Mapping of Antigenic Determinants in Androctonus Australis Hemocyanin: Preliminary Results

Lamy, Jean; Billiald, Philippe; Sizaret, P.Y.; Taveau, Jean-Christophe; Boisset, Nicolas; Frank, Joachim; Motta, Geneviève

doi:10.1007/978-3-642-71481-8_33

Cited by 5 publications

(2 citation statements)

References 22 publications

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“…Obviously, the resolution is not adequate to validate this suggestion definitively. We aim, however, to resolve this issue by obtaining a higher resolution reconstruction, possibly combined with domain localization using, for instance, antibody labeling (42,43).…”

Section: Discussionmentioning

confidence: 99%

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Brink

Ludtke

Yang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites. O besity, a major health factor, is a measure of the fat deposited with adipose in consequence to food intake, fatty acid and triglyceride synthesis and oxidation, and energy homeostasis (1). Excess food provides not only the energy needs of the body but promotes the synthesis of fatty acids and triglycerides and storage of the latter in liver and adipose tissues. The de novo synthesis of fatty acids from acetyl-CoA and malonylCoA in the presence of the reducing substrate NADPH is catalyzed by the enzyme fatty acid synthase (FAS, ref. 2). Malonyl-CoA is the donor of the C 2 units required for chain elongation and is generated by acetyl-CoA carboxylase reactions, the rate-limiting step in the synthesis of fatty acids. In animal tissues including that of humans, FAS is a homodimer of a multifunctional protein (M r 272,000; refs. 2 and 3). Each subunit protein contains seven catalytic activities plus the acyl carrier protein (ACP). The organization of the catalytic activities along the polypeptide chain from the N to the C termini is as follows: ␤-ketoacyl synthase, acetyl͞malonyl transacylases, ␤-hydroxyacyl dehydratase, enoyl reductase, ␤-ketoacyl reductase, ACP, and thioesterase. Throughout the elongation process, the fatty acyl groups are linked as a thioester to the 4Ј-phosphopantetheinyl-SH, the prosthetic group of the ACP component of FAS. This prosthetic group is Ϸ20 Å long and is an essential flexible linker for the catalytic activities of FAS (3, 4). The organization of the partial activities, as mentioned above, is based on proteolytic mapping of the subunit and is confirmed by the predicted amino acid sequences based on the cloning and nucleotide sequences of the cDNA of the FAS component enzymes (2, 5-11). Moreover, the multifunctional human FAS and its subdomains were cloned and expressed in Escherichia coli; the recombinant proteins were catalytically active, and their orders on the FAS subunit were established (12, 13). Based on these studies, the component activities were grouped into three functional subdomains: domain I, containing ␤-ketoacyl synthase, acetyl͞malonyl transacylases, and ␤-hydroxyacyl dehydratase; domain II, containing enoyl reductase, ␤-ketoacyl reductase, and ACP; and domain III, containing thioesterase. Moreover, an intermediate polypeptide containing 640 amino acids, located between domains I and II and designated as the int...

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Section: Discussionmentioning

confidence: 99%

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Brink

Ludtke

Yang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…A schematic of an arthropod hemocyanin dodecamer. Positions of the subunits were taken from Lamy et al 36 The lower hexamer is shown parallel to the 3-fold axis with the subunits labeled A being the ones involved in trimer interactions. The upper hexamer is vie wed ~90°to the lower hexamer.…”

Section: Cooperative Ligand Bindingmentioning

confidence: 99%

Recent Structural Work on the Oxygen Transport Protein Hemocyanin

Magnus¹,

Ton-That²,

Carpenter³

1994

Chem. Rev.

383

227

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Intermolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing

et al. 1988

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Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.

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Mapping of Antigenic Determinants in Androctonus Australis Hemocyanin: Preliminary Results

Cited by 5 publications

References 22 publications

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Recent Structural Work on the Oxygen Transport Protein Hemocyanin

Intermolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing

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