New structural insights into anomeric carbohydrate recognition by frutalin: an α-d-galactose-binding lectin from breadfruit seeds.

Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (frutalin-d-Gal complex) of resolution. The lectin exhibits post-translational cleavage yielding an α- (133 amino acids) and ß-chain (20 amino acids), presenting a homotetramer when in solution, with a typical JRL ß-prism. The ß-prism was composed of three 4-stranded ß-sheets forming three antiparallel Greek key motifs. The carbohydrate-binding site (CBS) involved the N-terminus of the α-chain and was formed by four key residues: Gly25, Tyr146, Trp147 and Asp149. Together, these results were used in molecular dynamics simulations in aqueous solutions to shed light on the molecular basis of FTL-ligand binding. The simulations suggest that Thr-Ser-Ser-Asn (TSSN) peptide excision reduces the rigidity of the FTL CBS, increasing the number of interactions with ligands and resulting in multiple-binding sites and anomeric recognition of α-d-galactose sugar moieties. Our findings provide a new perspective to further elucidate the versatility of FTL in many biological activities.

A natural cell-penetrating nanopeptide combined with pentavalent antimonial as experimental therapy against cutaneous leishmaniasis.

The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.

In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy.

Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted in-silico structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv's structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.

Structural evolution of an amphibian-specific globin: A computational evolutionary biochemistry approach.

Studies on the globin family are continuously revealing insights into the mechanisms of gene and protein evolution. The rise of a new globin gene type in Pelobatoidea and Neobatrachia (Amphibia:Anura) from an α-globin precursor provides the opportunity to investigate the genetic and physical mechanisms underlying the origin of new protein structural and functional properties. This amphibian-specific globin (globin A/GbA) discovered in the heart of Rana catesbeiana is a monomer. As the ancestral oligomeric state of α-globins is a homodimer, we inferred that the ancestral state was lost somewhere in the GbA lineage. Here, we combined computational molecular evolution with structural bioinformatics to determine the extent to which the loss of the homodimeric state is pervasive in the GbA clade. We also characterized the loci of GbA genes in Bufo bufo. We found two GbA clades in Neobatrachia. One was deleted in Ranidae, but retained and expanded to yield a new globin cluster in Bufonidae species. Loss of the ancestral oligomeric state seems to be pervasive in the GbA clade. However, a taxonomic sampling that includes more Pelobatoidea, as well as early Neobatrachia, lineages would be necessary to determine the oligomeric state of the last common ancestor of all GbA. The evidence presented here points out a possible loss of oligomerization in Pelobatoidea GbA as a result of amino acid substitutions that weaken the homodimeric state. In contrast, the loss of oligomerization in both Neobatrachia GbA clades was linked to independent deletions that disrupted many packing contacts at the homodimer interface.

GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling.

Cryptococcus spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target Cryptococcus spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3ζ. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to Cryptococcus spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of Cryptococcus spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling.

2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface;2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands;18B7-GXMR-CAR provided highest levels of tonic signaling;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A2 by site-directed mutagenesis and molecular dynamics simulation.

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the hsPLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors.

Selective xyloglucan oligosaccharide hydrolysis by a GH31 α-xylosidase from Escherichia coli.

Xyloglucan is ubiquitous in the cell walls of land plants and is also an essential storage polymer in seeds of many species. We studied the hydrolysis of the non-reducing end xylosyl residue of xyloglucan oligosaccharides (XGOs) by the Escherichia coli α-xylosidase (YicI). Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and ion fragmentation analysis together with high performance anion exchange chromatography with pulsed amperometric detection revealed that YicI preferentially removes the xylosyl residue from the glycosyl residue of non-galactosylated oligosaccharides. The YicI shows decreasing activity against the galactosylated oligosaccharides XXXG>XXLG≥XLXG. Studies of the XGOs interaction with active site residues by molecular dynamics simulations suggested that hydrogen bond interactions between the D49 and galactosylated oligosaccharides play an important role in enzyme-XGO interactions. This was confirmed by site-directed mutagenesis, where the D49A mutant affected catalytic efficiency against galactosylated XGOs. Our findings advance xyloglucan disassembly models and highlight the importance of YicI for biotechnology applications.

Molecular dynamics simulations of human L-asparaginase1: Insights into structural determinants of enzymatic activity.

The l-asparaginase enzyme is used in cancer therapy, mainly acute lymphoid leukemia (ALL). Commercial enzymes (EcASNase2) cause adverse reactions during treatment, such as immunogenicity. A human enzyme could be a non-immunogenic substitute. However, no candidate was found showing efficient kinetic properties. HASNase1 is an l-asparaginase that comes from the N-terminal domain of a protein called 60 kDa-lysophospholipase and its 3D structure has not been resolved. HASNase1 is homologous to EcASNase1 and gpASNase1, and this last one has shown efficient kinetic properties. Homology modeling was used to find the 3D structure of hASNase1, so one could submit it to Molecular Dynamics (MD), in order to understand structural differences that lead to different catalytic efficiency compared to EcASNase2 and gpASNase1. The interaction potential between L-Asn and active site residues showed that the substrate can rotate in the site when Region1 is open. Region1 residues sequence favors deformations and movements as shown in MD. Region2-A is linear in gpASNase1, and it features a helix portion in hASNase1, which leaves the Tyr308 position projected to the active site ratifying its role in catalytic efficiency. Analysis of Lys188 orientation and movement showed the effect of positive cooperativity in hASNase1. It was found that the presence of Asn at the allosteric site helps, not only in Region1 stabilization, but also in Lys188 stabilization for the maintenance of the triad. Despite structural similarities in hASNase1, gpASNase1, and EcASNase2, there are differences in structural determinants that, in addition to allosterism, may explain the different kinetic properties.

Alemtuzumab scFv fragments and CD52 interaction study through molecular dynamics simulation and binding free energy.

Specific antibody-antigen recognition is crucial for the immune response. Knowledge of molecular interaction details in the recognition process is fundamental for the rational design of antibodies with improved properties. We used state-of-the-art computer simulation tools to deepen the molecular-level understanding of the interactions between the monoclonal antibody Alemtuzumab and its antigen, the CD52 membrane receptor, of great biotechnological importance. Thus, we seek such responses by modeling the interaction of native and known mutants single-chain fragment variable (scFv) of Alemtuzumab with CD52 inserted in a membrane model to mimic the physiological conditions of antibody-antigen binding. Extensive molecular dynamics simulations of the interaction between Alemtuzumab's scFvs and CD52 promoted greater understanding of the structural and energetic bases, which can be translated into the biological action and affinity of this antibody. The quantification of the scFv-CD52 complexes binding free energy (ΔGbind) by Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) correlated with the experimental binding energies described before. Thus, the mutants D53K, K54D, and K56D resulted in less attractive ΔGbind, therefore lower scFv-CD52 affinity than the native scFv. On the other hand, K56D and K54D/K56D showed lower binding to CD52. These Results revealed that the model system mimicking an environment close to the physiological with the presence of the CD52 in a membrane model proved essential for this system's study. The present study allowed to unveil the molecular mechanisms involved in antigen-antibody interaction and the effects of mutations. Thus, these mechanisms may be explored in the Alemtuzumab variants' rational design with enhanced properties.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A2.

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phospholipase A(2) inhibitor.

Hydrogel and membrane scaffold formulations of Frutalin (breadfruit lectin) within a polysaccharide galactomannan matrix have potential for wound healing.

Plant lectins are carbohydrate-binding proteins, which can interact with cell surfaces to initiate anti-inflammatory pathways, as well as immunomodulatory functions. Here, we have extracted, purified and part-characterized the bioactivity of Jacalin, Frutalin, DAL and PNA, before evaluating their potential for wound healing in cultured human skin fibroblasts. Only Frutalin stimulated fibroblast migration in vitro, prompting further studies which established its low cytotoxicity and interaction with TLR4 receptors. Frutalin also increased p-ERK expression and stimulated IL-6 secretion. The in vivo potential of Frutalin for wound healing was then assessed in hybrid combination with the polysaccharide galactomannan, purified from Caesalpinia pulcherrima seeds, using both hydrogel and membrane scaffolds formulations. Physical-chemical characterization of the hybrid showed that lectin-galactomannan interactions increased the pseudoplastic behaviour of solutions, reducing viscosity and increasing Frutalin's concentration. Furthermore, infrared spectroscopy revealed -OH band displacement, likely caused by interaction of Frutalin with galactose residues present on galactomannan chains, while average membrane porosity was 100 µm, sufficient to ensure water vapor permeability. Accelerated angiogenesis and increased fibroblast and keratinocyte proliferation were observed with the optimal hybrid recovering the lesioned area after 11 days. Our findings indicate Frutalin as a biomolecule with potential for tissue repair, regeneration and chronic wound healing.

Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides.

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide ß-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesis generated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for ß-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.

The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis.

A thermostable variant of the mesophilic xylanase A from Bacillus subtilis (BsXynA-G3_4x) contains the four mutations Gln7His, Gly13Arg, Ser22Pro, and Ser179Cys. The crystal structure of the BsXynA-G3_4x has been solved, and the local environments around each of these positions investigated by molecular dynamics (MD) simulations at 328K and 348K. The structural and MD simulation results were correlated with thermodynamic data of the wild-type enzyme, the 4 single mutants and the BsXynA-G3_4x. This analysis suggests that the overall stabilizing effect is entropic, and is consistent with solvation of charged residues and reduction of main-chain flexibility. Furthermore, increased protein-protein hydrogen bonding and hydrophobic interactions also contribute to stabilize the BsXynA-G3_4x. The study revealed that a combination of several factors is responsible for increased thermostability of the BsXynA-G3_4x; (i) introduction of backbone rigidity in regions of high flexibility, (ii) solvation effects and (iii) hydrophobic contacts.

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights.

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration.