List of genes used in phylogenetic analysis.

<p>List of genes used in phylogenetic analysis.</p

Splice Variants of Perlucin from <i>Haliotis laevigata</i> Modulate the Crystallisation of CaCO₃

<div><p>Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone <i>Haliotis laevigata.</i> A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone <i>H. laevigata</i>. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in <i>E. coli</i> showed strong differences in their influence on precipitating CaCO₃ and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.</p></div

MALDI-ToF MS results.

<p>Trypsin digested peptides extracted from 2D electrophoresis were analysed by MALDI-ToF MS. Masses of peptides were used to search an in house SwissProt database in all entries mode. Sequences of peptides are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone-0097126-g001" target="_blank">Figure 1</a>.</p

Electrophoretic analysis of recombinant and native Perlucin preparations.

<p>A) Western blot of cell culture supernatants derived from COS-7 cells ectopically over expressing the indicated Strep-tagged recombinant Perlucins, which were detected using a polyclonal anti-Strep-tag antibody as described in the Methods section. B) SDS-PAGE of native Perlucin purified from abalone shell of <i>H. laevigata</i>, stained with Coomassie brilliant blue showed one distinct band at approx. 25<b> </b>kDa, one at 20<b> </b>kDa, and one at approx. 15<b> </b>kDa. C) 2D electrophoresis of native Perlucin purified from abalone shell of <i>H. laevigata</i>, stained with Coomassie Brilliant Blue. The indicated spots were cut out and analysed by MALDI-ToF MS as described in the Methods section. Spot 7 was used as control.</p

Perlucin concentration dependency of CaCO₃ precipitation.

<p>In order to obtain equal protein concentrations in all assays, the following mixtures of MBP and MBP-Perlucin-R8 were used in CaCO₃ precipitation assays as described in the Methods section: 50<b> µ</b>g MBP with 25<b> µ</b>g MBP-Perlucin-R8 (red); 25<b> µ</b>g MBP with 50<b> µ</b>g MBP-Perlucin-R8 (black); 75<b> µ</b>g MBP-Perlucin-R8 (blue).</p

Phylogenetic tree of Perlucin and related proteins.

<p>DNA Fragments coding for proteins related to Perlucin were selected as described in the Methods section. The phylogenetic tree was generated using Geneious <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Drummond1" target="_blank">[36]</a> plugin MrBayes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Huelsenbeck1" target="_blank">[43]</a> with HKY85 substitution model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Hasegawa1" target="_blank">[41]</a> and 4 invariant gamma rate categories, (HKY85 G+I) and type II antifreeze glycoprotein from the Atlantic herring (<i>Clupea harengus</i>) as out-group. Highlighted in pink are the putative Perlucins, in blue, Perlucin–like proteins and in green, proteins with C-type lectin domain from <i>Haliotis</i> spec. Asialo = asialoglycoprotein receptor H1, <i>Homo sapiens</i>; CD209 = DC-SIGN, <i>Homo sapiens</i>; collectin12 = collectin sub-family member 12, <i>Homo sapiens</i>; DCSIGNR = sDC-SIGN1A type III, <i>Homo sapiens</i>; LSIGN, <i>Homo sapiens</i>; macrophlec2 = macrophage lectin 2, <i>Homo sapiens</i>; REG1B = regenerating islet-derived 1 beta, <i>Homo sapiens</i>; Scavrec = Scavenger receptor, <i>Homo sapiens</i>; versican = chondroitin sulfate proteoglycan 2, <i>Homo sapiens</i>; HlaevPerl0* = Perlucin R0, <i>Haliotis laevigata</i>; HvarPerl = putative Perlucin, <i>Haliotis varia</i>; Hasi551 = <i>Haliotis asinina</i>; Hasi499 = <i>Haliotis asinina</i>; Hddperl1 = putative Perlucins 1, <i>Haliotis discus discus</i>; HddPerl3 = putative Perlucin 3, <i>Haliotis discus discus</i>; HddPerl4 = putative Perlucin 4, <i>Haliotis discus discus</i>; HddPerl5* = putative Perlucin 5, <i>Haliotis discus discus</i>; HddPerl6 = putative Perlucin 6, <i>Haliotis discus discus</i>; HddPerl7 = putative Perlucin 7, <i>Haliotis discus discus</i>; HddPerl8 = putative Perlucin 8, <i>Haliotis discus discus</i>; HdivPerl = putative Perlucin, <i>Haliotis diversicolor</i>; HdivPerl1 = putative Perlucin 1, <i>Haliotis diversicolor</i>; HdivPerl4 = putative Perlucin 4, <i>Haliotis diversicolor</i>, HdivPerl5 = putative Perlucin 5, <i>Haliotis diversicolor</i>; HdivPerl6 = putative Perlucin 6, <i>Haliotis diversicolor</i>; Hmid1a1 = putative Perlucin, <i>Haliotis midae</i>; Venom1 = Venome gland lectin 1, <i>Bungarus fasciatus;</i> Venom2 = Venome gland lectin 2, <i>Bungarus fasciatus</i>; Venom3 = Venome gland lectin 3, <i>Bungarus fasciatus</i>.</p

Enhancement of CaCO₃ precipitation by MBP-Perlucin.

<p>250<b> µ</b>L of 20<b> </b>mM NaHCO₃, 500<b> </b>mM NaCl, pH 8.7 containing 100<b> µ</b>g/mL of the indicated protein were mixed with 250<b> µ</b>L 20<b> </b>mM CaCl₂, 500<b> </b>mM NaCl, pH 8.7 and the pH was recorded to monitor CaCO₃ precipitation as described in the Methods section. The average pH values of three independent measurements (coloured) with standard deviations (grey) are shown. A) Control reactions with MBP (pink), BSA (brown), or without protein (black). B) MBP-Perlucin-R0 (blue), MBP-Perlucin-R5 (red), MBP-Perlucin-R8 (black).</p

Amino acid sequences of Perlucin from <i>H. laevigata</i>.

<p>Perlucin splice variants (Perlucin-R0, Perlucin-R5, Perlucin-R8) are indicated as grey bars above the sequence. The following characteristics of the proteins are marked: Amino acid exchanges in Perlucin-R0 (M89I, V129D, R149L), signal peptide, C-type lectin domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Drickamer1" target="_blank">[60]</a>, repeat units (light blue bars). Peptides identified from 2D electrophoresis spots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone-0097126-g003" target="_blank">Figure 3</a>) by MALDI-ToF MS (black bars) or ESI-MS (red bar). *Predicted glycosylation and phosphorylation (NetPhos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Blom1" target="_blank">[50]</a>) sites.</p

Hydrodynamic diameter of recombinant Perlucin splice variants.

<p>Hydrodynamic diameters were determined as described in the Methods section after storing in 20<b> </b>mM NaHCO₃ 500<b> </b>mM NaCl pH 8.7 for one or two months. Theoretical hydrodynamic diameters were calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Fu2" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097126#pone.0097126-Wilkins1" target="_blank">[48]</a>.</p

Biochemical Diversity in the <i>Trypanosoma congolense</i> Trans-sialidase Family

<div><p>Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in <i>Trypanosoma congolense</i>. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in <i>T. congolense</i> will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.</p></div