Synthetic Peptide Constructs Research Articles

Monoclonal antibody 7H2.2 binds the C-terminus of the cancer-oocyte antigen SAS1B through the hydrophilic face of a conserved amphipathic helix corresponding to one of only two regions predicted to be ordered.

The structure of the antigen-binding fragment (Fab) of mouse monoclonal antibody 7H2.2 in complex with a 15-residue fragment from the metalloproteinase sperm acrosomal SLLP1 binding protein (SAS1B), which is a molecular and cellular candidate for both cancer therapy and female contraception, has been determined at 2.75 Å resolution by single-crystal X-ray diffraction. Although the crystallization conditions contained the final 148 C-terminal residues of SAS1B, the Fab was observed to crystallize in complex with a 15-residue fragment corresponding to one of only two elements of secondary structure that are predicted to be ordered within the C-terminal region of SAS1B. The antigen forms an amphipathic α-helix that binds the 7H2.2 combining site via hydrophilic residues in an epitope that spans the length of the antigen α-helix, with only two CH-π interactions observed along the edge of the interface between the antibody and antigen. Interestingly, the paratope contains two residues mutated away from the germline (YL32F and YH58R), as well as aProH96-ThrH97-AspH98-AspH99 insertion within heavy chain CDR3. The intact 7H2.2 antibody exhibits high affinity for the SAS1B antigen, with 1:1 binding and nanomolar affinity for both the SAS1B C-terminal construct used for crystallization (3.38 ± 0.59 nM) and a 15-amino-acid synthetic peptide construct corresponding to the helical antigen observed within the crystal structure (1.60 ± 0.31 nM). The SAS1B-antibody structure provides the first structural insight into any portion of the subdomain architecture of the C-terminal region of the novel cancer-oocyte tumor surface neoantigen SAS1B and provides a basis for the targeted use of SAS1B.

Multiple Antimicrobial Effects of Hybrid Peptides Synthesized Based on the Sequence of Ribosomal S1 Protein from Staphylococcus aureus

The need to develop new antimicrobial peptides is due to the high resistance of pathogenic bacteria to traditional antibiotics now and in the future. The creation of synthetic peptide constructs is a common and successful approach to the development of new antimicrobial peptides. In this work, we use a simple, flexible, and scalable technique to create hybrid antimicrobial peptides containing amyloidogenic regions of the ribosomal S1 protein from Staphylococcus aureus. While the cell-penetrating peptide allows the peptide to enter the bacterial cell, the amyloidogenic site provides an antimicrobial effect by coaggregating with functional bacterial proteins. We have demonstrated the antimicrobial effects of the R23F, R23DI, and R23EI hybrid peptides against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus. R23F, R23DI, and R23EI can be used as antimicrobial peptides against Gram-positive and Gram-negative bacteria resistant to traditional antibiotics.

Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two NaV1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

A SARS-CoV-2 Vaccination Strategy Focused on Population-Scale Immunity.

Here we propose a vaccination strategy for SARS-CoV-2 based on identification of both highly conserved regions of the virus and newly acquired adaptations that are presented by MHC class I and II across the vast majority of the population, are highly dissimilar from the human proteome, and are predicted B cell epitopes. We present 65 peptide sequences that we expect to result in a safe and effective vaccine which can be rapidly tested in DNA, mRNA, or synthetic peptide constructs. These include epitopes that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor, and within a novel furin cleavage site thought to increase membrane fusion. This vaccination strategy specifically targets unique vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the human population.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Reversal of Aging-Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein-Protein Interfaces.

BackgroundThe proximal aorta normally functions as a critical shock absorber that protects small downstream vessels from damage by pressure and flow pulsatility generated by the heart during systole. This shock absorber function is impaired with age because of aortic stiffening.Methods and ResultsWe examined the contribution of common genetic variation to aortic stiffness in humans by interrogating results from the AortaGen Consortium genome‐wide association study of carotid‐femoral pulse wave velocity. Common genetic variation in the N‐WASP (WASL) locus is associated with carotid‐femoral pulse wave velocity (rs600420, P=0.0051). Thus, we tested the hypothesis that decoy proteins designed to disrupt the interaction of cytoskeletal proteins such as N‐WASP with its binding partners in the vascular smooth muscle cytoskeleton could decrease ex vivo stiffness of aortas from a mouse model of aging. A synthetic decoy peptide construct of N‐WASP significantly reduced activated stiffness in ex vivo aortas of aged mice. Two other cytoskeletal constructs targeted to VASP and talin‐vinculin interfaces similarly decreased aging‐induced ex vivo active stiffness by on‐target specific actions. Furthermore, packaging these decoy peptides into microbubbles enables the peptides to be ultrasound‐targeted to the wall of the proximal aorta to attenuate ex vivo active stiffness.ConclusionsWe conclude that decoy peptides targeted to vascular smooth muscle cytoskeletal protein‐protein interfaces and microbubble packaged can decrease aortic stiffness ex vivo. Our results provide proof of concept at the ex vivo level that decoy peptides targeted to cytoskeletal protein‐protein interfaces may lead to substantive dynamic modulation of aortic stiffness.

Nanoporous Hydrogels for the Observation of Anthrax Exotoxin Translocation Dynamics.

The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements. Here, we utilize nanoporous hydrogel membranes to assess the translocation of full-length anthrax lethal and edema factors through nanopores similar in diameter to protective antigen translocons. We find that, relative to globular serum and other proteins that do not translocate natively through nanopores, anthrax exotoxins demonstrate significantly reduced barriers to pore entry. Computed free-energy barriers to the unfolding of proteins and the dissociation of macromolecular complexes are generally found to coincide with translocation. Finally, a nanopore-blocking strategy is developed that utilizes nonspecific synthetic peptide constructs and effectively prevents LF translocation within the nanoporous hydrogel.

The way to the peptide vaccine against hepatitis C

In order to overcome the problem of genetic variability of hepatitis C virus (HCV) envelope proteins during vaccine development, we have used the so-called “reverse vaccinology” approach defined as “from genome to vaccine.” In the context of this approach we have created a database of amino acid sequences of HCV proteins, performed an analysis of the viral genome, and identified several highly conserved regions in HCV envelope proteins. These regions demonstrated low antigenic activity within full-length proteins and viral particles: antibodies against these regions were found not in all hepatitis C patients. However, among highly conserved regions, two regions, containing a wide set of potential T-helper epitope motifs were identified. Using solid-phase peptide synthesis we have generated several synthetic peptide constructs, which consist of two highly conservative sites of the HCV envelope protein E2 joint by a linker; one of these sites contained a set of T-helper epitope motifs. In experiments on laboratory animals the resultant peptide constructs exhibited immunogenicity comparable to the immunogenicity of the protein molecules and were able to induce generation of antibodies, which specifically bound HCV envelope proteins. Immunization with certain constructs resulted in generation of antibodies that were able to bind HCV from the blood plasma of hepatitis C patients. Based on these peptide constructs an original preparation of the synthetic peptide vaccine against hepatitis C is now under development.

Synthetic peptides reproducing tissue transglutaminase-gliadin complex neo-epitopes as probes for antibody detection in celiac disease patients' sera.

Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients' sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients' sera before gluten-free diet. The peptide II, containing Ac-tTG(553-564)-NH2 sequence cross-linked with deamidated Ac-α2-Glia(63-71)-NH2, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553-564)-NH2 and Ac-α2-Glia(63-71)-NH2 and the corresponding cross-linked peptide II were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments.

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.

Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T‐helper epitopes

We describe a peptide-based strategy for hepatitis C virus (HCV) vaccine design that exploits synthetic peptides representing antibody epitopes of the hypervariable region 1 (HVR1) of the E2 glycoprotein and also less variable regions immediately downstream of HVR1. These epitopes were linked to a T-helper (T(h)) epitope (KLIPNASLIENCTKAEL) derived from the Morbillivirus canine distemper virus. Antibody titres induced by the two vaccine candidates T(h)-A (E2 amino acid 384-414) and T(h)-B (E2 amino acid 390-414) were significantly higher than those produced against vaccines lacking the T(h) epitope (P<0.05). Mice inoculated with the vaccine candidates T(h)-C (E2 amino acids 412-423) and T(h)-F (E2 amino acids 436-447) emulsified in complete Freund's adjuvant each elicited antibody titres that were significantly higher than those elicited by T(h)-E (E2 amino acids 396-407) and T(h)-D (E2 amino acids 432-443) (P<0.01). Antisera obtained from mice inoculated with the epitope vaccines T(h)-A, T(h)-B, T(h)-D and T(h)-E bound to E2 expressed at the surface of 293T cells that had been transfected with E1E2. Furthermore, IgG from the sera of mice inoculated with four of the vaccine candidates, T(h)-A, T(h)-C, T(h)-D and T(h)-E, inhibited the entry of HCV/human immunodeficiency virus pseudoparticles (HCVpps) into Huh-7 cells. These results demonstrate the potential of synthetic peptide-based constructs in the delivery of potential neutralizing epitopes that are present within the viral envelope of HCV.

Design of Peptide-Based Vaccines for Cancer

The immune system responds efficiently to bacteria, viruses and other agents however, the immune response to cancers is not as effective. In most cases other than specific genetic rearrangements leading to non-self proteins such as in leukemia and idiotypes in lymphoma, tumor associated proteins are self proteins and are not recognized by the immune system to prevent malignancy. In most cancers, patients develop antibodies and/or CTL-precursors to tumor associated antigens but are not effective in generating a therapeutic immune response. Adjuvants have been used with either whole tumors, subunits or peptides with the aim of increasing their immunity. Whole tumor antigens have certain advantages associated with it, such as ready availability as recombinant proteins, potential epitopes that can be presented by a number of MHC class I/II alleles and antibody development. The methods of identification of CD8 and CD4 epitopes either by use of epitope prediction algorithms or use of transgenic mice has made the use of defined synthetic peptides more attractive. The possibility to synthesize long peptides and introduce multiple epitopes (CD4 or CD8) from single or multiple antigens makes peptide a viable alternative to whole proteins. As an alternative to totally synthetic peptide constructs or polymers, polytopes have been generated by genetic engineering methods. In addition, to deliver immunogens to and to activate DC, receptor-mediated delivery of peptides using antibodies, cytokines and carbohydrates have been used. This review will encompass the various strategies, preclinical and clinical applications in designing peptide-based vaccines for cancer.

Peptide immunogens based on the envelope region of HIV-1 are recognized by HIV/AIDS patient polyclonal antibodies and induce strong humoral immune responses in mice and rabbits

Peptide immunogens produced by novel synthesis techniques and based on envelope proteins have repeatedly proven successful in inducing anti-HIV and anti-SIV responses in addition to producing anti-peptide immunity [Vaccine 12 (8) (1994) 736; AIDS Res. Hum. Retroviruses 14 (9) (1998) 751; Vaccine (2002) 2680]. We report here the design and evaluation of synthetic peptide constructs mimicking the variability of the third variable (V3) loop, or representing the conservation of the CD4 region of HIV-1 envelope. Three peptides based on the V3 region of HIV-1 subtype C gp120 and designated multiple epitope immunogens (MEI) differ in that one was randomly branched containing a spacer (b-MEI-s), another conjugated to a tetrameric lysine core (MEIV3b 4) and the third conjugated to poly- l-lysine (poly- l-MEI). The method of synthesis employed produced peptides that were either linear, dimeric or tetramerically branched thereby providing differing levels of conformation. In addition the peptides also differed in their levels of variability. A fourth peptide designated B138 was based on a conserved region of HIV-1 envelope. The aim of this work was to evaluate the effect of variability and level of conformation on the immunogenicity of variable region peptides (MEI’s) when compared to that of a known immunogenic and conserved region peptide (B138). Anti-peptide humoral immunity induced in BALB/c mice and New Zealand White rabbits produced antigen-recognizing antibody titres of ≤5000 in enzyme linked immunosorbent assays (ELISA) and stimulation indices (SI) of 7 in cell proliferation assays. Plasma polyclonal antibodies collected from HIV/AIDS patients (from South Africa, Botswana and Puerto Rico) recognized the peptide constructs at antibody titres of ≤5000. As demonstrated by mouse, rabbit and human antibody and proliferation responses, the immunogenicity and antigenicity of the peptides varied, with MEIV3b 4 responding better than the other three, at times even surpassing the conserved region peptide B138.

Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2

Matix protein 2 (M2) is a transmembrane protein of influenza type A virus. It contains a 23 aa long ectodomain (M2e) that is highly conserved amongst human influenza type A viruses. M2e-specific antibodies have been shown to restrict virus growth in vitro and in vivo and thus have the potential of providing cross-reactive resistance to influenza type A virus infection. We attempted to induce M2e-specific protection with synthetic multiple antigen peptide (MAP) constructs that contained covalently linked M2e- and Th-determinant peptides. Mice, vaccinated twice by the intranasal (i.n.) route with adjuvanted M2e–MAPs exhibited significant resistance to virus replication in all sites of the respiratory tract. Compared to mice primed by two consecutive heterosubtypic infections, resistance was of similar strength in nasal and tracheal tissue but lower in pulmonary tissue. Importantly, the protection in M2e–MAP- and infection-immunized mice appeared to be mediated by distinct immune mechanisms. This suggests that stronger protection may be achievable by combining both protective activities.

Identification of antigenically active tryptic fragments of apical membrane antigen-1 (AMA1) of Plasmodium chabaudi malaria: strategies for assembly of immunologically active peptides

Apical membrane antigen-1 (AMA1) is a prime vaccine candidate for inclusion in a vaccine against malaria. It is known that the disulphide bond stabilised conformation of this antigen is important for eliciting a protective antibody response, however little is known about the epitopes within this molecule that are targeted by the immune response. We have used a peptide approach for the identification and characterisation of such regions. In this study, the in vitro refolded, recombinant ectodomain of AMA1 from the D strain of Plasmodium chabaudi adami, was digested with trypsin and individual peptide fragments examined for antigenic activity. We found that a tryptic fragment, which was derived from a loop-like structure within the putative domain I of the intact AMA1 molecule, was highly reactive with antibodies from the sera of hyperimmune mice. Two different synthetic peptide constructs incorporating this antigenically active fragment were assembled. The first consisted of two separate peptide chains which were linked through a disulphide bond formed using chemo-selective chemistry. A larger 45-mer loop peptide, generated by the oxidation of two cysteine residues close to the N- and C-termini of the 45-mer, represented the complete loop structure and incorporated the tryptic fragment. Each peptide construct was also able to elicit production of high titres of antibodies in mice and furthermore, the 45-residue loop peptide elicited antibodies capable of binding to AMA1 with titres comparable to those present in a mouse which had recovered from multiple exposures to P. chabaudi adami parasites. Passive immunisation with anti-loop antibodies did not suppress the development of parasitaemia in mice challenged with P. chabaudi adami suggesting that although highly immunogenic, the peptides represented inadequate or inappropriate epitopes for vaccination purposes.

Branched synthetic peptide constructs mimic cellular binding and efflux of apolipoprotein AI in reconstituted high density lipoproteins

This study investigates the suitability of the trimeric apolipoprotein (apo) AI(145–183) peptide that we recently described, to serve as a model to probe the relationship between apo AI structure and function. Three copies of the apoAI(145–183) unit, composed each of two amphipatic α-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102–140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)–apoAI complexes. The DMPC–trimeric-apoAI(145–183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149–186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145–183) bind specifically to HeLa cells with comparable affinity to the DMPC–apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145–183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145–183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102–140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102–140) construct had low affinity for lipid compared with the (145–183) construct. After association with lipids, it was a poor competitor of DMPC–apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure–function relationship of apoAI.

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.

Pseudopeptide TASP Inhibitors of HIV Entry Bind Specifically to a 95-kDa Cell Surface Protein

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction.

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid. Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides.