Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

Abstract

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.

Figure 1

Negative-ion ESI mass spectra of (A) fish pepsinogen A1 and (B) activated fish pepsin A1. The measured molecular weights were 38,845 Da for the pepsinogen form (theoretical mass: 38,850.6 Da) and 34,250 Da for the activated form (theoretical mass: 34,241.2 Da). The conversion of pepsinogen to pepsin leads to a mass shift of ~ 4600 Da due to the removal of the N-terminal residues 1–38. (C) Negative-ion ESI mass spectra of pig pepsin A from Sigma, as a control (theoretical mass: 34,583.8 Da; measured mass: 34,584 Da). (D). Commassie-stained SDS-PAGE gel of fish pepsin A1 before (lane 1) and after (lane 2) activation.

Figure 2

(A) Alignment of amino acid sequences of the Antarctic recombinant pepsins with pig pepsin. Multialignment was achieved using the program Clustal W version 1.83 [44]. The two aspartic acid residues present in the catalytic site are marked in gray. The missing residues in fish pepsin isoforms are boxed. (B) Model of fish pepsin A1 based on the crystal structure of pig pepsin (PDB code 5PEP, [21]), prepared as described previously [17]. The rendering on the right has been turned 90 degrees towards the observer, as shown, to look down onto the top of the active site. The conserved residues between pig and fish pepsin, as determined by the alignment, are colored green. The extra residues in pig pepsin that are boxed in panel A are shown in the position they occupy in the crystal structure of pig pepsin (5PEP) and are colored red. The remained of the pig pepsin residues are virtually superimposable on the fish pepsin A1 model. The two conserved aspartic acid residues present in the catalytic site are drwan as sticks and labeled. The synthetic phosphonate inhibitor IVA-Val-Val-Leu^P-(O)Phe-Ala-Ala-OMe (IVA = isovaleryl; Leu^P = phosphinic acid analogue of leucine; (O)Phe = L-3-phenyllactic acid) in the active site in this PDB file is shown in stick form with the N- and C-termini labeled. Modeling of the inhibitor binding to the fish pepsin model was obtained with overlaying the crystal structure of the model with human pepsin 3A in complex with the synthetic phosphonate inhibitor (PDP code 1QRP, [24]).

Figure 3

(A) Effect of pH on the activity of commercial pig pepsin and recombinant fish pepsin isoforms A1 and A2. Active fish isoenzymes were obtained as described in “Materials and Methods” (see section “Conversion of pepsinogen in the active form”). The relative activity has been expressed as a percentage of the highest activity over the pH range examined. Each data point represents the mean of three determinations. (B) Influence of temperature on enzymatic activity. Protease activity was determined using denatured hemoglobin as a substrate. Pepsin obtained from pepsinogen activation was added to the reaction mixture containing the buffer sodium citrate, pH 2.0. The assay was performed at various temperatures as described in “Material and Methods” The results are representative of three independent sets of measurements. Relative activity has been expressed as a percentage of the highest activity over the temperature range examined for the enzymes considered. (C) Effect of pepstatin A on fish and pig pepsins. Enzymes were assayed in the presence of increasing concentrations of pepstatin A using a 2.5 % hemoglobin as substrate. Pepsin activity was determined at pH 2.0 and 37 °C The fish and pig pepsin concentration in each assay was 0.021 µM. Legend: activity of commercial pepsin (■); activity of fish pepsin A1 (▲); activity of fish pepsin A2 (○).

Figure 4

Analysis of pepsin digestion of synthetic test peptides. (A) Amino-acid sequence of the two synthetic peptides used in this study. The P4 to P’3 nomenclature refers to the position of each residue. The arrow indicates the position of the scissile peptide bond between P1 and P’1. (B) Example of the HPLC elution profile observed at 280 nm for A1 digestion of synthetic peptide #1 (Legend: S = Substrate; P = Product). The digestion was performed in 100 mM sodium acetate buffer, pH 2.5, 0 °C using a 1 to 10 protease/substrate ratio (w/w). At defined time points, 600 pmoles were loaded onto a Jupiter 4u Proteo 90A column and peptides were eluted using a 5 to 70 % ACN gradient in 20 min. (C) Digestion of synthetic peptide #1 by pig pepsin, A1 and A2. Solid and empty symbols correspond to the substrate and the product, respectively. Arrows indicate time points at which additional amounts of fish pepsins were added [60 min: 4 µg (A1 and A2); 160 min: 4 µg (A2)]. (D) Digestion of synthetic peptide #2 by pig pepsin, A1 and A2. No digestion was observed with A1 and A2. Solid and empty symbols correspond to the substrate and the product, respectively.

Figure 5

(A) Comparison of the total ion chromatograms (TIC) obtained after digestion of CK-MM by pig pepsin, A1 and A2. The gray box corresponds to the region that was selected to compare the CK-MM peptide fragments generated after 3.5 min digestion on ice (see below). (B) Representative mass spectra comparing the different peptide fragments eluted after 12 min elution time. Identical ions are noted with a circle; non identical ions with a diamond. (C) MS-MS of the [M + 3H]³⁺ ion (m/z 850.49) of the peptide Met360-Lys381, generated after A2 digestion.

Figure 6

CK-MM peptide map generated after 3.5 min digestion on ice (protease/substrate ratio: 1/1 (w/w)) by the pig pepsin (black arrows), A1 (red arrows) and A2 (blue arrows). Secondary structural elements of CK-MM determined by X-ray crystallography [45] are shown above the sequence.

Figure 7

(A) Cleavage preference of pig, A1 and A2 pepsins at the P1, P1’ positions. The size of letters indicates the probability of cleavage. (B) Three dimensional structures of the monomeric form of CK-MM showing the regions that were extensively covered by the pig pepsin [Tyr39-Asp90 (green); Leu193-Phe250 (orange)], A1 [Asp335-Lys381 (red)] and A2 [Leu193-Val237 (blue)].