Dimerization of the immunosuppressive peptide fragment of HLA-DR molecule enhances its potency

Our previous studies revealed that the nonapeptide fragment of HLA-DR molecule, located in the beta chain 164-172 with the VPRSGEVYT sequence, suppresses the immune responses. The sequence is located on the exposed molecule loop, therefore it may be involved in the interactions with other proteins. We suggested that the loop may serve as a functional epitope on the HLA class II surface for intermolecular binding, and that possible mechanism of biological action of the synthesized peptides is associated with interfering of adhesion of HLA class II molecules to their coreceptors. It has been postulated that oligomerization of the coreceptors is required for stable binding to class II HLA. Based on the crystal dimeric structure of HLA-DR molecules, we designed, and synthesized molecules able to induce the putative coreceptors dimerization. The synthesized series of compounds consisted of two VPRSGEVYT sequences linked through their C-termini by spacers of different length: (VPRSGEVYTGn)2K-NH2 ( n = 4-6). The results demonstrate that the dimerization of the nonapeptide fragment of HLA-DR results in enhanced immunosuppressory properties.     

Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N‐terminus to N‐terminus, C‐terminus to C‐terminus, and N‐terminus to C‐terminus (head‐to‐tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α‐amino groups of the C‐terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head‐to‐tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16-21 and ubiquitin 52-57 fragments, and second designed to mimic the ubiquitin 5-13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.     

New conformationally restricted analog of the immunosuppressory mini-domain of HLA-DQ and its biological properties

Our previous studies revealed that the nonapeptide fragment of HLA-DQ located in the beta 164-172 loop of the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr sequence suppresses the immune humoral and cellular responses [30]. Based on the crystal structure of HLA-class II molecules we designed and synthesized a cyclic analog with restricted conformation, cyclo(Suc-Thr-Pro-Gln-Arg-Gly-Asp-Val-Lys)-Thr-OH (Suc = succinyl) by reacting a Lys side chain with a succinylated N-terminus. The cyclization product more potently suppresses the cellular immune response than its linear counterparts and is efficiently cleaved by trypsin. The results indicate that the beta 164-172 loop may serve as a functional epitope on the HLA class II surface for intermolecular binding.     

A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16–21 and ubiquitin 52–57 fragments, and second designed to mimic the ubiquitin 5–13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.     

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti‐inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin^50–59 sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well‐defined conformation in methanol, its structure was distinct from the corresponding 50–59 fragment in the native ubiquitin molecule. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 423–431, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: energetics of peptide association and folding

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.     

Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab

The crystal structure of an intact molecule of HIV-1 capsid protein (p24) in complex with a monoclonal antibody fragment recognizing an epitope on the C-terminal domain has been determined at 3 A resolution. The helical N- and C-terminal domains of p24 are linked by an extended peptide forming a flexibly linked dumb-bell-shaped molecule 75 A in overall length. The p24 construct used is a variant with an N-terminal extension that mimics to some extent the Gag context of p24. We observed a novel head-to-tail dimer of p24 molecules which occurs through the formation of a substantial intermolecular interface between the N- and C-terminal domains. Comparison with previously observed p24 dimers shows that the same residues and secondary structural elements can partake in different interfaces revealing a remarkable stickiness and plasticity of the p24 molecule, properties which, combined with the inter-domain flexibility, are presumably important in the assembly and maturation of viral particles. Previous mutagenesis studies designed to test specific N-N and C-C homodimer interfaces do not discriminate fully against the possibility of the observed N-C interface.     

A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.     

Epitope mapping of the phosphorylation motif of the HIV-1 protein Vpu bound to the selective monoclonal antibody using TRNOESY and STD NMR spectroscopy

The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.     

Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies

Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression system is generally applicable for biosynthesis of V3-related peptides and was also used to prepare the V3JR-FL. The 447-52D Fab fragment was obtained by a short enzymatic papain cleavage of the whole antibody. Preliminary NMR spectra demonstrate that full structural analysis of the V3MN complexed to the 447-52D Fab is feasible. This system enables studies of the same epitope bound to different HIV-1 neutralizing antibodies.     

Synthesis and immunochemical properties of peptides corresponding to sequences 59-72 and 25-36 of human interleukin-2

Synthesis of peptides corresponding to the 59-72 and 25-36 sequences of human IL-2 is reported. The former peptide, which had been shown to be immunogenic in the protein molecule, was prepared to obtain antipeptide antibodies for isolation and purification of the recombinant IL-2. We located the epitope at the C-terminus of this peptide. In accordance with the IL-2 secondary structure and hydrophilicity profile analysis, the 25-36 fragment was chosen as the potential epitope. The peptides were synthesized by conventional methods in solution, conjugated with a protein carrier, and polyclonal rabbit antisera were obtained. Antibodies against both peptides were shown to be specific to human IL-2 in ELISA. Besides, monoclonal antibodies to IL-2 recognized in ELISA the 59-72 peptide, suggesting the epitope located in this region to be main one in the protein molecule.     

Immunosuppressory activity of the cyclodimeric peptide with RGD-sequences.

Our previous studies showed that the nonapeptide fragment of HLA-DQ of the sequence H-Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr-OH, located in the beta164-172 loop, strongly suppresses the humoral and cellular immune responses, while its shorter analogs, H-Arg-Gly-Asp-Val-OH, H-Arg-Gly-Asp-Val-Tyr-OH and H-Gln-Arg-Gly-Asp-Val-Tyr-OH show only a weak stimulatory activity in respect to the humoral immunological response. These fragments contain the Arg-Gly-Asp (RGD) sequence, known for its importance for cellular association phenomena. Based on the crystal structure of HLA-DR1, we also designed and synthesized a cyclic analog H-Cys-Arg-Gly-Asp-Val-Tyr-Cys-OH with restricted conformation, which strongly suppresses the immune response and selectively inhibits the alphavbeta3 integrin, suggesting that the mechanism of the immunosuppressory action of the peptide is associated with inhibition of the integrin. In this paper we present the design and synthesis of the cyclodimeric peptide, Arg-Gly-Asp-Arg-Gly-Asp, which is also known as a selective alphavbeta3 inhibitor. The synthesized peptide strongly suppresses both the humoral and cellular immune response. The results support our hypothesis that the immunomodulatory activity of HLA-DQ fragments may be connected with their interactions with some particular integrins on the cell surface.     

Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.     

Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48.

The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).     

Investigation of the solid- and solution-phase binding reactivities of continuous epitopes recognized by polyclonal guinea-pig anti-recombinant bovine growth hormone antisera.

We have used the technique of multiple pin peptide synthesis to identify three major continuous epitopes in the recombinant bovine (rb) GH molecule. We have synthesized these peptides, residues 24-40, 139-152 and 179-189, as N-terminally acetylated, C-terminal amides and confirmed their reactivity in a standard solid-phase ELISA. Subsequently, for epitope 139-152, we have synthesized a peptide affinity column and used this to isolate antibodies with this epitope specificity from whole antiserum. In addition, we demonstrate that under native conditions in a liquid phase RIA, these antibodies will precipitate [125I]rbGH. Further, peptide 139-152 itself also cross-reacts in an rbGH RIA inhibiting binding by up to 20%. Our data suggest that during the immune response to rbGH in guinea-pigs a substantial part of the B-cell response is directed to the 139-152 region and that this part of the protein is a native epitope.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

The C-terminal binding domain of hirullin P18. Antithrombin activity and comparison to hirudin peptides

Hirullin P18 is a 61-amino acid hirudin-related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C-terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C-terminal fragment acetyl-hirullin P18(41-62) [corrected] possesses an antithrombin potency similar to that of acetyl-desulfatohirudin(45-65). Additionally, like the hirudin fragment analog, it inhibits fibrin-clot formation by binding to a non-catalytic site on thrombin. Sequential shortening of the hirullin P18 C-terminal fragment demonstrates the critical nature of Phe51, which corresponds to the important Phe56 residue of hirudin. Although the sequences of hirullin P18(54-61) and hirudin(59-65) have substantial differences, the C-terminal functional domain represented by hirullin P18(50-61) appears to be comparable to hirudin(55-65) in terms of its functional role in antithrombin activity.     

HIV-1 encoded virus protein U (Vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites Ser52 and Ser56

Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.     

Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin.

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.