Marc H.V. Van Regenmortel

Monoclonal antipeptide antibodies: Affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.

Localization of two antigenic determinants in histone H4

Four overlapping synthetic peptides corresponding to the carboxy-terminal region 80-102 of histone H4 were prepared by solid-phase peptide synthesis. Their antigenic activity was analysed by inhibition of the H4-anti-H4 reaction in complement fixation and enzyme-linked immunosorbent assay. One antigenic determinant was localized in residues 88-96 of the H4 molecule. No antigenic activity was found in peptides 80-89 and 97-102. Antibodies induced by peptide 85-102 were found to bind to free H4 in solution but not to chromatin subunits, suggesting a lack of accessibility of the C-terminal region of H4 in nucleosomes. A second epitope was found to be situated in the N-terminal region 1-53 of histone H4.

Accessibility and Structural Role of Histone Domains in Chromatin. Biophysical and Immunochemical Studies of Progressive Digestion with Immobilized Proteases

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.

[12] Use of antihistone antibodies with nucleosomes

Publisher Summary Antibodies directed against defined regions of histone molecules represent one of the most specific probes for studying the surface topography of nucleosomes and chromatin and for monitoring post-translational histone modifications. Antibodies directed against the epitopes can be used to determine the regions of histone molecules that are exposed at the surface of nucleosomes and are useful probes for analysing chromatin structure and function. Four approaches have been used to obtain antibodies suitable for these types of studies: (1) immunization with histone molecules, (2) immunization with synthetic peptide fragments of histones, (3) immunization with nucleosomes, and (4) preparation of hybridomas from nonimmunized, autoimmune mice. The method used for purifying histones and the immunization protocol, using histones complexed with RNA, influence the conformation of histone molecules and will thus affect their antigenic determinants. There is also evidence that polymerization of histones into chromatin components produces novel antigenic determinants that derive their specificity from the tertiary and quaternary structure. Changes in environmental conditions that alter the structure of chromatin or posttranslational modifications can thus be expected to modify its antigenic reactivity.

Immunochemical studies of turnip yellow mosaic virus III. Localization of two viral epitopes in residues 57–64 and 183–189 of the coat protein☆

Abstract The antigenic activity of the C-terminal region of the coat protein of turnip yellow mosaic virus was investigated with two synthetic peptides corresponding to residues 183–189 and 187–189. The inhibitory activity of these peptides was measured by complement fixation, ELISA and RIA, using either antibodies directed to the depolymerized viral subunits or antibodies to the capsid. Although the activity of the peptides varied markedly in the different assays, the results clearly demonstrated that the C-terminal residues 183–189 correspond to an antigenic determinant present at the outer surface of the assembled capsid. Another synthetic peptide corresponding to residues 57–64 of the coat protein was also prepared and found by inhibition of complement fixation to correspond to an antigenic determinant of the virus. These results bring to three the number of antigenic determinants of the virus particle that have been located.

Anti-cyclosporine monoclonal antibodies and their anti-idiotopic counterpart: Structure and biological activity

In order to study the structural and functional mimicry of an antigen by anti-idiotypic antibodies, we generated anti-idiotopic monoclonal antibodies (anti-Id mAbs) against a mAb (R45-45-11) with specificity for the immunosuppressive cyclic undecapeptide cyclosporine (Cs; Sandimmune). Three out of five anti-Id mAbs inhibited the binding of Cs to the anti-Cs mAb R45-45-11. All anti-Id mAbs cross-reacted only with one (anti-Cs mAb V45-271-10) out of 19 anti-Cs mAbs. The anti-Cs mAb V45-271-10 recognizes an epitope on the Cs molecule which is very similar to that recognized by R45-45-11. R45-45-11 and V45-271-10 differ only by one amino acid in the variable region. The anti-Id mAbs which recognize combining site-associated idiotopes (Ids) reverse the blocking effect of the anti-Cs mAb R45-45-11 on Cs immunosuppression in vitro. The sequences of the variable regions of heavy and light chain of one anti-Id mAb were determined. X-ray analysis of the corresponding Fab fragment, either alone or complexed with the Fab fragment of the Id, is currently in progress.

Role of Conformation on the Antigenic Determinants of Tobacco Mosaic Virus Protein

The majority of viruses have a fairly simple architecture consisting of a number of identical protein subunits assembled into a capsid with icosahedral or helical symmetry. The polymerization of subunits leads to the effective disappearance of certain surfaces of the protein that are turned inward, a feature common to all macro-molecular structures built up from monomers. Epitopes situated on these hidden surfaces have been called cryptotopes1; they become antigenically expressed only when the capsids have been disassembled.