Identifikation und Charakterisierung von Perlucin Spleißvarianten der marinen Abalone Haliotis laevigata.

Abstract

Mother of pearl is a polymer of significance for material sciences due to its fascinating properties. A frequently investigated protein in mother of pearl from the Australian Abalone Haliotis laevigata is the protein Perlucin. Despite its importance relatively little is known about the role of Perlucin in detail in the biomineralisation of mother of pearl. After SDS-PAGE and staining with Coomassie the native protein from H. laevigata shows a heterogenic band pattern (Blank et al., 2001 [1] und Weiss et al., 2000 [2]) postulated to be an expression of Perlucin isoform variability. Focus of the submitted thesis is to test this hypothesis by synthesizing cDNA from the mantle epithelium of the Australian Abalone H. laevigata. Assembling degenerated primer based on the amino acid sequence of the native Perlucin of H. laevigata (Mann et al., 2000 [3]) seven previously unknown splice variants of the protein have been identified. These splice variants differ mainly in the number of a ten amino acid long peptide (repeat) with zero, two, three, five, six or eight repetitions at the C-terminus (of the protein). Three of the seven splice variants have been characterised in detail: Perlucin-R0 (no repeat), Perlucin-R5 (five repeats) and Perlucin-R8 (eight repeats). The splice variants as well as the Repeat-peptide have been identified in the organic matrix of the shell from H. laevigata using MALDI-ToF MS. For subsequent experiments the Perlucin splice variants were fused with the maltose-binding protein (MBP) using a bacterial expression system. After tandem purification via the N-terminal His-Tag and a C-terminal Strep-Tag very pure MBP-Perlucin splice variants were obtained. Native Perlucin can lead to a faster precipitating of CaCO3 (Weiss et al., 2000 [2]) consisting of a heterogeneous protein mixture potentially containing the here newly identified splice variants. The here presented thesis demonstrates that the different variants expressed as MBP fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3. These differences might be due to a splice variant specific formation of large protein aggregates influenced by the number of the 10mer peptide. In the presence of the MBP-Perlucin splice variants, the precipitation of CaCO3 leads to precipitates with well-defined surface structures, which might grew first followed by a tree structure, but the passing into a very regular layer-by-layer structural pattern. The newly discovered Perlucin splice variant could potentially have multiple functions during the mineralisation of CaCO3. First, they could act as a nucleation-seed by oligomerisation and formation of aggregates of up to 1 µm and second, they could have a modulatory effect on the mineralisation of CaCO3, influenced by the differing length of the repeat-region. Phylogenetic comparison of H. laevigata Perlucin with other related proteins from the GenBank database showed that all Perlucins differ substantially from C-type lectins of nematodes, snakes, fish and human. The phylogenetic study in this thesis defined five proteins as Perlucin. Based on a subsequent phylogenetic classification in the C-type lectin group, these five proteins can be assigned to the Tetranectin-family and Collectins.