3D-Elektronenmikroskopie (3,5 Å) und gerichtete Mutagenese des dodekaedrischen Acetylcholin-Bindeproteins der Posthornschnecke Biomphalaria glabrata
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Abstract
Acetylcholine binding proteins (AChBP) occur in the hemolymph of molluscs and annelids. AChBP is structurally and functionally homologous to the extracellular ligand-binding domain of the nicotinic acetylcholine receptor (nAChR). The tropical freshwater snail Biomphalaria glabrata (Planorbidae) has a special AChBP, which forms 25 nm regular dodecahedron out of twelve pentamers. In the present study, a 3D-reconstruction with a resolution of 3.5 Å was generated by recombinant BgAChBP1-dodecahedrons dissolved in pure water solved by 3D-electron microscopy. Applying site directed mutagenesis and subsequent electron microscopy, the importance of certain amino acids for the contacts between the pentamers was investigated. Further evidence was gained on the existence of a dipentamer in BgAChBP2; This aspect could not be finally clarified. In addition, evidence has been gathered that recombinant BgAChBP1 binds amorphous calcium carbonate and is maybe involved in the formation of the snail shell. Recombinant BgAChBP2 appears to bind amorphous calcium carbonate with low affinity.
In the native planorbid snail Planorbarius corneus AChBP dodecahedra were detected in the hemolymph and biochemically enriched. Transcriptome analysis provided three AChBP sequences, two of them were complete and the third was almost complete. As declined from phylogenetic and biochemical analysis, no protein in P. corneus has been found which is orthologous to BgAChBP1. The dodecahedra obviously has been formed by an AChBP similar to BgAChBP2. This suggests that the BgAChBP2 also forms dodecahedra in the haemolymph of B. glabrata. However the recombinantly expressed BgAChBP2 is (so far) unable to form dodecahedra. This could be due to a certain disulfide bridge, which could be crucial for the oligomerization of two pentamers in BgAChBP2.