The role of structure in antibody cross-reactivity between peptides and folded proteins

Peptides have the potential for targeting vaccines against pre-specified epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced fit of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with sub-micromolar affinities. The strong preference for a type II beta-turn predicted for one peptide was confirmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.     

Can active immunization redirect an anti-IgE immune response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.     

Perceived importance of clinical teaching characteristics for nurse anesthesia clinical faculty

Cancer-related, mucin-type carbohydrate epitopes, principally mannose and sialo-syl residues, are expressed on the envelope protein gp 160 of the human immunodeficiency virus (HIV). Anticarbohydrate antibodies directed toward these and other carbohydrate epitopes are known to neutralize HIV-1 infection by cell-free virus. Carbohydrates, however, being T cell-independent antigens, typically elicit diminished immune responses. To overcome this potential draw back, we have examined the ability of peptides that mimic such epitopes to elicit immune responses that cross-react with carbohydrate structures. We report that mouse polyclonal antisera generated against peptides that mimic mucin-related carbohydrate epitopes have anti-HIV-1 activity. Generation of antibodies was not lr-gene restricted, as at least two different strains of mice. Balb/c (H-2d) and C57Bl/6 (H-2b), responded equally to the peptides. The antipeptide sera displayed neutralizing activity against HIV-I/MN and HIV-I/3B viral strains. This neutralization was as good as human anti-HIV sera. These results indicate that peptide mimics of carbohydrates provide a novel strategy for the further development of reagents that elicit immune responses to carbohydrate epitopes associated with many infectious organisms and tumor cells.     

Influence of the T-helper epitope on the titre and affinity of antibodies to B-cell epitopes after co-immunization

We have assessed the influence of different T-helper cell epitopes on the level and affinity of antibody to B-cell epitopes induced following co-immunization with free peptides mimicking epitopes from measles and respiratory syncytial virus envelope proteins. The responses obtained following co-immunization have been compared to those obtained following immunization with chimeric synthetic peptide immunogens in which the epitopes were covalently coupled. The results show that covalent linkage of the B- and T-cell epitopes is not necessary for the generation of T-cell dependent antibody responses to non-immunogenic B-cell epitopes. In addition the induction of memory B-cells required adjuvant but subsequent stimulation of these memory cells did not. The responses obtained were non-MHC restricted since co-immunization resulted in the production of antibody responses to B-cell epitopes in a panel of five inbred mouse strains but there were differences in the ability of different T-cell epitopes to provide help for antibody production to the same B-cell epitope. The affinity of antibodies to the B-cell epitopes induced following immunization with chimeric T:B peptides was higher than that obtained following co-immunization. These results indicate the value of co-immunization for the induction of antibody responses to B-cell epitopes across MHC differences and suggest that this strategy may be of value in the development of synthetic peptide vaccines. However, modifications of the approach need to be developed to ensure the production of antibody of the highest possible affinity.     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation.     

Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.     

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.     

Mapping a conserved conformational epitope from the M protein of group A streptococci

The carboxyl terminus of the M protein of group A streptococci (GAS) is highly conserved and contains epitopes that have been shown to induce opsonic antibodies and protection against GAS infection. This region of the protein can also stimulate T cells, which can react in vitro with heart antigens. Since different segments of the carboxyl terminus may be involved in immunity to GAS and in the pathogenesis of autoimmune disease (rheumatic heart disease), it is important to precisely define critical epitopes. However, the M protein is known to be a coiled coil, and a critical immunodominant antibody-binding epitope within this region (peptide 145, a 20-mer with the sequence LRRDLDASREAKK-QVEKALE) is shown here to be conformational. Thus, small synthetic overlapping peptides of 8-12 amino acids in length that span peptide 145 (p145) were unable to capture antibodies present in p145-immune mouse sera or in endemic human sera, even though antibodies raised to these small peptides coupled to diphtheria toxoid could bind the smaller peptides and, in some cases, p145. A series of mutated peptides in which every residue of p145 was sequentially altered also failed to identify critical residues for antibody binding. We thus devised a strategy to produce chimeric peptides in which small peptides copying the M protein sequence were displayed within a larger 28-mer peptide derived from the sequence of the GCN4 leucine zipper DNA binding protein of yeast. A 12-amino-acid window of the p145 sequence was inserted into the GCN4 peptide in such a way as to preserve any potential helical structure. The window was moved along one residue at a time to give a series of peptides representing p145. Circular dichroism demonstrated that these larger chimeric peptides and p145, but not a shorter 12-mer peptide, displayed alpha-helical potential in 50% trifluoroethanol. Certain chimeric peptides efficiently captured antibodies specific for p145 and thus enabled us to map the minimal antibody-binding sequence. RRDLDASREAKK, referred to as J(1)2. The chimeric peptide containing this sequence, referred to as J2, was able to inhibit opsonization of GAS by human antisera containing anti-peptide 145 antibodies. The T-cell response from p145-immunized responder B10.BR mice to J2 and J(I)2 was much lower than the response to p145 and mapped to a different peptide.     

Molecular recognition of a peptide mimic of the Lewis Y antigen by an anti-Lewis Y antibody

Peptides as mimics of carbohydrates display a distinct advantage in vaccine design because of ease of synthesis and their inherent T cell-dependent nature as immunogens. While peptides that mimic carbohydrates have been described, it is not clear how they do so. To further our insight into structural relationships between peptide-mimics and carbohydrate structures, we have analyzed a potential recognition scheme between the murine monoclonal antibody, B3, directed against the tumor-associated antigen Lewis Y oligosaccharide and a peptide identified from phage display screening with B3. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. The B3 antibody binds to the peptide sequence APWLYGPA in which the putative sequence APWLY is critical for binding to the antibody. Not having experimental structural information for B3, the crystal structure of another anti-Lewis Y antibody, BR96, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, provides a molecular basis for LeY antigen recognition and specificity, and how this binding relates to peptide binding. As a guide to place the APWLY motif in the B3 combining site, a fragment library was searched for analogous compounds that have the potential to bind to B3. Our modeling study shows that the B3-peptide complex shares similar recognition features for the difucosylated type 2 lactoseries' structure. This analysis provides a molecular perspective for peptide mimicry of a carbohydrate epitope.     

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.     

Reactivity of antibodies against conserved regions of pilins of Haemophilus influenzae type b

To identify epitopes on pilins of Haemophilus influenzae type b (Hib) that may also be immunologically available on assembled pili, antisera were developed against eight synthetic peptides that represent conserved and hydrophilic regions of Hib pilin. Seven of the eight peptides were immunogenic. Binding of the anti-peptide antibodies to purified pili of Hib strain Eagan was weak. However, when the purified pili were denatured by heating, binding of the anti-peptide antibodies improved considerably, suggesting that the epitopes defined by the peptides were more available for anti-peptide antibody binding on the denatured pilins than on purified pili. On Western blot analysis, strain variation was seen in the binding of some of the anti-peptide antibodies, notably those directed against peptides in the N-terminal half of the pilin. Thus, when pilins are assembled into pili, the epitopes defined by the seven immunogenic peptides appear to be altered so that binding of the anti-peptide antibodies is greatly reduced.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.     

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.     

Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles

To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides. An HLA-A1-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK). CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV). HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC). Replacement of either cysteine residue with alpha-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.     

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.     

Recognition of three epitopic regions on invasion plasmid antigen C by immune sera of rhesus monkeys infected with Shigella flexneri 2a

The invasive ability of Shigella spp. is correlated with the expression of several plasmid-encoded proteins, including invasion plasmid antigen C (IpaC). By characterizing the antigenic structure of IpaC with monoclonal antibodies and convalescent-phase sera, it may be possible to determine the physical location of specific epitopes as well as the involvement of epitopes in a protective immune response or the host's susceptibility to disease. By using overlapping octameric synthetic peptides, which together represent the entire IpaC protein, the precise linear sequence of four surface-exposed epitopes was defined for four IpaC monoclonal antibodies. Furthermore, 17 unique peptide epitopes of IpaC were mapped by using 9-day-postinfection serum samples from 13 rhesus monkeys challenged with Shigella flexneri 2a. Each individual recognized a somewhat different array of IpaC peptide epitopes after infection with shigellae. However, the epitopes were clustered within three regions of the protein: region I (between amino acid residues 1 and 61), region II (between amino acid residues 177 and 258), and region III (between amino acid residues 298 and 307). Region II was recognized by 92% of S. flexneri-infected individuals and was considered to be a highly immunogenic region. Animals asymptomatic for shigellosis after challenge with S. flexneri recognized peptide epitopes within all three epitopic regions of IpaC, whereas symptomatic animals recognized peptides in only one or two of the epitopic regions. Antibody from monkeys challenged with S. sonnei recognized IpaC peptide epitopes which fell within and outside the three S. flexneri epitopic regions. While numerous potential epitopes exist on the IpaC protein, the identification of three regions in which epitopes are clustered suggests that these regions are significant with respect to the immune response and to subsequent pathogenesis postinfection.     

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.     

Involvement of amino acid residues 423-429 of human protein S in binding to C4b-binding protein

Human protein S binds to C4b-binding protein (C4BP) both in plasma and in a system using purified proteins. Amino acid residues 420-434 of the first disulfide loop of the sex hormone binding globulinlike domain of protein S are involved in the interaction of protein S with C4BP. To define the involvement of specific polar amino acids within residues 420-434, we studied in parallel synthetic protein S peptides and recombinant protein S variants containing the same amino acid replacements, K423E, E424K, Q427E and K429E. Synthetic peptide analogs of peptide PSP-420 (residues 420-434) were assayed for binding C4BP and as inhibitors of complex formation. The PSP-420 peptide and the analogous peptide with the substitution E424K, but not the peptides containing the substitutions K423E and K429E, were able to bind C4BP. Recombinant proteins with mutations of K423E, Q427E and K429E showed reduced affinity for C4BP compared to plasma protein S, recombinant wild type protein S, or E424K-protein S. These results suggest that Lys-423, Gln-427 and Lys-429 of protein S are important for normal binding to C4BP. The anti-protein S monoclonal antibody LJ-56, raised against peptide PSP-420, recognizes only free protein S and inhibits complex formation with C4BP. Antibody LJ-56 recognized the E424K and Q427E peptides but not the K423E or K429E peptides. Similarly, the E424K and Q427E protein S mutants were recognized by LJ-56, whereas the K423E and K429E protein S mutants were not recognized. This suggests that both in the peptide PSP-420 and in protein S, Lys-423 and Lys-429 significantly contribute to binding to antibody LJ-56. These results demonstrate that protein S residues 423, 427 and 429, but not residue 424, are involved in binding to both the antibody LJ-56 and to C4BP. When peptides PSP 420 and SL-6 (residues 447-460) with carboxyterminal amide or carboxylate moieties were compared to their ability to inhibit C4BP-protein S complexation, PSP-420-amide was the most potent. This finding together with the other results described here supports the hypothesis that the residues 420 and 434 in protein S provides a major binding site for C4BP.     

Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin

We have generated rat monoclonal antibodies (MoAbs) against cell surface antigens of the mouse endothelioma cell line bEND.3. Three antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which recognize a 75-kD antigen on bEND.3 cells and bind selectively to endothelial cells in cryostat sections of mouse tissues. A cDNA for the antigen was isolated from a bEND.3 pCDM8 expression library by using transient expression in COS-7 cells and immunoselection with the three MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine residues (35%). The protein is sensitive to O-sialoglycoprotein endopeptidase, indicating that it belongs to the class of sialomucin-like proteins. Therefore, we suggest the name endomucin. Treatment of isolated endomucin by sialidase and O-glycosidase reduced the apparent molecular weight to 45 kD and abolished binding of all three antibodies, indicating that carbohydrates are directly or indirectly involved in the formation of the antibody epitopes. Immunohistological analysis of all examined mouse tissues showed that endomucin is an endothelial antigen found in venous endothelium as well as in capillaries, but not on arterial endothelium. Interestingly, high endothelial venules of peripheral and mesenteric lymph nodes as well as of Peyers's patches were negative for staining with the three MoAbs.     

Molecular dynamics simulation of MHC-peptide complexes as a tool for predicting potential T cell epitopes

The class I major histocompatibility complex-encoded HLA-B*2705 protein was simulated in complex with six different peptides exhibiting unexpected structure-activity relationships. Various structural and dynamical properties of the solvated protein-peptide complexes (atomic fluctuations, solvent-accessible surface areas, hydrogen bonding pattern) were found to be in qualitative agreement with the available binding data. Peptides that have been experimentally shown to bind to the protein remained tightly anchored to the MHC molecule, whereas nonbinders were significantly more weakly complexes to the protein and progressively dissociate from it at their N- and C-terminal ends. The molecular dynamics simulations emphasize the unexpectedly important role of secondary anchors (positions 1 and 3) in influencing the MHC-bound conformation of antigenic nonapeptides. Furthermore, it confirms that dominant anchor residues cannot solely account for peptide binding to a class I MHC molecule. The molecular dynamics method could be used as a complementary tool to T cell epitope predictions from the primary sequences of proteins of immunological interest. It is better suited to MHC proteins for which a crystal structure already exists. Furthermore, it may facilitate the engineering of T cell epitopes as well as the rational design of new MHC inhibitors designed to fit optimally the peptide binding cleft.