Biomedical peptides, proteins & nucleic acids : structure, synthesis & biological activity最新文献

The fine specificity of two different monoclonal antibodies raised against synthetic peptides, each representing one of the two Arg-Gly-Asp (RGD) sequences in fibrinogen, was examined using synthetic combinatorial libraries (SCLs). The monoclonal antibodies (mAb), mAb LJ-134B/29 and mAb LJ-155B/16, recognize both the immunogenic peptide and native fibrinogen. The specificity of mAb LJ-134B29 was mapped using hexa- and decapeptide positional scanning SCLs (PS-SCLs) and competitive ELISA. The most active amino acids at each position of the two libraries were identified from a single screening. Individual hexa- and decapeptides were synthesized and assayed to determine their binding affinities. The 16 individual hexapeptides represented single and multiple substitutions of the antigenic determinant sequence, -GDSTFE-, eight of which had affinities less than 10nM. Four of the twelve individual decapeptides were found to have binding affinities of approximately 300nM, or nearly three-fold less than the peptide immunogen. A dual-defined hexapeptide library was screened against mAb LJ-155B/16, and individual peptides were obtained through an iterative selection and synthesis process. Surprisingly, one of the most active sequences was Ac-WWYESW-NH2 (IC50 = 40nM), which showed no similarity to the sequence of the immunizing peptide. Further mapping of the specificity of this antibody revealed that the antigenic determinant within the peptide immunogen was not completely linear. Recognition of this unrelated sequence by mAb LJ-155B/16 was confirmed in a direct binding assay using biotinylated peptide. The use of SCLs for the elucidation of high affinity peptides recognized by these two antibodies may provide additional information on the molecular mechanisms of fibrinogen binding to different integrin receptors.

Synthetic peptides are frequently used to mimic the antigenic sites of proteins. In order to increase the level of mimicry between the peptide and the protein, it is important to understand the structural basis of protein antigenicity. A review of recent crystal structures of antigen-antibody complexes shows that important conformational rearrangements occur in both antigen and antibody during complexation and that many water molecules are located at the complex interface. Both these features are responsible for the low success rate of antigen-antibody docking. The complementarity observed in the complex cannot be predicted from the structure of the free molecules before the occurrence of induced fit and mutual adaptation. The structuralist paradigm assumes that it is possible to understand complex biological recognition phenomena solely in terms of structural data. However, the dynamic component of protein structure requires that both space and time dimensions be included in the description of antigenic specificity. This means that both structural data and activity measurements are required for understanding immunological interactions and for designing synthetic epitopes. The recently developed biosensor technology should greatly facilitate the quantitative measurement of binding interactions and the design of synthetic peptide epitopes.

Human T-lymphotropic virus type 1 (HTLV-I) causes adult T-cell leukemia/lymphoma (ATLL) and has been associated with a variety of immunologically-mediated diseases. Recently, the immunodominant epitopes of HTLV-I have begun to be defined through the utilization of synthetic peptides and recombinant proteins. Strategies to define the conformational features of immunogenic peptides and design chimeric and multivalent constructs that mimic native viral proteins have provided the opportunity to create an effective synthetic vaccine against HTLV-I infection. An ideal peptide vaccine to be universally immunogenic must incorporate rationally designed antigenic determinants that accurately mimic the corresponding structural architecture found in native proteins and elicit relevant components of the immune system. We have recently designed and tested chimeric and beta-sheet template constructs containing HTLV-I immunodominant peptide motifs that elicit neutralizing antibody responses and overcome genetically restricted immune responses. To further illustrate putative vaccine candidates, HTLV-I env and tax proteins were analyzed using various computer-predicted correlates of protein antigenicity, secondary structural predictions, and major histocompatibility complex class I binding motifs. These approaches provide the opportunity to design synthetic peptide vaccines against HTLV-I infection that are based on structurally defined criteria, as well as test the influence of glycosylation on peptide conformation and immunogenicity.

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.

Four synthetic lipopeptides, (K-pm 19,31), (K-pm 19,21,31), (K-pm 19,28,31) and (K-pm 19,21,28,31) with the lysine-palmitoyl (K-pm) residue as a lipophilic moiety, based on the pseudosubstrate sequence 19RFARKGALRQKNV31 (R19-V31), were found to be potent protein kinase C (PKC) inhibitors. However, the lipopeptides (K-pm 19,21,31), (K-pm 19,28,31) and (K-pm 19,21,28,31) were also found to act as protein kinase cAMP-dependent (PKA) inhibitors. Peptide (K-pm 19,31), the least water soluble, is marginally selective towards PKC, unlike the other palmitoyl derivatives studied here. Since the non-palmitoylated analogues (K 19,31), (K-ac 19,31), (K 19,21,31) and (K-ac 19,21,31) were inhibitors of PKC but not of PKA, the palmitoyl moiety must play a role in the specificity of protein kinase inhibition. In vitro, the lipophilic peptides showed greater stability to protease-mediated hydrolysis than the pseudosubstrate peptide depending upon the number of lipophilic (K-pm) residues. CD studies showed that in comparison with the peptide analogues, the remarkable resistance of the pseudosubstrate (R19-V31) to adopt an alpha-helix conformation in TFE, known to be strongly alpha-helix inducing, rules out this structure as the peptide binding conformation to PKC. By contrast, in aqueous media all the peptides show an extended conformation that correlates well with their inhibitory activity. This is in compliance with the crystallographic observation that an extended structure has been observed for the (5-24) PKI peptide inhibitor bound to PKA.

Presentation of T-cell determinants to the immune system in multimeric form has clear advantages and the production of synthetic peptide-based polymers using the solubilisable KS resin described by Goddard et al. [1] provides a method of assembling such polymers and also offers the means for making heteropolymers. The present study investigates the potential of polymeric synthetic peptide constructs in eliciting proliferative T-cell responses to determinants of the influenza virus hemagglutinin. The induction of vigorous CD4+ T-cell immunity was achieved with a polymeric construct containing two different T-cell determinants. The data presented here also highlight the fact that distancing the determinant from the support backbone with appropriate amino acid residues is an important consideration for the success of these polymeric immunogens. This approach may be readily applied in other systems where induction of helper T-cell responses are required.

In order to elucidate structural and biological properties required for an optimal immunological carrier function and to provide a rational basis for its selection, two new groups of synthetic branched polypeptides with a general formula poly[Lys-(X(i)-DL-Ala(m))][XAK] or poly[Lys-(DL-Ala(m)-X(i))][AXK], where m approximately 3 and i < 1 were introduced by our laboratory. Here we review our recent results on the application of these polypeptides as biodegradable carriers for constructing synthetic immunogens/antigens with a well-known phenyl oxazolone hapten, peptide epitopes of epithelial mucin [MUCI] or herpes simplex virus [HSV 1] glycoprotein D. Observations collected during the last five years with the conjugates presented serve to illustrate the usefulness of branched polypeptides as carriers for the rational design of synthetic immunogens for the development of vaccines or clinically relevant immunodiagnostics. Furthermore, this polypeptide model system enables the analysis and potentially reliable interpretation of the correlation between chemical structure and immunogenic/antigenic features.

Treatment of 2'-O-silyl-oligoribonucleotides with triethylamine trihydrofluoride in DMF at 55 degrees C for 1 h effected complete desilylation. The product was isolated by a single addition of 1-butanol to the reaction mixture. The resulting RNA was found to be identical with that obtained by traditional desilylation methods as analyzed by HPLC, enzyme digest and ribozyme-substrate assays.

The identification and characterization of epitopes of human T-lymphotropic virus type 1 (HTLV-I), which elicit an effective humoral or cell-mediated immune response, remains a central obstacle to the development of a peptide-based vaccine against the virus infection. The objective of the studies presented here was to examine the influence of N-linked glycosylation on peptide structure and immunogenicity. We engineered the 233-253 sequence of gp46 of HTLV-I to contain an N-acetylglucosamine (GlcNAc) residue at Asn244. Secondary structure prediction using computer algorithms indicated that this peptide may contain a beta-turn at residues 242-246. Recent work with model glycopeptides suggests that beta-turn conformation in peptides may be induced, and probably is stabilized, by the presence of even a single sugar residue. In the present study, the structures of the 233-253 peptide, SC1, and the 233-253(Asn244-GlcNAc) glycopeptide, SC2, were determined. Similar conformation was exhibited by both the glycosylated and nonglycosylated peptide displaying a beta-turn at residues 243-246 and extended-chain structure at the peptide/glycopeptide termini. Both peptides were engineered into chimeric constructs with a promiscuous T-cell epitope from measles virus and were used as immunogens in rabbits. Both chimeric peptides were highly immunogenic in rabbits, producing high-titered antibodies as early as primary + three weeks. The antibodies generated against either construct were able to bind to whole virus (ELISA) and to gp46 (radioimmunoprecipitation assay). Additionally, human sera of individuals known to be positive for HTLV-I recognized both the glycosylated and nonglycosylated constructs. It appears that the 233-253 peptide is able to adopt a conformation that mimics the structure in native gp46, and addition of a GlcNAc residue at Asn244 does not affect the conformational preference or stability of this construct; nor does glycosylation alter immunogenicity but instead appears to enhance immune recognition.

Conformationally restricted cyclic peptide mimics of the antigenic site A of foot-and-mouth disease virus serotype C-S8c1 have been designed, first by comparison to the three-dimensional structure of the O1BFS serotype, later more accurately on the basis of X-ray diffraction data from a complex between a linear peptide reproducing site A and an FMDV-derived monoclonal antibody Fab fragment. A variety of cyclization strategies have been attempted, both in solution and in the solid phase, involving disulfide, side chain lactam and head-to-tail arrangements. Preliminary immunological results have shown one of the cyclic disulfide mimics to be a better immunogen than its linear counterpart.