Jean Lamy

Immunological correlates between the multiple hemocyanin subunits of Limulus polyphemus and Tachypleus tridentatus

Abstract The 60 S hemocyanins of the horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus, are of interest as models of how structurally diverse subunits interact in the assembly of high molecular weight proteins. Subunit diversity is a general characteristic in hemocyanins of the Chelicerates (horseshoe crabs, spiders, and scorpions) and is expressed to a lesser extent in hemocyanins of the crustaceans (crabs, shrimp, and lobsters). In order to better characterize the hemocyanins of Limulus and Tachypleus, their subunits were fractionated by ion-exchange and thin-layer gel chromatography. Immunological techniques were used extensively in isolation and characterization of the subunits. Both hemocyanins yield six chromatographic zones at pH 8.9. The Limulus pattern is due to the existence of seven monomers and a heterodimer. The heterodimer contains one additional monomer for a total of eight distinct subunits. Limulus monomer IIA is antigenically related to IIIa but is antigenically deficient. Dissociation of Tachypleus hemocyanin at pH 8.9 yields five monomers and a heterodimer that contains two additional monomers. One monomer in the dimeric component of Limulus hemocyanin is antigenically related to a monomer of the dimeric component of Tachypleus and the other two monomers in the dimers are antigenically identical. It is proposed that dimeric components link the hexameric “building blocks” of the Chelicerate hemocyanins and thus play specific functional and structural roles.

Three-dimensional reconstruction of the hexagonal bilayer hemoglobin of the hydrothermal vent tube worm Riftia pachyptila by cryoelectron microscopy

A frozen‐hydrated specimen of the V1 hemoglobin of the hydrothermal vent tube worm Riftia pachyptila was observed in the electron microscope and subjected to three‐dimensional reconstruction by the method of random conical tilt series. The 3D volume possesses a D6 point‐group symmetry. When viewed along its 6‐fold axis the vertices of its upper hexagonal layer are 16° clockwise rotated compared to those of the lower layer. A central linker complex is decorated by 12 hollow globular substructures. The linker complex comprises (i) a central hexagonal toroid, (ii) two internal bracelets onto which the hollow globular substructures are built, and (iii) six structures connecting the two hexagonal layers. The hollow globular substructures, related to the dodecamers of globin chains resulting from the dissociation of the hexagonal bilayer hemoglobin, have a local pseudo 3‐fold symmetry and are composed each of three elongated structures visible when the volume is displayed at high threshold. At a resolution of 36 Å, the 3D volumes of the hexagonal bilayer hemoglobins of Riftia pachyptyla and of the leech Macrobdella decora look almost perfectly identical.

Overabundant single-particle electron microscope views induce a three-dimensional reconstruction artifact

In the field of single-particles 3D reconstruction, the development of angular assignment techniques has allowed the collection of a large amount of cryoelectron microscope images of untilted-specimens. When particles have a preferential orientation within the ice layer, one type of electron microscope (EM) view is oversampled and produces a new type of 3D reconstruction artifact. This oversampling artifact is observed with simultaneous iterative reconstruction techniques (SIRT) but the theoretical analysis of the problem indicates that similar effects could also occur with other reconstruction algorithms.

Three-dimensional reconstruction of the αD and βC-hemocyanins of Helix pomatia from frozen-hydrated specimens

Abstract The three-dimensional (3D) reconstructions of the di-decameric forms of αDand βC-hemocyanins of the Roman snail Helix pomatia and of the decameric half molecules of αD-hemocyanin were carried out on frozen-hydrated specimens observed in the electron microscope by using the random conical tilt series method. The three 3D volumes were examined by computing solid-body surface representations and slices through the volume and by eroding the structure progressively through raising of the threshold. The di-decameric molecule of αD and βC-hemocyanins, reconstructed from side views, are very similar and are composed of a cylindrical wall, comprising ten oblique wall units, and of two collar complexes located at both ends of the cylinder, comprising each five arches and an annular collar made up of five collar units. Erosion of the structure reveals that the wall looks like a segment of a five-stranded right-handed helix and that each oblique wall unit resembles a figure 8 inclined to the right. The decameric half molecule of αD-hemocyanin, reconstructed from end-on views, resembles the whole molecule, except that the collar is thinner and appears composed of five independent collar complex units. It is suggested that the difference in structural appearance of the collar complex between the whole and the half α D -hemocyanin may be due to the missing cone artifact, induced by the angular limitations imposed by the goniometer of the electron microscope. The comparison between the α D -hemocyanin and the βC-di-decameric hemocyanin at high thresholds suggests that in the βC-hemocyanin the oblique wall units of each half molecule may be linked by two connections, whereas in α D -hemocyanin there may be only one. This difference in the number of connections may be responsible for the lower stability of the α D molecule at high salt concentration.

Lumbricus terrestris hemoglobin: A comparison of small‐angle X‐ray scattering and cryoelectron microscopy data

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of clylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm-1 and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm-1) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex.

Immunological correlates between multiple isolated subunits of Androctonus australis and Limulus polyphemus hemocyanins: an evolutionary approach.

Immunological cross-reactivities between isolated subunits of the scorpion Androctonus australis (Aa) and of the horseshoe crab Limulus polyphemus (Lp) hemocyanins were studied using subunit-specific antibodies prepared through immunoadsorption to pure immobilized subunits. Rocket immunoelectrophoreses of the various subunits of both hemocyanins were carried out at constant antigen concentration against the various subunit-specific antibody preparations. Then the data were analyzed through factorial correspondence analysis and compared to the respective intramolecular locations of the subunits in both hemocyanins. The results show that the dimeric subunits located in the central part of each (4 X 6)meric structure (Aa whole molecule and Lp half molecule) were strongly preserved. In addition, the (8 X 6)mer-forming subunit of Lp hemocyanin (LpIV) and the subunit occupying the same intramolecular position in Aa hemocyanin (Aa5A) were also strongly preserved. Besides the strong antigenic relatedness, less pronounced crossed immunoprecipitations or no precipitation at all were observed between subunits with homologous positions suggesting a minor structural and/or functional roles for these subunits. All the antigen-antibody combinations leading to an absence of immunoprecipitation were screened for the presence of soluble immunocomplexes by radioimmunological tests. In all cases, soluble immunocomplexes were observed. These results suggest the following evolution scenario. First, the central dimeric subunits, responsible of the dodecamer aggregation (Aa3C and 5B and LpV and VI) were already differentiated when Merostomata diverged from Arachnida. Second, the differentiation of the (8 X 6)mer-forming subunit occurred in the Merostomata ramification in a preserved subunit already possessing a functional advantage. Third, the differentiation of subunits Aa3A and Aa3B recently occurred in the scorpion ramification.

Three-dimensional reconstruction of a complex of human α2-macroglobulin with monomaleimido nanogold (Au1.4nm) embedded in ice

Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.

Image analysis and three‐dimensional model of chymotrypsin‐transformed human alpha2‐macroglobulin complexed with a monoclonal antibody specific for this conformation

Alpha2‐macroglobulin (α2M) is a plasma inhibitor of proteinases, the steric mechanism of which is based on a considerable conformational change. The typical and distinct H‐like shape of α2M‐chymotrypsin (α2M‐chy) complexes seen by electron microscopy led us to an ultrastructural study of the binding of a monoclonal antibody (Mab) specific for this conformation of α2M. The epitope of this Mab is located near the extremities of the 4 arms of the H‐like α2M‐chy, at a site that is not accessible on the native molecule. The identical binding of the Mab on the 4 arms of the tetrameric molecule demonstrates that these arms are equivalent portions of the 4 monomers. Various types of immune complexes between α2M and IgG are described, and images of individual immune complexes are processed by correspondence analysis. This extracts new information concerning the organization of chymotrysin‐transformed α2M. The molecule appears asymmetrical, presents 2 conformational states (which we describe as relaxed and twisted), and has flexible arms. These intramolecular motions are supposed to be related to IgG binding. The results are discussed in comparison with previously published models of proteinase‐transformed α2M.

Crystals of the carboxyl-terminal functional unit from Octopus dofleini hemocyanin

The carboxyl-terminal oxygen-binding unit of the polypeptide from Octopus dofleini hemocyanin has been crystallized in a form suitable for three-dimensional X-ray analysis. This proteolytic fragment has a molecular weight of 47 kDa and reversibly binds O2 while exhibiting a slight Bohr effect. Two types of crystals have been grown. Type I crystals, currently under analysis, belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of 92.6 A x 167.4 A x 59.2 A. A composition of two protein molecules per asymmetric unit and 50% solvent content is consistent with a self-rotation function that identifies a non-crystallographic 2-fold axis of symmetry relating these molecules. Diffraction extending beyond 1.9 A Bragg spacings can be detected with synchrotron X-radiation.

Preliminary Results on the Structure of Octopus Dofleini Hemocyanin

The hemocyanin of Octopus dofleini has provided an excellent model for the study of the behavior of multi-subunit proteins (1–3). Sedimentation experiments indicated that the native molecule (Mr = 3.6 × 106, S20,w =51S) is a decamer of polypeptide chains (Mr = 3.6 × 105, S20,w = 11.1S). Preliminary electrophoretic studies suggested that there might be only a single kind of polypeptide chain, making this structure simpler than that of most molluscan hemocyanins. Furthermore, the molecular weight of the subunit is smaller than that of gastropod hemocyanins, suggesting that it might contain fewer than eight oxygen binding domains. We report here on our investigations of the structure of O. dofleini hemocyanin.