Table 1.
List of genes used in phylogenetic analysis.
Figure 1.
Amino acid sequences of Perlucin from H. laevigata.
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 [60], repeat units (light blue bars). Peptides identified from 2D electrophoresis spots (Figure 3) by MALDI-ToF MS (black bars) or ESI-MS (red bar). *Predicted glycosylation and phosphorylation (NetPhos [50]) sites.
Figure 2.
Phylogenetic tree of Perlucin and related proteins.
DNA Fragments coding for proteins related to Perlucin were selected as described in the Methods section. The phylogenetic tree was generated using Geneious [36] plugin MrBayes [43] with HKY85 substitution model [41] and 4 invariant gamma rate categories, (HKY85 G+I) and type II antifreeze glycoprotein from the Atlantic herring (Clupea harengus) 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 Haliotis spec. Asialo = asialoglycoprotein receptor H1, Homo sapiens; CD209 = DC-SIGN, Homo sapiens; collectin12 = collectin sub-family member 12, Homo sapiens; DCSIGNR = sDC-SIGN1A type III, Homo sapiens; LSIGN, Homo sapiens; macrophlec2 = macrophage lectin 2, Homo sapiens; REG1B = regenerating islet-derived 1 beta, Homo sapiens; Scavrec = Scavenger receptor, Homo sapiens; versican = chondroitin sulfate proteoglycan 2, Homo sapiens; HlaevPerl0* = Perlucin R0, Haliotis laevigata; HvarPerl = putative Perlucin, Haliotis varia; Hasi551 = Haliotis asinina; Hasi499 = Haliotis asinina; Hddperl1 = putative Perlucins 1, Haliotis discus discus; HddPerl3 = putative Perlucin 3, Haliotis discus discus; HddPerl4 = putative Perlucin 4, Haliotis discus discus; HddPerl5* = putative Perlucin 5, Haliotis discus discus; HddPerl6 = putative Perlucin 6, Haliotis discus discus; HddPerl7 = putative Perlucin 7, Haliotis discus discus; HddPerl8 = putative Perlucin 8, Haliotis discus discus; HdivPerl = putative Perlucin, Haliotis diversicolor; HdivPerl1 = putative Perlucin 1, Haliotis diversicolor; HdivPerl4 = putative Perlucin 4, Haliotis diversicolor, HdivPerl5 = putative Perlucin 5, Haliotis diversicolor; HdivPerl6 = putative Perlucin 6, Haliotis diversicolor; Hmid1a1 = putative Perlucin, Haliotis midae; Venom1 = Venome gland lectin 1, Bungarus fasciatus; Venom2 = Venome gland lectin 2, Bungarus fasciatus; Venom3 = Venome gland lectin 3, Bungarus fasciatus.
Figure 3.
Electrophoretic analysis of recombinant and native Perlucin preparations.
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 H. laevigata, stained with Coomassie brilliant blue showed one distinct band at approx. 25 kDa, one at 20 kDa, and one at approx. 15 kDa. C) 2D electrophoresis of native Perlucin purified from abalone shell of H. laevigata, 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.
Table 2.
MALDI-ToF MS results.
Figure 4.
Purification of Perlucin splice variants fused with MBP.
Recombinant proteins were expressed in E. coli Rosetta (DE3) pLacI and purified via N-terminal His-tag and C-terminal Strep-tag of MBP-Perlucin-R0 (60 kDa), MBP-Perlucin-R5 (68 kDa) and MBP-Perlucin-R8 (72 kDa) and MBP (42 kDa) as described in the Methods section. (A) Samples representing the purification steps. (B) Purified proteins after dialysis.
Figure 5.
Enhancement of CaCO3 precipitation by MBP-Perlucin.
250 µL of 20 mM NaHCO3, 500 mM NaCl, pH 8.7 containing 100 µg/mL of the indicated protein were mixed with 250 µL 20 mM CaCl2, 500 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).
Figure 6.
Perlucin concentration dependency of CaCO3 precipitation.
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 µg MBP with 25 µg MBP-Perlucin-R8 (red); 25 µg MBP with 50 µg MBP-Perlucin-R8 (black); 75 µg MBP-Perlucin-R8 (blue).
Table 3.
Hydrodynamic diameter of recombinant Perlucin splice variants.