Biomedical subjects

H M Geysen

Publications and source records attributed to H M Geysen.

66 records · Page 4Linked to original sources

PubMed1987

Mechanisms of antibody binding to a protein.

The mechanisms of antibody binding to a protein were studied by an analysis of specific amino acid residues critical to nine antigenic sites on myohemerythrin. Rabbit antisera to the whole protein were assayed for binding to more than 1500 distinct peptide analogs differing from the protein sequence by single amino acid replacements. The results, combined with information from the three-dimensional crystallographic structure, were used to evaluate probable mechanisms of antibody binding at individual sites. The data from all sites examined indicate that initial binding to solvent-exposed amino acid residues may promote local side-chain displacements and thereby allow the participation of other, previously buried, residues.

Animals↗

PubMed1987

Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti-peptide antibodies with a protein alpha-helix.

Monoclonal antibodies against an alpha-helical region of the iron-containing, oxygen-binding protein myohemerythrin were isolated following immunization of mice with either the whole protein or a peptide homolog of the helix. Three distinct epitopes within the myohemerythrin helix were identified. The individual residues within two of these epitopes that were essential for antibody binding were determined by measuring antibody binding to a set of peptides in which each amino acid of the epitope was replaced in turn by each of the other 19 amino acids. Hydrophilic residues that are exposed in the native conformation and buried, hydrophobic residues were both shown to be irreplaceable, suggesting their direct involvement in antibody binding. The influence of antigen conformation on antibody binding to these amphipathic epitopes was assessed by measuring the relative affinities of the antibodies for peptides, intact protein, and apoprotein. All of the antibodies bound to apoprotein better than to native protein, indicating that relaxation of the native structure by removal of the iron center increases antibody affinity for myohemerythrin. However, not all of the antibodies tested bound to peptides better than to protein, suggesting that increased antigen flexibility is not always sufficient to maximize antibody binding. Antibody binding to peptides appeared to also be influenced by the ability of the peptides to attain secondary structure at the epitopes, either alone or due to carrier influences.

Antibodies, Monoclonal↗

PubMed1986

The delineation of peptides able to mimic assembled epitopes.

Present methods allow a detailed study of the immune system's recognition of sequential epitopes. The results so far suggest that peptides homologous with these epitopes may not fulfil the early promise of synthetic vaccines. A procedure is described which now allows the study and evaluation of assembled epitopes. Using a monoclonal antibody which had been shown both to strongly neutralize foot-and-mouth disease virus, and to bind to a discontinuous epitope, peptides mimicking this epitope were determined a priori. An iterative procedure based on the progressive identification of amino acids in a random mixture of antibody-binding octapeptides was used. Strongly binding peptides consisted of the two elements W-Q-M (Trp-Gln-Met) and H-S (His-Ser) separated by a spacer. It was also shown that element W-Q-M was best composed of D-isomers, and the element H-S of L-isomers. Comparison of the sequence of these peptides with that of the immunologically important coat protein of the virus leads to the prediction that the epitope recognized by this monoclonal antibody consists of the residues occurring at positions 29-30, 54-55 and 88. Application of this general approach will answer questions about the nature of discontinuous epitopes and the stereochemical requirements for antigen-antibody interactions, as well as defining useful peptide immunogens.

Amino Acid Sequence↗

PubMed1986

A priori delineation of a peptide which mimics a discontinuous antigenic determinant.

A technique was developed for identifying peptides with high affinity for a given antibody. By testing a monoclonal antibody directed against a discontinuous antigenic determinant on foot-and-mouth disease virus, peptides mimicking the determinant were identified even though the tertiary structure of the proteins comprising the virus capsid is unknown. The allowable variations in spacing and stereochemistry of the peptides shown to mimic this epitope suggest protein folding in which amino acid residues from three regions, distant from one another in the primary sequence, are brought into close proximity at this epitope. The technique has potential for identification of peptides which will bind with high affinity to receptors other than antibody molecules.

Amino Acids↗

PubMed1986

The antibody response to myoglobin--I. Systematic synthesis of myoglobin peptides reveals location and substructure of species-dependent continuous antigenic determinants.

Sets of peptides representing all the possible hepta-, octa-, nona- and decapeptides of sperm whale myoglobin were synthesized. An ELISA method was used to detect the ability of antibodies, present in antisera raised against native sperm whale myoglobin, to bind to these peptides. Antisera made in two species were compared. It was found that the peptides recognized by the antibodies were a function of the species in which the antiserum was prepared and of the individual outbred member of that species. Peptides corresponding to surface epitopes of the native antigen were identified by reacting the antisera with native antigen prior to ELISA testing on peptides. More detailed analysis of one epitope revealed that, for some sera, a leucine residue which is facing inwards in the crystal structure is critical for the binding of antibody to the peptide. This suggests that binding between native antigen and antibody can require a restructuring of the native antigen.

Animals↗

PubMed1985

Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein.

Antisera were raised against the chemically synthesized peptide corresponding to each epitope of three foot-and-mouth disease virus strains. Peptide synthesis was further used to determine which amino acid residues in each epitope are important for the specificity of antisera raised against the whole virus. The specificity of the antibody paratope for its epitope was shown to depend on structure as well as sequence. Anti-virus sera demonstrated a greater specificity for the homologous peptide than did the anti-peptide sera. Two of the three peptides were able to induce neutralizing antibodies against the homologous virus. The specificities of the antibodies present in the anti-peptide sera were also inferred from the reactions of each with related sets of peptides. The cross-reactions observed for the anti-peptide sera were readily explained in terms of the antibody specificities determined to be present. The findings also suggest that the diversity of antibodies raised against small peptides is limited and is determined by the immune system. A similar limited response to the native protein was observed, which may account for the high frequency with which anti-peptide sera react with the native homologous protein.

Amino Acid Sequence↗

PubMed1984

Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid.

A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.

Amino Acid Sequence↗

PubMedDate not supplied

Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structure.

Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.

Amino Acid Sequence↗

PubMedDate not supplied

Use of the multipin peptide synthesis technique for the generation of antipeptide sera.

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pin-synthesized peptides and beta-amide peptides and the protein antigen coated to microtiter plates.

Amino Acid Sequence↗

PubMedDate not supplied

High-throughput purity estimation and characterisation of synthetic peptides by electrospray mass spectrometry.

High-throughput analysis for purity and molecular weight determination of synthetic peptides including characterisation of any peptidic byproducts arising from synthesis is described. The data from electrospray mass spectrometry are processed with an algorithm that calculates the contribution of the target peptide and each of the identifiable contaminants to the total ionizable material in a sample of synthetic peptide. All essential data are obtained by one instrumental technique in < 3 min per sample. The technique has distinct advantages in the rapid analysis of the many hundreds of peptides/peptidomimetics required in systematic quantitative structure-activity relationship and other investigations.

Algorithms↗

PubMedDate not supplied

Systematic screening for bioactive peptides.

Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of end groups on the stimulatory capacity of minimal length T cell determinant peptides.

We studied the effect of end groups on the capacity of two closely related peptides to stimulate a human T cell clone. Using multipin peptide synthesis technology, we generated solution phase peptides with six combinations of end groups for each sequence. The end modifications examined were amino-terminal acetylation, carboxy-terminal methylamidation or the addition of a dipeptide containing a diketopiperazine ring. The response to the less stimulatory of the two peptide sequences was significantly increased by acetylation of the amino-terminus, a finding which was consistent at different peptide doses. Amino-terminal acetylation was found to be more significant in affecting responses than any of the carboxy-terminal modifications tested. The use of peptides with a diketopiperazine ring structure at the carboxy-terminus did not interfere with presentation and recognition of peptides and may enhance the effectiveness of peptides for T cell epitope scanning.

Amino Acid Sequence↗