Diana Tello

The Crystal Structure of the Mouse Apoptosis-Inducing Factor Aif

Mitochondria play a key role in apoptosis due to their capacity to release potentially lethal proteins. One of these latent death factors is cytochrome c, which can stimulate the proteolytic activation of caspase zymogens. Another important protein is apoptosis-inducing factor (AIF), a flavoprotein that can stimulate a caspase-independent cell-death pathway required for early embryonic morphogenesis. Here, we report the crystal structure of mouse AIF at 2.0 Å. Its active site structure and redox properties suggest that AIF functions as an electron transferase with a mechanism similar to that of the bacterial ferredoxin reductases, its closest evolutionary homologs. However, AIF structurally differs from these proteins in some essential features, including a long insertion in a C-terminal β-hairpin loop, which may be related to its apoptogenic functions.

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy

Using synthetic Tn (GalNAc‐O‐Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti‐Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

The crystal structure of a plant lectin in complex with the Tn antigen

The structure of the tetrameric Vicia villosa isolectin B4 (VVLB4) in complex with a cancer antigen, the Tn glycopeptide (GalNAc‐O‐Ser), was determined at 2.7 Å resolution. The N‐acetylgalactoside moiety of the ligand binds to the primary combining site of VVLB4 in a similar way as observed for other Gal/GalNAc‐specific plant lectins. The amino acid moiety of the Tn antigen is largely exposed to the solvent and makes few contacts with the protein. The structure of the complex provides a framework to understand the differences in the strength of VVLB4 binding to different sugars and emphasizes the role of a single protein residue, Tyr127, as a structural determinant of Tn‐binding specificity.

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Amino acid sequence and three‐dimensional structure of the Tn‐specific isolectin B4 from Vicia villosa

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three‐dimensional structure solved by molecular replacement techniques and refined at 2.9 Å resolution to a crystallographic R‐factor of 21%. Each subunit displays the thirteen‐stranded β‐barrel topology characteristic of legume lectins. The amino acid residues involved in metal‐ and sugar‐binding are similar to those of other GalNAc‐specific lectins, indicating that residues outside the carbohydrate‐binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

Crystallization and preliminary X-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes☆

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.

Three-dimensional structure and antigen binding specificity of antibodies

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.

Crystallization of antibody fragments and their complexes with antigen

Abstract Immunoglobulins, myeloma light chains and their fragments, and Fab fragments from monoclonal antibodies of predefined specificity have been crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab and Fc fragments separately. Intrasegmental mobility in Fabs has not been an obstacle to their crystallization, although this has been a low frequency event, occuring in about 1 in 25 to 1 in 50 trials with different Fabs. However, the immune system provides a large functional and structural diversity of antibody molecules so that an active search may eventually reveal antibodies of the desired specificity suitable for crystallization and X-ray diffraction studies.

Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freunds complete adjuvant.