Quaternary structure of scorpion (Androctonus australis) hemocyanin. Localization of subunits with immunological methods and electron microscopy

Lamy, Jean; Bijlholt, Mmc; Sizaret, P.Y.; Vanbruggen, Efj

doi:10.1021/bi00510a021

Cited by 72 publications

(50 citation statements)

References 26 publications

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“…Thus, U. pusilla Hc can be defined as a tetramer of 1 × 6-meric building blocks, in contrast to the 4 × 6-meric Hc of Chelicerates where strong interactions exist between hexamers to build up the 2 × 6-meric half molecules. In the case of tarantula, scorpion and horseshoe crab Hcs the quaternary organization depends on a precise topology of the distribution of the various subunits within the oligomer (Lamy et al, 1981;Markl et al, 1981;Markl and Decker, 1992;Voit et al, 2000;Martin et al, 2007). In particular, linker subunits are responsible for the aggregation of the two 2 × 6-mers and for the highly hierarchical order of interactions (Voit et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

“…Two models are known for the ikosatetrameric Hc: the first one refers to chelicerate 4 × 6-mers, fully described for the scorpion Androctonus australis (Lamy et al, 1981) and the tarantula E. californicum (Erker et al, 2006;Voit et al, 2000); the second model has been proposed to describe the thalassinidean shrimps Hc, notably in Callianassa californiensis (Cavellec et al, 1990). It was shown by multivariate statistical analysis applied to computer aligned electron microscopic images that the 4 × 6 Hcs of A. australis and E. californicum are very similar (Bijholt et al, 1982) and fit the model earlier derived for the 4 × 6 half molecules of Limulus polyphemus.…”

Section: Introductionmentioning

confidence: 99%

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The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

Paoli

Giomi

Hellmann³

et al. 2007

Gene

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

Paoli

Giomi

Hellmann³

et al. 2007

Gene

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“…It is noteworthy that these values concerning three different cheliceratan species are significantly smaller than those (44-47 %) existing between the subunits a, d, and e of a single species, E. cal~fofnmicurn. This suggests a close stuctural relationship between the three subunits (A. uustralis chain Aa6, E. calfornicum chain e, and 7: tridentutus chain a ) which are known to occupy the same intramolecular location in the native hemocyanin oligomers (Lamy et al, 1981 ;Markl et al, 1981 ; Lamy, J., unpublished results) and to be immunologically related (Lamy et al, 1979b;Markl et al, 1984). The same observation has already been made, concerning the merostom L. polyphemus chain I1 and the arachnid E. californicum chain a, which show 40% difference between them (Kempter et al, 1985;Beintema et al, 1994).…”

Section: G R T D a A L D D L H E N T K F I H Y G ---Y D R A Y P D Kmentioning

confidence: 83%

“…Using an immunolabelling method with subunit-specific polyclonal Fab fragments followed by an observation of the immunocomplexes in the electron microscope, the intramolecular locations of the various subunit types composing the hemocyanin of the scorpion Androc.torzu.s australis were determined (Lamy et al, 1981). This work was repeated with the hemocyanins of the spider Eurypelmu culifiwnicum (Mark1 et al, 1981) and of the horse shoe crab L. pnlyphenzus (Lamy et al, 1983b;Lamy, 1987).…”

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confidence: 99%

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

Buzy

Gagnon

Lamy

et al. 1995

European Journal of Biochemistry

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The primary structure of the hemocyanin Aa6 subunit from the scorpion Androctonus u~istralis 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 8 9 0 k 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. uustmlis shares 405 identical residues with chain e of another arachnid, Eurypelrnu calijornicurn, and 399 with chain IX of the merostom Ec1zypleu.q trirleiztatus. 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. cal$ornicurn. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms.

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“…This method has supplied important information for other protein assemblies like the ribosome (Stoffler-Meilicke and Stoffler, 1987) or viruses (Wang et al, 1992). Electron microscopy of the multi-hexameric arthropod hemocyanins has yielded much insight relating to their quaternary structures (Lamy et al, 1980(Lamy et al, , 1981(Lamy et al, , 1982Markl et al, 1981 ;Bijlholt et al, 1982;Boisset et al, 1990;de Haas et al, 1991) and the methods developed for these investigations can also be applied to studies of interactions of hemocyanins with antibodies. A study by Lamy et al (1990) revealed four different epitopes by a combination of electron microscopy and classical immunological mapping for 13 different monoclonal antibodies binding to subunit Aa6 of scorpion (Androctonus australis) hemocyanin.…”

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confidence: 99%

Identification of two antibody‐interaction sites on the surface of Panulirus interruptus hemocyanin

Haas

Perton

Breemen

et al. 1994

European Journal of Biochemistry

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Negatively stained complexes of Panulirus interruptus (spiny lobster) hemocyanin with two different monoclonal antibodies, named E and J, were studied by electron microscopy and image processing. The attachment site of the antibodies to the hexameric hemocyanin molecule was deduced from two perpendicular views of hernocyanidantibody complexes, in which either the threefold axis or one of the twofold axes was oriented perpendicular to the supporting film. Images of complexes in these orientations were searched with reference images simulated from the known X-ray structure of P. interruptus hemocyanin.The two sites were further characterized by combining our results from electron microscopy with structural data obtained by X-ray diffraction and other methods. These two antibodies recognize different non-overlapping epitopes. The epitope for clone E is located on domain 3 at the surface of the p barrel and consists of certain loops, which form connections between /3-strand structures.The epitope for clone J is situated on domain 1 at the surface of an a-helical region and consists mainly of certain a-helices connecting loops.The orientation of the hemocyanin hexamers in the two complexes is very different, as is demonstrated most clearly when they form chains. Clone E forms complexes with the threefold axes perpendicular to the chain direction, while for clone J the threefold axes seem to be parallel to the main direction. The angle between the Fab part of an IgG molecule and the threefold axis of the hexamer is 60 5 5" for clone E and 35 5 7" for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.Hemocyanin is an extracellular, copper containing protein that occurs in high concentrations (10-100 mg/ml) in the blood of molluscs and certain arthropods belonging to the chelicerata (spiders, scorpions, horseshoe crabs) and crustacea (spiny lobsters, crabs; Van Holde and van Bruggen, 1971;Van Holde and Miller, 1982;Ellerton et al., 1983;Markl and Decker, 1991). It serves as an oxygen carrier, functionally similar to hemoglobin, but binds oxygen between two copper ions. The architecture of arthropod hemocyanin multimers consists of discrete assemblies of hexameric building blocks (Mr approximately 450000). The threedimensional X-ray structures of the hexameric hemocyanins from the spiny lobster (Panulirus interruptus ; Gaykema et al., 1984Gaykema et al., , 1986Volbeda and Hol, 1989) and of subunit I1 from the horseshoe crab (Limulus polyphemus; Hazes et al., 1993) are known in detail. Roughly, six bean-shaped subunits (Mr approximately 75000) occupy the corners of a trigonal antiprism.Hemocyanin has powerful antigenic properties used in many medical tests. Our interest concerns the structural features which cause these strong immunogenic reactions.Correspondence to E. F. J. van Bruggen, BIOSON Research Institute, Rijksuniversiteit Groningen, Nijenborgh 4, NL-9747 AG

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Quaternary structure of scorpion (Androctonus australis) hemocyanin. Localization of subunits with immunological methods and electron microscopy

Cited by 72 publications

References 26 publications

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

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

Identification of two antibody‐interaction sites on the surface of Panulirus interruptus hemocyanin

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