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M Sollazzo

Publications and source records attributed to M Sollazzo.

At least 37 records · Page 2Linked to original sources
PubMed1993

A designed metal-binding protein with a novel fold.

A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.

Amino Acid Sequence↗
PubMed1993

Chemical synthesis of a designed beta-protein through the flow-polyamide method.

A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a beta-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70% beta structure. Accordingly, it was anticipated, and actually found, to be a "difficult sequence" for all its length. Comparison of the standard, "minimum redundancy" protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected Ion Spray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.

Amino Acid Sequence↗
PubMed1993

Affinity purification of a difficult-sequence protein. Implications for the inclusion of capping in synthetic protocols.

A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41, 385-393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimensional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.

Amino Acid Sequence↗
PubMed1990

Structural definition by antibody engineering of an idiotypic determinant.

Using computer-aided techniques for predicting molecular structure, we constructed an atomic model of the variable domain of a murine anti-thyroglobulin antibody whose immunodominant idiotypic determinant (Id62) was mapped by site-directed mutagenesis and immunochemical analysis. We previously showed that under experimental conditions this idiotype activates anti-idiotypic B cells and T cells, and modulates the response to thyroglobulin in mice. Because idiotype interactions are considered of physiological importance for immune regulation, we studied this idiotype as a model to understand the relationship between function and structure. To determine the contribution of heavy- and light-chain variable domains to the idiotype structure, we constructed chimeric expression vectors and introduced them into the (non-secreting) P3X63Ag8.653 myeloma cell line. Mutants of the heavy-chain variable domain were obtained by site-directed mutagenesis and transfected into the murine (lambda 1) light-chain producer J558L cell line. The expressed proteins were purified from culture supernatants of transfected cells and characterized. We provide evidence that the third hypervariable loop (D region) of the heavy-chain variable domain is the structural correlate of the idiotypic determinant of this autoantibody and is independent from the nature of the associated light chain. Substitution of residues of the first and second complementarity-determining regions do not affect idiotype expression. The results described here are discussed in relation to our understanding, at a molecular level, of the interaction of idiotopes with B- and T-cell compartments.

Amino Acid Sequence↗
PubMed1990

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding.

Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti-(NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis.

Amino Acid Sequence↗
PubMed1990

A breakdown in macromolecular synthesis preceding differentiation in Streptomyces coelicolor A3(2).

A transitory cessation of growth was recorded in Streptomyces coelicolor A3(2) at the end of vegetative mycelium formation on solid medium. In the same phase a striking reduction in protein and nucleic acid synthesis was detected. Growth and macromolecular synthesis resumed, nearly reaching the original values, when morphological differentiation occurred. It is concluded that a physiological stress occurs within the bacterial population just before the onset of the morphological differentiation.

Cell Cycle↗
PubMed1989

Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALB/c mice.

We report on the molecular characterization of the heavy (H) chain variable (V) region of two murine autoantibodies reacting with a conventional self antigen, thyroglobulin (Tg), originated from unimmunized/unstimulated neonatal mice (clone B10H2) and from an adult hyperimmunized mouse (clone 62), respectively. Serologically, both hybridoma antibodies express the same idiotype (Id). By cloning and sequencing we demonstrated that their VH regions are encoded by identical VDJ gene elements including the VD and DJ joints. This VH belongs to the VH 7183 gene family, the most 3'-end proximal family in the murine genome. The D segment is unique and only 50% similar to any murine D segment. The J segment utilized is germ-line JH4. The cloned DNA rearrangement was transfected into the J558L myeloma cells and the secreted antibody was found to express the reference Id on the H chain, hence proving that the productive VDJ rearrangement had been identified. These results show that a spontaneously arising and an antigen-induced autoantibody use an identical VDJ gene rearrangement and that after hyperimmunization somatic mutation did not occur. The significance of this finding with respect to ontogeny of the B cell repertoire is discussed.

Animals↗
PubMed1989

DNA sequence and pattern of expression of the sea urchin (Paracentrotus lividus) alpha-tubulin genes.

To study the molecular aspects of the regulation of transcription of a multigene family, we have isolated and sequenced cDNA and genomic clones coding for the alpha-tubulin of the sea urchin Paracentrotus lividus. Two cDNA clones, P alpha 10 and P alpha 4, contain respectively the coding information for 391 C-terminal and for 338 N-terminal amino acids of the 452 residues that constitute the complete protein. They show silent nucleotide substitutions only, suggesting that P alpha 10 and P alpha 4 represent the cloned copies of two allelic gene transcripts, which encode for two alpha-tubulin isoforms with identical amino acid sequence in the region of the overlap. The comparison of the predicted amino acid sequence of the composite P alpha 4-10 and of the mouse M alpha-6 (Villasante et al., Mol Cell Biol 1986; 6:2409-2419) reveals a conservation of 97% between the two polypeptides. By RNA blotting hybridization six major alpha-tubulin transcripts were identified. Two, of 3.5 kb and 2.0 kb, are expressed in the unfertilized eggs and during early cleavage. The other two maternal mRNAs, of 2.4 kb and 1.8 kb, are expressed in both early and late cleavage embryos, but in the intestine the 1.8 kb RNA, which specifically reacted with the 3' specific probe of the P alpha 10 cDNA, is the only transcript detected. Finally, the 1.5 kb and 1.9 kb mRNAs represent the transcription of stage- and tissue-specific genes, respectively. In fact, the former becomes detectable at blastula stage and accumulates during late development, whereas the latter is found in the testis only. The sequence data of the 3' terminus of the alpha-3 genomic clone suggests that it encodes for a divergent alpha-tubulin, and it most probably corresponds to the testis-specific gene.

Amino Acid Sequence↗
PubMed1989

Autoantibody idiotypy and neonatal B cell repertoire.

During ontogeny, antibody variable (V) regions are subjected to selection events at the level of B-cell clones bearing on their surfaces Ig molecules useful to the developing organism. Antigenic determinants of immunoglobulin V regions (idiotypes) are believed to play an essential role in molecular recognition and immune responsiveness to exogenous and self antigens. Recent data indicate that reactivity with self-antigens is prevalent within the natural neonatal repertoire, suggesting that self-antigens are involved in the selection and shaping of the immunologic repertoire. One implication of the above considerations is that V regions having regulatory idiotypes are borne preferentially on antibodies that react with self-antigens. Here, we report on the molecular characterization of the idiotype 62 expressed by BALB/c autoantibodies to a classic self-antigen, thyroglobulin. We show that under appropriate experimental conditions, Id62 can modulate the autoantibody response through a process requiring the activation of regulatory T cells. We also demonstrate that self--reactive V regions bearing Id62 are present in newborn mice and report on the primary structure of the V-region genes encoding Id62. Lastly, we provide evidence that hybridomas derived from unstimulated splenocytes of normal neonatal BALB/c mice express Ig molecules that react prevalently with self-antigens.

Amino Acid Sequence↗
PubMed1989

The management of 138 psoriatic arthritic patients.

Clinical experience obtained in the management of 138 patients of psoriatic arthritis is reported. The correct recognition of arthritic subtype (according to Moll and Wright classification) always resulted essentially in the choice of the therapy. Our programme included rehabilitative, pharmacologic and surgical approaches. Our data suggest that psoriatic arthritis is a mild articular disease when compared to other affections such as rheumatoid arthritis or ankylosing spondylitis. Notwithstanding an accurate therapeutical programme, it is necessary to control atypical cases which not infrequently can occur.

Adult↗
PubMed1988

Isotype, VH genes, and antigen-binding analysis of hybridomas from newborn normal BALB/c mice.

In this study we report on the characterization of a panel of 62 hybridomas generated by fusing unstimulated spleen cells from neonatal (less than 24 hr old) normal BALB/c mice with the non-secreting Sp 2/0 cell line. The vast majority (98%) of these hybridomas secreted Ig but only 20% produced IgM. The isotype of the remaining hybridomas was determined as being IgG2b. Interestingly, when splenocytes from 1-day-old mice were stimulated with LPS for 48 h prior to the fusion event, 84% of the hybridomas were secreting IgM. The hybridoma supernatants were screened either by ELISA or RIA for binding reactivity using a panel of 17 Ag, proportionally divided between self and non-self. A binding reactivity could be assigned in 44% of cases. Of these, 29% were monoreactive, i.e., reactivity occurred with one Ag only, while the remaining 15% were multireactive. The majority (21 of 27) of hybridomas with a defined reactivity were directed against self-Ag. These included autologous red blood cells, DNA, histone H1, thyroglobulin, and Ag of the cell surface of T cells. The frequency of utilization of VH genes was determined using DNA probes for eight VH gene families. While all VH gene families appeared to have been used, one, VH 7183, had a slight but significant (p less than 0.02) higher utilization than expected by random expression. The frequency of all the other VH gene families was not significantly different from random utilization. No correlation was found between Ag reactivity in the supernatants and the utilization of a particular VH gene family. These findings indicate that early in the ontogeny the predominant reactivity of B cells is for self-Ag and, unlike what it is commonly believed, the IgM isotype is not dominant within these endogenously activated B cells at this time of ontogeny when genes from all VH families are utilized.

Animals↗
PubMed1987

Idiotype regulation of self responses, autoantibody V regions and neonatal B cell repertoire.

The role of autoantibodies themselves in immune regulation is still unknown. There is evidence that some autoantibody idiotypes (Id) may play a regulatory role in physiologic and possibly pathologic situations of the immune system. Here we present evidence that (1) certain autoantibody Id modulate the immune response to self antigens (regulatory Id), and (2) that the neonatal preimmune repertoire is primarily devoted to self recognition. The humoral and cellular events following immunization with regulatory Id suggest an active participation of T cells in the regulatory events. Based on immunochemical, structural and molecular genetics analysis, it appears that this regulatory Id is of germline origin as it exists within the neonatal preimmune repertoire. B cell hybridomas generated from unstimulated splenocytes of neonatal mice are primarily self-reactive, suggesting that the immune system essentially begins as a self-recognizing system. The findings are discussed with regard to the relationship between germline repertoire, autoantibodies, and regulatory idiotypes.

Animals↗
PubMed1987

Characterization of human ferritin H chain synthetized in Escherichia coli.

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

Cloning, Molecular↗
PubMed1985

High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences.

We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.

Chromosome Deletion↗