A型肉毒毒素表位多肽免疫和人源噬茵体单链抗体文库的构建

采用表位合成多肽免疫获得编码人源抗体的基因材料,抗体轻重链可变区基因通过剪接重叠延伸(SOE)技术进行组装后,亚克隆入噬菌体粒中构建免疫库。以重组制备的A型肉毒毒素保护性抗原为配体筛选结合子,ELISA鉴定,获得具有较高抗原结合力的噬菌体克隆,进行基因测序和家族定位分析。结果显示,表位合成多肽免疫,成功构建了库容大于10^8的重组抗体库。体外3轮免疫淘筛,筛选到人源A型肉毒抗毒素克隆,基因结构分析表明,其中ScFvB17全长750bp,可编码250个氨基酸残基：VH属于VH4家族,369bp,编码123个氨基酸残基;Vκ属于κ链Ⅱ家族,336bp,编码112个氨基酸残基。基因的同源分析表明,这是一株特异的单链抗体新基因。     

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.     

A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1-A4

Botulinum neurotoxin (BoNT) is a category A toxin that has been classified within seven serotypes, designated A-G. Recently, it has been discovered that sequence variability occurs in BoNTs produced by serotype A (BoNT/A) variant strains, designated as subtypes A1 and A2, which have significantly different antibody-binding properties. We have therefore made efforts to understand at the molecular level the diversity and its effects on the biological actions of the toxin, including receptor binding, substrate recognition, and catalysis. We provide the results of these studies, including the analysis of two newly sequenced BoNT/A variants, Loch Maree (A3) and 657Ba (A4), and their comparison to A1 and A2. Using sequence analysis, available functional data, molecular modeling, and comparison of models with the crystal structures of BoNT/A1 and the light chain of BoNT/A2, we conclude that these sequence differences within subtypes will impact development of broad-spectrum antibody and small ligand therapeutics, and suggest dissimilarities in binding affinity and cleavage efficiency of the SNAP-25 substrate. In particular, sequence variation in subtypes BoNT/A3 and BoNT/A4 will likely effect alpha-exosite and S1' subsite recognition, respectively.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain

Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion.     

The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by selectively cleaving core components of the vesicular fusion machinery. The synaptic vesicle proteins Synaptotagmin-I and -II act as receptors for BoNT/B and BoNT/G. Here we show that BoNT/A also interacts with a synaptic vesicle protein, the synaptic vesicle glycoprotein 2C (SV2C), but not with the homologous proteins SV2A and SV2B. Binding of BoNT/A occurs at the membrane juxtaposed region preceding transmembrane domain 8. A peptide comprising the intravesicular domain between transmembrane domains 7 and 8 specifically reduces the neurotoxicity of BoNT/A at phrenic nerve preparations demonstrating the physiological relevance of this interaction.     

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C–D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.     

A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC₅₀ values ?1 μM and 11 effective at ?2 μM in an ex vivo assay.     

Delineating the susceptibility of botulinum neurotoxins to denaturation through thermal effects

Botulinum neurotoxins (BoNT) are the etiological agents responsible for botulism and are acknowledged terrorist threat agents. Passive immunotherapy may provide one countermeasure. Importantly, in the virtually unlimited repertoire of antibody specificities, enzyme linked immunosorbent assays (ELISA) has become an indispensable method for antibody selection. We report that of the BoNTs, BoNT/E is highly susceptible to polystyrene induced denaturation. To further dissect this result and the potential susceptibility of other BoNTs to denaturation we selected a thermal platform, which could be readily quantified using surface plasmon resonance (SPR), a primary rat spinal cord cell-based assay and an animal lethality model.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.     

Differential role of molten globule and protein folding in distinguishing unique features of botulinum neurotoxin

Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37 °C. LCE, on the other hand, was although optimally active at 37 °C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics.     

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-Å X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.     

Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of its six hypervariable loops

The enhancement of antibody affinity by mutagenesis targeting only complementarity determining regions has the advantage of respecting the framework regions, which are important for tolerance if clinical use is envisaged. Here, starting from a Fab (antigen-binding fragment; 35PA₈₃) capable of neutralizing the lethal toxin of anthrax and having an affinity of 3.4 nM for its antigen, a phage-displayed library of variants where all six complementarity determining regions (73 positions) were targeted for mutagenesis was built. This library contained 5 × 10⁸ variants, and each variant carried four mutations on average. The library was first panned with adsorbed antigen and washes of increasing stringency. It was then screened in parallel with either small concentrations of soluble biotinylated antigen or adsorbed antigen and long elution times in the presence of soluble antigen. The stringencies of both selections were pushed as far as possible. Compared with 35PA₈₃, the best selected clone had an affinity enhanced 19-fold, to 180 pM, and its 50% inhibitory concentration was decreased by 40%. The results of the two selection methods were compared, and the generality of these methods was considered.     

A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic α-Exosite Binding Region

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K_d) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K_d for BoNT/A Lc of 1.47 × 10^− 10 M and an IC₅₀ (50% inhibitory concentration) of 4.7 × 10^− 10 M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 Å resolution. The structure reveals that the Aa1 VHH binds in the α-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc α-exosite as a target for inhibitor development.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

Ycf1-dependent cadmium detoxification by yeast requires phosphorylation of residues Ser908 and Thr911

Yeast cadmium factor (Ycf1), an ATP-binding cassette (ABC) protein of the multidrug resistance protein subfamily, is a vacuolar GS-conjugate transporter required for heavy metal and drug detoxification. There is evidence that phosphorylation may play a critical role in the function of ABC transporters from higher organisms. In this work, the possibility of Ycf1 phosphorylation was examined using site-directed mutagenesis. We demonstrate that Ser⁹⁰⁸ and Thr⁹¹¹, within the regulatory domain (R domain), are functionally important for Ycf1 transport activity and likely sites for phosphorylation. Mutation of these residues to alanine severely impaired the Ycf1-dependent cadmium detoxification capacity and transport activity, while replacement by acidic residues (mimicking phosphorylation) significantly suppressed the cadmium resistance and transport defects. Both in vitro treatment of Ycf1 with alkaline phosphatase and changes in the electrophoretic mobility of the S908A, T911A and double mutant S908A/T911A proteins supported the conclusion that Ycf1 is a phosphoprotein. The screening of the yeast kinome identified four protein kinases affecting cadmium detoxification, but none of them was involved directly in the phosphorylation of Ycf1. Our data strongly implicate Ycf1 phosphorylation as a key determinant in cadmium resistance in yeast, a significant finding given that very little is known about phosphorylation of ABC transporters in yeast.     

Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies

The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the V_H-V_L interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V_H-V_L interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.     

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.     

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display,in silico loop reconstruction and molecular dynamics simulations

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.     

Localization of subunit C (Vma5p) in the yeast vacuolar ATPase by immuno electron microscopy

Vacuolar ATPases (V1V0 -ATPases) function in proton translocation across lipid membranes of subcellular compartments. We have used antibody labeling and electron microscopy to define the position of subunit C in the vacuolar ATPase from yeast. The data show that subunit C is binding at the interface of the ATPase and proton channel, opposite from another stalk density previously identified as subunit H [Wilkens S., Inoue T., and Forgac M. (2004) Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking. J. Biol. Chem. 279, 41942-41949]. A picture of the vacuolar ATPase stalk domain is emerging in which subunits C and H are positioned to play a role in reversible enzyme dissociation and activity silencing.