Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans

Abstract

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.

Figure 1. Purification and characterization of recombinant shewasin D.

Wild-type shewasin D was produced in E. coli in a soluble form, fused to an N-terminal His-tag. A purification protocol was optimized as described in Methods. (A) Anion exchange chromatogram. Purified recombinant shewasin D (dotted lines, sample 1) was subsequently used in all characterization assays. (B) SDS-PAGE and Western blot (anti-His) analyses of sample 1, panel (A). (C) Analytical SEC of purified recombinant shewasin D. The dots indicate the elution volumes of molecular mass markers used for calibration (from left to right: aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; ribonuclease A, 13.7 kDa). (D) Effect of pH on the activity of recombinant shewasin D. Shewasin D was tested for activity using the fluorogenic peptide (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP) as substrate; activities were measured by incubating shewasin D at 37 °C with buffers between pH 2.5 and pH 5.5 containing 0.1 M NaCl. (E) Effect of temperature on the activity of recombinant shewasin D. Activity studies were performed by incubating shewasin D at temperatures between 5 and 50 °C, for 8 min, towards the same substrate. (F) Effect of protease inhibitors on the activity of recombinant shewasin D. The effect of different compounds was tested using the same fluorogenic substrate by preincubating the enzyme with each inhibitor for 8 min at 37 °C. ****P < 0.0001 compared with control using one-way ANOVA followed by Tukey’s post hoc test. The error bars represent standard deviation of the mean.

Figure 2. Shewasin D and shewasin A specificity preferences profiled by PICS.

Graphical representation of shewasin D (A,C) and shewasin A (B,D) specificity profiles by Heatmaps and IceLogos. Results are from Tryptic and GluC peptide libraries derived from a Homo sapiens proteome (THP1 cells) incubated with recombinant shewasin D or shewasin A at a ratio of 1:40 (enzyme/library). The analytical strategy applied was similar to that described in ref. . The average amino acid occurrences in P4–P4′ were calculated from one experiment for the trypsin library and two experiments for the GluC library and are shown in the form of two-dimensional heatmaps of log(2) transformed values of fold-enrichment over natural abundance of amino acids (A,B) and % difference IceLogos (C,D). Both tryptic and GluC display consistency between them. In IceLogos representation, horizontal axis represents the amino acid position and vertical axis denotes the over- and under-representation of amino acid occurrence compared with the Swiss-Prot Homo sapiens protein database. Cysteines are carboxyamidomethylated and lysines are dimethylated.

Figure 3. Differential IceLogo representation of shewasin D versus shewasin A specificity profile using trypsin PICS library.

The differential IceLogo represents the enriched and depleted residues for shewasin D (upper part of the IceLogo) using as reference set the peptides identified for shewasin A. Residues that are over- or under-represented in the experimental set are respectively shown in the top or bottom part of the IceLogo.

Figure 4. Partial purification of native shewasin D.

A soluble protein extract of S. denitrificans overnight cultures was prepared and subjected to two anion exchange chromatographic steps. (A) High Q chromatogram of soluble extract fractionation. Dotted lines delimit the fractions staining positive in the Western blot analysis with anti-shewasin D antibody. (B) Western blot analysis of samples 1 and 2 (panel A), with the anti-shewasin D antibody. (C) Mono Q chromatogram. Fractions 1 and 2 from (A) were pooled, diluted and subsequently applied to a Mono Q column. The enzymatic activity was tested towards the substrate (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP) and is represented by a discontinuous line. (D) Western blot analysis of fractions 13–18 eluted from Mono Q (C) with anti-shewasin antibody. (E) Effect of protease inhibitors on the activity of partially purified fractions of native shewasin D. The effect of different compounds was tested by preincubating protein sample (fraction 13, panel C) with each inhibitor for 8 min at 37 °C. ****P < 0.0001 compared with control using one-way ANOVA followed by Tukey’s post hoc test. (F) Effect of pH on the activity of partially purified fractions of native shewasin D. The assays were performed by incubating fraction 13 at 37 °C with buffers between pH 3.0 and 4.0. The error bars represent standard deviation of the mean.

Figure 5. Shewasin D is preferentially accumulated in the cytoplasm in Shewanella denitrificans.

Representative iEM images of Shewanella denitrificans immunogold labeled using anti-shewasin D (1:200) as primary antibody (A–D) or pre-immune serum (1:16 000) (E,F). Both membranes (arrowheads), the periplasm between them as well as the gold particles (arrows) can be discerned (B). Gold particles within 25 nm from the membrane bilayers are encircled (C,D). The white spaces surrounding the cells correspond to bacterial capsules. Scale bar represents 2 μm (A,E), 1 μm (B,F), 100 nm (B, inset), 500 nm (C) and 200 nm (D).