Design and Synthesis of Lipopolysaccharide-Binding Antimicrobial Peptides Based on Truncated Rabbit and Human CAP18 Peptides and Evaluation of Their Action Mechanism.

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4-128 µg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.

Construction of silica-enhanced S-layer protein cages.

The work presented here shows for the first time that it is possible to silicify S-layer coated liposomes and to obtain stable functionalized hollow nano-containers. For this purpose, the S-layer protein of Geobacillus stearothermophilus PV72/p2 was recombinantly expressed and used for coating positively charged liposomes composed of dipalmitoylphosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4. Subsequently, plain (uncoated) liposomes and S-layer coated liposomes were silicified. Determination of the charge of the constructs during silicification allowed the deposition process to be followed. After the particles had been silicified, lipids were dissolved by treatment with Triton X-100 with the release of previously entrapped fluorescent dyes being determined by fluorimetry. Both, ζ-potential and release experiments showed differences between silicified plain liposomes and silicified S-layer coated liposomes. The results of the individual preparation steps were examined by embedding the respective assemblies in resin, ultrathin sectioning and inspection by bright-field transmission electron microscopy (TEM). Energy filtered TEM confirmed the successful construction of S-layer based silica cages. It is anticipated that this approach will provide a key to enabling technology for the fabrication of nanoporous protein cages for applications ranging from nano medicine to materials science.

HNA-3a-specific antibodies recognize choline transporter-like protein-2 peptides containing arginine, but not glutamine at Position 154.

BACKGROUND: Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter-like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope. STUDY DESIGN AND METHODS: Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a-specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154. RESULTS: Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2 peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide. CONCLUSION: The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a-specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood.

Solid-phase synthesis and evaluation of glycopeptide fragments from rat epididymal cysteine-rich secretory protein-1 (Crisp-1).

Three 18-residue peptides with the sequence Glp-Asp-Thr-Thr-Asp-Glu-Trp-Asp-Arg-Asp-Leu-Glu-Asn-Leu-Ser-Thr-Thr-Lys, taken from the N-terminus of the rat epididymal cysteine-rich secretory protein (Crisp-1) that is important in the fertilization process, were prepared by Fmoc solid-phase synthesis using a convergent strategy. These peptides were the parent sequence, plus two possible α-O-linked T(N) antigen-containing glycopeptides with a Thr(α-D-GalNAc) residue in place of either Thr3 or Thr4. During chain assembly, two deletion peptides [des-Asp2 and des-Thr(Ac(3)-α-D-GalNAc)] and one terminated peptide [N-acetylated 14-mer] arose, as did several peptides in which aspartimide formation had occurred at each of the four possible positions in the sequence. These by-products totaled ~20% of the desired product; they were recognized by HPLC and ESI-MS and removed during the intermediate purifications. Final products, obtained in 15-21% overall yields, were characterized by HPLC purities and ESI-MS. Circular dichroism (CD) spectra for all three purified peptides, recorded in pure water and in trifluoroethanol-H(2)O (1:1), revealed that the presence of a sugar moiety does not significantly impact the sampled conformations. Future biological evaluation could elucidate the nature and locus of sugar modification of Crisp-1, and provide insight as to why Crisp-1 protein E binds sperm irreversibly, in contrast to protein D that lacks a sugar near the N-terminus and only binds sperm loosely.

Engineered carbohydrate-recognition domains for glycoproteomic analysis of cell surface glycosylation and ligands for glycan-binding receptors.

Modular calcium-dependent carbohydrate-recognition domains (CRDs) of mammalian glycan-binding receptors (C-type lectins), engineered to have novel glycan-binding selectivity, have been developed as tools for the study of glycans on cell surfaces. Structure-based specificity swapping between domains can be complemented by empirical characterization of ligand-binding specificity using glycan arrays. Both natural and modified CRDs can be used as probes for detecting and isolating glycoproteins that bear specific glycan epitopes and that act as target ligands for glycan-binding receptors. CRD-based affinity chromatography facilitates proteomic and glycomic analysis of such ligands.

Sulfated and non-sulfated glycopeptide recognition domains of P-selectin glycoprotein ligand 1 and their binding to P- and E-selectin.

Total synthesis through block glycosylation and selective chemical O-sulfation of tyrosine residues yielded the glycopeptide recognition domain A (X=SO(3) (-)) of the P-selectin glycoprotein ligand 1, in which the terminal sialic acid of the complex hexasaccharide side chain was replaced by (S)-cyclohexyl lactic acid. In binding assays the O-sulfated structure A showed high affinity towards P-selectin, the non-sulfated towards E-selectin.

Synthesis and antigenic analysis of the BclA glycoprotein oligosaccharide from the Bacillus anthracis exosporium.

The glycoprotein BclA is an important constituent of the exosporium of Bacillus anthracis spores. This glycoprotein is substituted with an oligosaccharide composed of a beta-L-rhamnoside substituted with the previously unknown terminal saccharide, 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-D-glucopyranose, also referred to as anthrose. Anthrose has not been found in spores of B. cereus and B. thuringiensis, making it a potential species-specific marker for B. anthracis. In order to study the antigenicity of anthrose, efficient syntheses of an anthrose-containing trisaccharide and a series of structurally related analogues were developed. The analogues lacked either the methyl ether at C-2 or contained modified C-4 amino functionalities of anthrose. The synthetic compounds were equipped with an aminopropyl spacer to facilitate conjugation to the carrier proteins mariculture Keyhole Limpet Hemocyanin (mcKLH) and bovine serum albumin (BSA). Serum antibodies of rabbits immunized with live or irradiated spores of B. anthracis Sterne 34F(2) were able to recognize the synthetic trisaccharide-mcKLH conjugate. The specificity of the interaction was confirmed by competitive inhibition with the free- and BSA-conjugated trisaccharides. Inhibition using the trisaccharide analogues demonstrated that the isovaleric acid moiety of anthrose is an important structural motif for antibody recognition. These data demonstrate that 1) anthrose is a specific antigenic determinant of the B. anthracis Sterne spore; 2) this antigen is presented to the immune system of rabbits receiving the anthrax live-spore vaccine; 3) synthetic analogues of the oligosaccharide retain the antigenic structure; and 4) the antigenic region is localized to specific terminal groups of the oligosaccharide. Collectively these data provide an important proof-of-concept step in the synthesis and development of spore-specific reagents for detection and targeting of non-protein structures in B. anthracis.

Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state.

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.

Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus.

This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1-Sp6), one in the S2 (Sp7) and four in the N protein (Np1-Np4) were synthesized and coupled to either diphtheria toxoid (dt) or biotin (bt). Bt-peptides were used to assess if selected regions were antigenic and contained B- or T-cell epitopes and dt-peptides if regions induced an antibody response and protection against virulent challenge. All S1 and S2 peptides were antigenic, being recognised by IBV immune sera and also induced an antibody response following inoculation into chicks. Three S1-and one S2-bt peptides also induced a delayed type hypersensitivity response indicating the presence of T-cell epitopes. The S2 peptide Sp7 (amino acid position 566-584) previously identified as an immundominant region, was the most antigenic of all peptides used in this study. Two S1 (Sp4 and Sp6) and one S2 peptide (Sp7), protected kidney tissue against virulent challenge. From four N peptides located in the amino-terminal part of the N protein, only one, Np2 (amino acid position 72-86), was antigenic and also induced a delayed type hypersensitivity response. None of the N peptides induced protection against virulent challenge. The results suggest that the S1 glycoprotein carries additional antigenic regions to those previously identified and that two regions located in the S1 and one in the S2 at amino acid positions 294-316 (Sp4), 532-537 (Sp6) and 566-584 (Sp7) may have a role in protection.

Characterization of the heptad repeat regions, HR1 and HR2, and design of a fusion core structure model of the spike protein from severe acute respiratory syndrome (SARS) coronavirus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emergent virus responsible for a worldwide epidemic in 2003. The coronavirus spike proteins belong to class I fusion proteins, and are characterized by the existence of two heptad repeat (HR) regions, HR1 and HR2. The HR1 region in coronaviruses is predicted to be considerably longer than that in other type I virus fusion proteins. Therefore the exact binding sequence to HR2 from the HR1 is not clear. In this study, we defined the region of HR1 that binds to HR2 by a series of biochemical and biophysical measures. Subsequently the defined HR1 (902-952) and HR2 (1145-1184) chains, which are different from previously defined binding regions, were linked together by a flexible linker to form a single-chain construct, 2-Helix. This protein was expressed in Escherichia coli and forms a typical six-helix coiled coil bundle. Highly conserved HR regions between mouse hepatitis virus (MHV) and SARS-CoV spike proteins suggest a similar three-dimensional structure for the two fusion cores. Here, we constructed a homology model for SARS coronavirus fusion core based on our biochemical analysis and determined the MHV fusion core structure. We also propose an important target site for fusion inhibitor design and several strategies, which have been successfully used in fusion inhibitor design for human immunodeficiency virus (HIV), for the treatment of SARS infection.

Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines.

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S(603-634), which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.

A facile synthetic method to prepare fluorescently labeled ROMP polymers.

[structure: see text] To probe the activities of sperm ADAM protein (fertilinbeta), we devised a general synthetic strategy to generate fluorescently labeled fertilinbeta oligopeptide polymers. Immunofluorescence studies with these polymers demonstrated that fertilinbeta polymers bind specifically to a protein receptor on the mouse egg plasma membrane.

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor.

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.

Structural characterization of the SARS-coronavirus spike S fusion protein core.

The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes. Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein. Our results show that in isolation the HR-N region forms a stable alpha-helical coiled coil that associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled coil. When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable alpha-helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916-950 of HR-N and residues 1151-1185 of HR-C. Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel. Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.

Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules.

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.

The (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-linker in the synthesis of glycopeptide partial structures of complex cell surface glycoproteins.

The (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL) linker represents a novel fluoride-sensitive anchor for the solid-phase synthesis of protected peptides and glycopeptides. Its cleavage is achieved under almost neutral conditions using tetrabutylammonium fluoride trihydrate in dichloromethane thus allowing the construction of complex molecules sensitive to basic and acidic media commonly required for the cleavage of standard linker systems. The advantages of the PTMSEL linker are demonstrated in the synthesis of glycopeptides from the liver intestine (LI)-cadherin and the mucin MUC1, bearing carbohydrate moieties such as N-linked chitobiose or O-linked sialyl-T(N)-residues. The synthesis of these types of glycopeptides is difficult because they are prone to secondary structure formation during the synthesis on the solid phase as well as in the completely deprotected form. Using the PTMSEL linker these molecules are accessible by automated synthesis according to the Fmoc strategy without frequently observed side reactions such as aspartimide or diketopiperazine formation.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter: Part 3. A potential 5-HT transporter imaging agent, 3-(3-[18F]fluoropropyl)-6-nitroquipazine.

3-(3-[18F]Fluoropropyl)-6-nitroquipazine ([18F]FPNQ) as a 5-HT transporter imaging agents was designed, synthesized, and evaluated. FPNQ was selected due to its potent in vitro biological activity (K(i)=0.32 nM) in rat brain cortical membranes. The 18F-labeled FPNQ was prepared by reaction of the propyl mesylate as a precursor with tetra-n-butylammonium [18F]fluoride generated under NCA conditions. The precursor mesylate was synthesized from commercially available hydrocarbostyril in nine steps in 21% overall yield. The specific activity of the [18F]FPNQ determined by radioreceptor assay was 27.0 GBq/micromol. Tissue distribution studies in mice showed the highest uptake in the frontal cortex (5.79 %ID/g) at 60 min post-injection.

Synthetic glycopeptides for the development of tumour-selective vaccines.

Based on structural information reported for the tumour-associated epithelial mucin MUC1, glycopeptides have been synthesized which contain tumour-associated saccharide antigens. such as the Thomsen-Friedenreich (T), TN or sialyl TN antigen. in combination with peptide sequences of the tandem repeat region of MUC1. Solid-phase syntheses have been carried out using N-Fmoc protected O-glycosyl serine and threonine building blocks and an allylic anchor which is stable to basic and acidic conditions, but can be cleaved under neutral conditions in a palladium(0)-catalysed allyl transfer reaction. In addition. a (2-3)sialyl T antigen threonine building block was prepared by a chemoenzymatic strategy and used in the synthesis of an N-terminal glycopeptide antigen of leukosialin (CD43). The proliferation of cytotoxic T cells could be induced using a construct consisting of a MUC1-glycopeptide antigen and a T cell epitope.

First total synthesis of a GPI-anchored peptide.

A GPI-anchored dipeptide of sperm CD52 antigen was prepared through a convergent synthesis. First, the dipeptide with its C-terminus free and the GPI with its nonreducing end phosphoethanolamine bearing a free amino group were synthesized separately. Then, the two building blocks were coupled with use of EDC/HOBt as the condensation reagent. Finally, the GPI-anchored peptide was deprotected to give the target molecule 1.

Evaluation of the performance of immobilized penicillin G acylase using active-site titration.

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.