Table 1.
Primers used in the real-time RT-PCR assay.
Fig 1.
Phylogenetic analysis of APEHs proteins in the vertebrata subphylum.
The cladogram includes the sequences retrieved from 22 organisms, two of which are invertebrates and were used as sister groups in the analysis. Numbers at nodes represent the confidence limits computed by the bootstrap procedure (100 replicates). All APEH sequences were found distributed into two distinct clusters: cluster 1 (C1) and cluster 2 (C2).
Fig 2.
Total exopeptidase activity of APEH measured in (A) liver and (B) blood of T. bernacchii, D. labrax and C. hamatus.
All the enzymatic activities were measured at 37°C using Ac-Met-AMC as substrate and expressed in arbitrary units. The results shown are representative of three independent experiments.
Fig 3.
SDS-PAGE and immunoblotting analyses of APEH isoforms from the Antarctic and temperate fishes C. hamatus and D. labrax.
(A) SDS-PAGE analysis of APEH-1Dl purified from blood. Lane 1, Broad range (6.5–200 kDa) molecular weight markers (MWM) (Sigma-Aldrich); lane 2, APEHpl from porcine liver used as control; lane 3, APEH-1Dl purified from blood; (B) Immunoblotting analysis of APEH-1Dl. Lane 4, purified APEH-1Dl from blood; lane 5, APEHpl; lane 6, APEH-1Dl partially purified from liver. (C) SDS-PAGE analysis of APEH-1Ch. Lane 1, MWM Color Plus Prestained (BioLabs); lane 2, APEH-1Ch purified from blood; lane 3, APEHpl. (D) Immunoblotting analysis of APEH-1Ch. Lane 4, APEH-1Ch purified from blood; lane 5, APEHpl. (E) SDS-PAGE analysis of APEH-2Ch. Lane 1, MWM as well as lane 1 in A; lane 2, APEHpl; lane 3, APEH-2Ch purified from blood. (F) Immunoblotting analysis of APEH-2Ch. Lane 4, APEH-2Ch purified from blood; lane 5, APEHpl. The results shown are representative of three independent experiments.
Fig 4.
Effects of temperature and pH on catalytic parameters of C. hamatus APEH isoforms as compared with those of APEH-1Dl from D. labrax.
(A) pH–activity profiles of APEH-1Ch (▲), APEH-2Ch (■) and APEH-1Dl (•). (B) Influence of temperature on enzyme activities. (C) Temperature–Km profiles and (D) pH–Km profiles of APEH-1Ch (▲), APEH-2Ch (■) and APEH-1Dl (•) compared with those of APEH-1Tb (△) and APEH-2Tb (□) previously described [11] from T. bernacchii. Effect of temperature on (E) kcat and (F) catalytic efficiency (kcat/Km) of analyzed APEHs. All experiments were performed in triplicate on three different protein preparations, using Ac-Met-AMC as substrate. Data are expressed as means ± standard deviations.
Table 2.
Optimal kinetics parameters of C. hamatus APEHs in comparison with those from D. labrax.
Fig 5.
Oxidised protein endohydrolase (OPEH) activity using BSA as substrate.
(A) SDS-PAGE analysis of oxidised BSA incubated at 37°C without enzyme used as control. (B) Representative SDS-PAGE profile of oxidised BSA treated with APEH-1Ch and APEH-1Dl at 37°C. The same results were obtained after incubation of unoxidised BSA with all APEH isoforms (data not shown). (C) SDS-PAGE analysis of oxidised BSA treated at 37°C with APEH-2Ch. (D) Electrophoretic data expressed as percentage density of BSA at the indicated incubation times versus time 0 obtained by densitometric analysis with CHEMIDOC XRS and QUANTITY ONE software. Oxidised BSA levels after incubation with APEH-2Tb were reported as comparative data [12]. (E) Exopeptidase (APEH) vs endoprotease (OPEH) activity of APEH-2Ch and APEH-2Tb. All the experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate are expressed as means ± standard deviation (values lower than 5% are not shown).
Fig 6.
3D models of APEHs from C. hamatus.
The structural architecture of APEH-1Ch (panel A) and APEH-2Ch (panel B) shows an N-terminal domains characterized by β strands (yellow arrows) while the C-terminal domains are characterized by β strands and α helices (red cylinders). Note that some non-structured segments on the top of the figure are segments modelled without reference in the template, due to gaps in the alignment. In panel C, we show the effects of rotations of 20°, 40°, 60° and 80° around a backbone bond of APEH-2Ch along the segment 451–460, which connects the two domains. In this specific example, the rotation has been performed at the N-Cα bond of 454 residue. Similar effects were observed by rotating other backbone bonds (both N-Cα or Cα-C) at different positions of the 451–460 segment.
Table 3.
Charges distribution, inter-domain salt bridges and H-bonds in the 3D models of APEHs from T. bernacchii and C. hamatus.
Fig 7.
Expression analysis of apeh genes from C. hamatus and D. labrax.
Analysis of the expression levels of apeh-1 and apeh-2 genes in cells and tissues of (A) C. hamatus and (B) D. labrax, normalised respect to the β-actin gene. The inset in the upper box of panel A contains a magnification for tissues with low expression levels. (C) The ratios of the expression folds obtained for the two apeh genes (apeh-2/apeh-1) in C. hamatus and D. labrax are shown compared to the relative values found in T. bernacchii [12]. All data are expressed as mean expression fold from triplicates.