Chemical modification of pseudo‐dimannoside ligands guided by fragment‐based design allowed for the exploitation of an ammonium‐binding region in the vicinity of the mannose‐binding site of DC‐SIGN, ...leading to the synthesis of a glycomimetic antagonist (compound 16) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo‐dimannoside derivatives as DC‐SIGN ligands, their synthesis, their evaluation as DC‐SIGN selective antagonists, the biophysical characterization of the DC‐SIGN/16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC‐SIGN carbohydrate recognition domain.
Fragment‐based design: A high affinity monovalent DC‐SIGN ligand was obtained by targeting an ammonium‐binding pocket identified by virtual screening. The structural basis of its activity was elucidated by X‐ray crystallography, also unveiling an unusual bridging interaction within the crystals.
MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It ...involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli. We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.
Due to their interactions with C‐type lectin receptors (CLRs), glycans from the helminth Schistosoma mansoni represent promising leads for treatment of autoimmune diseases, allergies or cancer. We ...chemo‐enzymatically synthesized nine O‐glycans based on the two predominant O‐glycan cores observed in the infectious stages of schistosomiasis, the mucin core 2 and the S. mansoni core. The O‐glycans were fucosylated next to a selection of N‐glycans directly on a microarray slide using a recombinant fucosyltransferase and GDP‐fucose or GDP‐6‐azidofucose as donor. Binding assays with fluorescently labelled human CLRs DC‐SIGN, DC‐SIGNR and MGL revealed the novel O‐glycan O8 as the best ligand for MGL from our panel. Significant binding to DC‐SIGN was also found for azido‐fucosylated glycans. Contrasting binding specificities were observed between the monovalent carbohydrate recognition domain (CRD) and the tetravalent extracellular domain (ECD) of DC‐SIGNR.
A series of parasite O‐glycans structures have been prepared by chemoenzymatic synthesis and their affinity towards several C‐type lectins screened using glycan arrays.
The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about ...the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLR
S
) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2
+
Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.
Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. ...Specific deuteration furnishes a “stealth” carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.
Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of N-glycans and smaller glycan ...structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galβ1,4GlcNAc (LN), GalNAcβ1,4GlcNAc (LDN), and GalNAcβ1,4Fucα1,3GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-LeX monoclonal IgM antibodies raised against S. mansoni glycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular recognition.
The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric ...flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47(phox), p67(phox) and p40(phox). p67(phox), due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67(phox) is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67(phox). Our data reveals that p67(phox) behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67(phox). We suggest that the dual character of p67(phox) conformation in solution is central to ensure the numerous interactions to be accommodated.
Precision Glycodendrimers for DC‐SIGN Targeting Goti, Giulio; Colombo, Cinzia; Achilli, Silvia ...
European journal of organic chemistry,
August 5, 2022, Letnik:
2022, Številka:
29
Journal Article
Recenzirano
Odprti dostop
Multivalent ligands of the C‐type lectin receptor DC‐SIGN have emerged as effective antiadhesive agents against various pathogens. Some years ago, we described a hexavalent DC‐SIGN ligand, ...Polyman‐26, designed to bridge two of the four binding sites displayed by the receptor. In this work, we present our efforts to accomplish simultaneous coordination of all four carbohydrate binding sites of DC‐SIGN through the synthesis of cross‐shaped glycodendrimers. The tailored rigid scaffold allowed multivalent presentation of glycomimetics in a spatially defined fashion, while providing good water solubility to the constructs. Evaluation of the biological activity by SPR assays revealed strong binding avidity towards DC‐SIGN and increased selectivity over langerin. Inhibition of DC‐SIGN binding to SARS‐CoV‐2 spike protein and of DC‐SIGN mediated Ebola virus trans‐infection testifies for the glycodendrimers potential application in infection diseases. The tetravalent platform described here is easily accessible and can be used in modular fashion with different ligands, thus lending itself to multiple applications.
Multivalent antagonists able to reach the four carbohydrate recognition domains (CRD) of DC‐SIGN have been prepared. The extended rigid core of these glycodendrimers allows multivalent presentation of glycomimetic molecules in a spatially defined fashion, providing high affinity towards DC‐SIGN and selectivity over other C‐type lectins featuring distinct CRD arrangements. The constructs successfully inhibit DC‐SIGN binding to SARS‐CoV‐2 spike protein and DC‐SIGN mediated trans‐infection by Ebola virus.
NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 ...residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.
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•NIS sequence is a flexible structure supporting a functional role in NOX enzymes.•NIS sequence of NOX2 regulates its NADPH oxidase activity in phagocytes.•ATM kinase is activated by ROS production in phagocytes after PMA stimulation.•Ser486 phosphorylation in NIS-NOX2 by ATM inhibits the NADPH oxidase activity.
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