Androctonus Australis Hemocyanin Research Articles

Complete Ammo Acid Sequence of the Aa6 Subunit of the Scorpion Androctonus australis Hemocyanin Determined by Edman Degradation and Mass Spectrometry

The primary structure of the hemocyanin Aa6 subunit from the scorpion Androctonus australis was resolved by using protein sequencing and mass spectrometry for analysis of the polypeptide chain and of fragments obtained by CNBr, trypsin, and chymotrypsin cleavage. Due to the high sensitivity of the methodologies used, only a small amount of material, less than 1 mg, was consumed. The complete sequence is composed of 626 amino acid residues and the protein is not glycosylated but probably phosphorylated at Ser374. Its molecular mass measured by mass spectrometry (71,890 +/- 7 Da) is about 30 Da higher than the mass calculated from the sequence data (71,860.1 Da). The origin of this difference is not clear but could result from minor molecular heterogeneities. Within the chelicerates, the Aa6 subunit of the arachnid A. australis shares 405 identical residues with chain e of another arachnid, Eurypelma californicum, and 399 with chain alpha of the merostom Tachypleus tridentatus. The degrees of identity between these three subunits, which are known to occupy the same location in the native hemocyanin oligomers, are significantly higher than those existing between the subunits a, d, and e of E. californicum. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms.

Production of a Functional Anti-Scorpion Hemocyanin ScFv in Escherichia coli

A58 is a murine monoclonal antibody raised against the internal dimeric subunit Aa 1 of the hemocyanin of the scorpion Androctonus australis. When A58 is tested against a range of related hemocyanins, it exhibits cross-reactivity with a limited number of scorpion species. All of them belong to a single Scorpion Family: the Buthidae. We have utilized a bacterial expression-export system in which the variable regions of A58 are expressed as a single-chain variable fragment (scFv). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into a scFv. After insertion into a vector which contains the pectate lyase signal sequence from Erwinia caratovora for periplasmic expression, a scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced A58 scFv has binding properties similar to those of the original murine antibody. It binds specifically the heterodimeric subunit of Androctonus australis hemocyanin, cross-reacts with only one subunit of each Buthidae investigated, and blocks the recognition of the hemocyanin by antibody A58 in competitive enzyme-linked immunosorbant assay. Previous reports have demonstrated the value of monoclonal antibodies in taxonomical and molecular evolution studies. A58scFv is the first of a new generation of antigen-binding proteins which should prove useful both in taxonomical studies and in the analysis of VH-VL structure-function relationships of antibodies.

Three-dimensional reconstruction of native Androctonus australis hemocyanin

A sample of native 4 × 6-meric hemocyanin of Androctonus australis was negatively stained with the double-layer technique, and was observed by transmission electron microscopy under low-dose conditions with a 50 ° and 0 ° tilt. The three-dimensional reconstruction method from “Single-exposure, random conical tilt series” was then applied. Independent three-dimensional reconstructions were obtained from the top, side and 45 ° views. Despite a pronounced flattening effect, presumably due to the specimen preparation technique, the positions of the 24 subunits composing the oligomer were unequivocally determined. This experiment definitely solves the problem of the architectural organization of the subunits in the cheliceratan 4 × 6-meric hemocyanins. Moreover, distinction between the flip and flop faces and an attenuated rocking effect were observed.

Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin

A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)

Arthropod hemocyanin structure: Isolation of eight subunits in the scorpion

Eight immunologically pure subunits were isolated from Androctonus australis hemocyanin. Antisera specific against each of these were prepared. Two subunits associate to form a heteroöligomer which is probably one of the bridges visible in electron microscopy of the native molecule. There is no cross-reactivity between native subunits. When denatured by 7 m urea, the specific antigenic determinants disappear and a broader specificity appears. This is probably due to the unfolding of the molecule which unmasks deeply buried antigenic sites.

Ultrastructure of 16 S substances obtained by reassociation using different combinations of three isolated subunits of scorpion hemocyanin ( Androctonus australis garzonii)

Different combinations of three subunits of Androctonus australis hemocyanin, isolated by the method of Lamy et al. (1976) and treated with divalent cations at pH 7.5, give reassociation products which, after isolation by thin layer gel chromatography, permit the determination of the subunit composition and the ultrastructure. Subunit 4 as well as its binary and ternary compositions containing subunit 2 give intermediate products. Hexamers corresponding to ternary mixtures are more stable than the others and tend to adhere to each other in two's or three's.