Alberto Chersi

Detection of complex alleles by direct analysis of DNA heteroduplexes.

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.

Human papillomavirus-16 E7 interacts with siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes†

The viral factor E7 plays a key role in the well‐established association between “high‐risk” Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano‐genital cancer. To delve into the molecular mechanisms of HPV‐mediated cell transformation, we searched for novel potential cellular targets of the HPV‐16 E7 oncoprotein, by means of the yeast two‐hybrid technique, identifying a protein–protein interaction between HPV‐16 E7 and the pro‐apoptotic cellular factor Siva‐1. Using co‐precipitation assays and the “PepSets” technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved. Additionally, we found that HPV‐16 E7 competed in vitro with the binding of the Bcl‐XL anti‐apoptotic factor to Siva‐1, an interaction that has a major inference in UV radiation‐induced apoptosis. In HaCaT immortalized human keratinocytes, forced HPV‐16 E7 expression by retroviral infection caused Siva‐1 transcript up‐regulation, detected by cDNA macroarray hybridization and real‐time quantitative PCR, paralleled by an increased amount of protein. Confirming the anti‐apoptotic role of HPV‐16 E7 in the HaCaT cellular model, evaluated by nuclear morphology, we also found that Siva‐1 expression produced a significant increase of the apoptotic rate in UV radiation‐exposed HaCaT cells, and that this effect resulted explicitly counteracted by HPV‐16 E7. Being apoptosis a key physiological process for the elimination of irreversibly injured cells, the anti‐apoptotic role of HPV‐16 E7, performed at least by its interference with Siva‐1, can be considered an additional mechanism for the survival of damaged, potentially transforming, cell clones. J. Cell. Physiol. 212: 118–125, 2007.

Anchor residue motifs of HLA class-I-binding peptides analyzed by the direct binding of synthetic peptides to HLA class I α chains

The binding characteristics of the primary anchor residue motifs reported for HLA-A2 (A*0201, A*0205) and HLA-B27 (B*2705) alleles were investigated by a direct binding assay of the pertinent synthetic peptides to HLA class I alpha chains derived from a panel of HLA homozygous B-cell lines of various HLA phenotypes, including four A2 subtypes. The assay is based on a serologic detection of the conformational change of HLA class I alpha chains induced by binding to specific peptides in the presence of beta 2m. It is applicable to test a large number of HLA allelic products and synthetic peptides. Assay data confirmed the high allele specificity of the anchor residue motifs tested, but also revealed the intra- and interlocus cross-reactivity of these motifs. In the case of A2 anchor motifs, not only a broad cross-reactivity within the A2 subgroup, but also cross-reactivities with A24, A26, A28, and A29 were observed. With B27 anchor motifs, an interlocus cross-reactivity with A3 and A31 was seen. Several peptides, even though they carried A2 or B27 major anchor residue motifs, failed to bind to the relevant alpha chains, suggesting that the presence of a primary anchor residue motif is necessary for HLA class-I-peptide binding but is not by itself sufficient to guarantee binding.

Unfolded HLA class I α chains and their use in an assay of HLA class-I-peptide binding

Abstract Unfolded HLA class I α chains were isolated from B-cell lysates by alkaline denaturation and subsequent gel filtration and used for the detection of HLA class-I-peptide binding. Binding to specific peptides in the presence of excess β 2 -microglobulin induced the unfolded α chains to refold and acquire a conformation that is specific to folded α chains. This conformational change was measured by a specific RIA that involves inhibition of the binding of 125 I-labeled HLA-A2 α/β dimers and rabbit anti-HLA-B7 serum absorbed with β 2 -microglobulin. This assay procedure does not require labeling of either test peptides or test class I proteins and does not seem to have specificity degeneracy. It is applicable to the detection of peptide binding by all HLA class I allelic proteins. Evaluation of the assay conditions and HLA allelic specificity of the peptide binding defined by the use of synthetic peptides are described here, including the technical details, specificity, and reproducibility.

Increased Resistance of Peptides to Serum Proteases by Modification of their Amino Groups

Abstract The ability of synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by slight modifications at their N-terminal alpha-amino group. This can be achieved by addition of beta-alanine or amino acids of the d-configuration, amino acids which are seldom found in a living organism. These modifications do scarcely modify the chemical and physical properties of the peptides, and should be preferrred, especially for in vivo tests, to drastic alterations of peptides as produced by dinitrophenylation or dansylation of the amino groups.

MHC-peptide binding : Dimers of cysteine-containing nonapeptides bind with high affinity to HLA-A2.1 class I molecules

Summary Small peptides, 8–10 aminoacids long, derived from degradation of cytoplasmic proteins by a proteasome–proteinase complex, are usually presented and recognized by CD8+ cytolytic T lymphocytes (CTLs) associated with major histocompatibility complex (MHC) class I molecules. Recently synthetic peptides were used for the in vitro induction of tumor-specific CTLs, offering another strategy in the study of the immune-response repertoire and providing a new tool in cancer vaccination and immunotherapy. Peptides derived from otherwise normal proteins, overexpressed in many tumors as products of the protooncogene, may represent a target for an immune response. This is the case of HER-2/neu gene (also known as ErbB-2), encoding a cysteine-rich glycoprotein transmembrane receptor with tyrosine kinase activity (gp185neu). Recent data, demonstrating that HLA-A2.1–related peptides are able to stimulate in vitro CD8+ lymphocytes, prompted us to study the binding to HLA-A2.1 molecules of several gp185 synthetic peptides containing a cystein residue and to define the relevance of this amino acid residue in the reduced or oxidated form of the sulfhydryl group. We found that monomers and their homodimers, linked by a disulfide bridge, bind to HLA-A2.1 molecules with overlapping affinity. These results suggest that additional amino acids of the nonapeptide do not prevent the binding and the HLA refolding through chemical or sterical interactions. This might be of particular relevance for the in vivo processing of cysteine-rich proteins. Because ErbB-2 molecules, as tumor-differentiation antigens in melanoma, are cysteine-rich molecules, it may be relevant to evaluate the possible role of the cystine residues interacting with the T-cell receptor. The recognition of these heterodimers by CD8+ lymphocytes will require functional in vivo studies.

Selective `in synthesis' labelling of peptides by fluorochromes

A new method is described for producing fluorescently-tagged peptides containing specific internal derivatives of lysyl residues. The technique employs the base-labile Boc-Lys(Fmoc)-COOH derivative with base-catalyzed removal of the Fmoc protecting group during peptide synthesis and subsequent fluorescent derivatization of the deprotected epsilon-amino group of lysine. By this technique, other lysine residues and the alpha-amino group of the fragment remain unmodified, which could have some value in studies where it might be required to tag a single individual lysine residue within the peptide, but not the amino terminus. In spite of the fact that poly-substituted peptides are badly soluble and might seldom find a practical application, this technique also allows the introduction of different fluorochromes at different lysyl residues within the peptide, thus obtaining double fluorescence. The method, fast and easy, requires a limited number of manual operations during the automatic synthesis of peptides. Although peptide synthesizers provided with an oscillating glass reactor are more suitable for the manual interventions described, this technique might be also adapted to the newer instruments utilizing continuous-flow columns.

Partial primary structure of the immunoglobulin light chain constant region of a single rabbit of b5 allotype

Abstract The partial amino acid sequence of the constant region of the b5 light chain from the normal IgG of a single rabbit is reported. For structure determination, the IgG light chain was fully reduced and carboxymethylated, then digested with chymotrypsin or trypsin. All chymotryptic peptides covering the constant region from positions 116–210 (117–214 in the standardized numbering system of Kabat et al. ∗ ) were isolated and purified by column and paper chromatography. Sequences were then determined using traditional sequencing methods. Overlapping was obtained by use of large tryptic peptides, covering positions 138–210 (139–214 ∗ ). The chymotryptic peptide ending with Leu 115(116 ∗ ) could not be obtained in a pure state owing to insolubility and perhaps heterogeneity. When the sequence of this light chain is compared to those of light chains of b4 and b9 allotypes, about 77% homology is found with b4 and 62% with b9. This confirms serological data which would indicate that b4 and b5 allotypes are more similar to each other in structure than they are to b9. The allotypic differences are distributed throughout the whole constant portion of the light chain.

Preparation and utilization of fluorescent synthetic peptides

In this study, several methods for controlled labelling of synthetic peptides by the use of fluorescent compounds (fluorescein isothiocyanate and dimethylaminonaphthalene sulfonyl chloride) were investigated. The first reagent yielded monofluoresceinated, active compounds only when the peptides lacked lysine residues. Monolabelling of peptides in solution with dimethylaminonaphthalenesulphonyl chloride was hindered by the broad reactivity of the reagent, but was achieved by reacting the fluorochrome on protected resin-bound peptides in solid-phase synthesis. The remarkable stability of the linkage allowed the cleavage of the peptide from the resin and deprotection of side-chain functions without hydrolysis of the labelled group. The binding of antipeptide antibodies to the labelled fragments was then estimated using different techniques.

Immunologic and functional characterization of anti-HLA-DR rabbit antibodies induced by synthetic peptides

Three peptides selected from the amino acid sequence of the alpha- and beta-chains of DR2 histocompatibility antigens were chemically synthesized and coupled to carrier proteins to be used as immunogens in rabbits. This immunization resulted in the production of specific antibodies that readily recognized the antigen. However, only one of the four antibody preparations, antibody 6148, elicited by a short peptide from the beta-chain (residues 61-73), reacts with native membrane glycoproteins as well as intact human lymphoblastoid cells in enzyme-linked immunosorbant assays. This antibody was found to react also with membrane glycoproteins solubilized by nonionic detergents from cells bearing a different HLA-DR specificity: therefore it is likely that the peptide responsible for eliciting antibody 6148 represents a common framework determinant of DR alloantigens that is accessible on the surface of lymphoblastoid cells. The ability of antibody 6148 to bind to intact cells was confirmed by indirect immunofluorescence and by fluorescein-activated cell sorter analysis. This antibody is also capable of mediating antibody-dependent cellular cytotoxicity as determined by a 51Cr-release assay.