosomes and H1/H5-depleted nucleosomes is predominantly of the induced dipole type.

Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis.

Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×107 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC50 of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC50 of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future.

Isolation and characterization of single-chain Fv genes encoding antibodies specific for Drosophila Poxn protein.

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single‐chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated α‐Poxn2 and α‐Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The α‐Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.

A nanobody-based tracer targeting DPP6 for non-invasive imaging of human pancreatic endocrine cells

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.

Molecular Imaging with Kupffer Cell-Targeting Nanobodies for Diagnosis and Prognosis in Mouse Models of Liver Pathogenesis.

PurposeKupffer cells (KCs), the liver resident macrophages, are important mediators of tissue homeostasis and pathogen clearance. However, depending on the inflammatory stimuli, KCs have been involved in divergent hepato-protective or hepato-destructive immune responses. The versatility of KCs in response to environmental triggers, in combination with the specific biomarkers they express, make these macrophages attractive in vivo targets for non-invasive monitoring of liver inflammation or pathogenicity. This study aims to determine whether V-set and Ig domain-containing 4 (Vsig4) and C-type lectin domain family (Clec) 4, member F (Clec4F) can be used as imaging biomarkers for non-invasive monitoring of KCs during distinct liver inflammation models.ProcedureFlow cytometry (FACS), immuno-histochemistry (IHC), and single-photon emission computed tomography (SPECT) with Tc-99m labeled anti-Vsig4 or anti-Clec4F nanobodies (Nbs) was performed to evaluate in mice KC dynamics in concanavalin A (ConA)-induced hepatitis and in non-alcoholic steatohepatitis induced via methionine choline deficiency (MCD).ResultsIn homeostatic mice, Nbs targeting Clec4F were found to accumulate and co-localize with Vsig4-targeting Nbs only in the liver. Upon induction of acute hepatitis using ConA, down-regulation of the in vivo Nb imaging signal was observed, reflecting reduction in KC numbers as confirmed by FACS and IHC. On the other hand, induction of steatohepatitis resulted in higher signals in the liver corresponding to higher density of KCs. The Nb-imaging signals returned to normal levels after resolution of the investigated liver diseases.ConclusionsAnti-Clec4F and anti-Vsig4 Nbs targeting KCs as molecular imaging biomarkers could allow non-invasive monitoring/staging of liver pathogenesis.

Novel half-life extended anti-MIF nanobodies protect against endotoxic shock

Sepsis—leading to septic shock—is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity‐matured nanobodies (Nbs; single‐domain antigen‐binding fragments derived from camelid heavy‐chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIFs conformation and receptor binding), the TNF‐inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half‐life extended construct (NbE10‐NbAlb8‐NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory endorgan damage.—Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.‐J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half‐life extended anti‐MIF nanobodies protect against endotoxic shock. FASEB J. 32, 3411–3422 (2018). www.fasebj.org

Abstract 4627: Generation and characterization of non-competitive furin-inhibiting nanobodies

Furin belongs to a family of seven closely related subtilisin-like serine endoproteases, known as proprotein convertases (PCs), PC1/3, PC2, PC4, PC5/6, PACE4 and PC7. The physiological role of furin is to cleave and hence activate a large variety of proproteins. Therefore, it is not surprising that it plays a major role in many pathologies such as cancer and infectious diseases. Furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although thus far none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy chain antibodies against catalytically active furin were developed as specific furin inhibitors. Nanobodies derived from these antibodies bind only to mouse and human furin but not to other PCs. In addition, upon overexpression in cell lines, these nanobodies can inhibit the cleavage of three different furin substrates, TGFβ, BAFF and GPC3. The purified nanobodies can also inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but do not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode of action based on steric hindrance. The inhibitory constant of the purified nanobodies was shown to be in the micromolar range and the nanobodies are non-competitive inhibitors as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies can protect HEK293T cells from diphtheria toxin-induced cytotoxicity as efficiently as the well characterized PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-chain nanobodies represent the first generation of highly specific, non-competitive furin inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4627. doi:1538-7445.AM2012-4627