Table 1.
Results for the Amphibalanus amphitrite shell using the XRF-technique (quantitative analysis).
Fig 1.
PAGE image showing protein extracts of the Amphibalanus amphitrite shell.
The gel was stained by SYPRO Ruby dye (Invitrogen) and the later coomassie blue staining confirmed the molecular weight of each band in the marker. Lane I: acetic acid fraction; Lane II: 1% SDS fraction; Lane III: 10% SDS fraction. Marker from top to bottom: 170, 130, 93, 70, 53, 41, 30, 22, 14 and 9 kDa.
Fig 2.
Comparison of an Asp-rich shell matrix protein from Amphibalanus amphitrite with Asprich (Atrina rigida; AAU04808.1) and Aspein (Pinctada fucata; AB094512).
The Asp-rich shell matrix protein from A. amphitrite mainly contained Asp punctuated with Glu or Gly, which are labeled in bold with a single underline. Asprich includes three poly(Asp) motifs (presented in block with a single underline) and two DEAD motifs (presented in bold with a double underline). Aspein consists of mostly poly(Asp) blocks punctuated with Ser-Gly dipeptides (presented in bold with a single underline).
Fig 3.
Sequence analysis of serine proteases and chorionic proteinase inhibitor from Amphibalanus amphitrite.
(A). Alignment of three serine proteases from A. amphitrite (I, II, and III) with Pacifastacus leniusculus (ACB41380), Drosophila sechellia (XP002036473.1), Nasonia vitripennis (NP001155060), Camponotus floridanus (EFN72618.1), and Cerapachys biroi (EZA53191.1). The three catalytic triad active-site residues (H, D and S) were all conserved in the three serine protease isoforms from A. amphitrite and marked with an asterisk. (B). Signal peptide prediction in serine protease I from A. amphitrite. The first 16 amino acids were predicted to be a signal peptide. C-score (raw cleavage site score), S-score (signal peptide score) and Y-score (combined cleavage site score) were calculated using the SingalP 4.1 Server. (C). Motif scanning revealed that the chorionic proteinase inhibitor from A. amphitrite contained 1 transmembrane domain (blue rectangle) and 5 WAP (whey acidic protein) domains (yellow triangle). (D). Signal peptide prediction suggested that the first 16 residues represented a signal peptide in the chorionic proteinase inhibitor.
Table 2.
Physical and chemical parameters of three serine protease isoforms and chorionic proteinase inhibitor in Amphibalanus amphitrite.
Fig 4.
Alignment of the carbonic anhydrase from Amphibalanus amphitrite (I, II and III), Daphnia pulex (EFX81683.1), Danio rerio (NP571185.1) and Hyriopsis cumingii (AHY35316.1).
Putative histidine residues that function as zinc ligands are marked with filled circles above the residues. The residues forming the hydrogen-bonded network to zinc-bound solvent molecules are depicted by open circles.
Fig 5.
Alignment of the deduced chorion peroxidase/peroxinectin from Amphibalanus amphitrite, Acyrthosiphon pisum (XP001946672.2), Eriocheir sinensis (ADF87945.1), Macrophthalmus japonicas (AID47197.1), and Penaeus monodon (AAL05973.1).
The putative integrin-binding motif is indicated by a red box, and in A. amphitrite, this motif was mutated to YGD (Tyr-Gly-Asp) rather than the canonical sequence RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp).
Fig 6.
Alignment of C-type lectin-like domains from Amphibalanus amphitrite (I and II), Scylla paramamosain (ADF27340.1), Fenneropenaeus indicus (ADV17348.1), Litopenaeus vannamei (AGV68681.1), and Danio rerio (XP005172687.1).
Similarly to S. paramamosain and F. indicus, the typical "EPD" or "EPN" motif in both C-type lectin-like domains (I and II) was mutated into "QPD" in A. amphitrite (boxed in red). At least two and four cysteines were conserved in the C-type lectin-like domains I and II, respectively. These conserved cysteines are indicated by arrows.
Fig 7.
Hematoxylin-eosin (A) and DAPI staining (B) showing cells in histological sections of the Amphibalanus amphitrite shell.
Arrows identify potential cell structures.