Human mesotrypsin defies natural trypsin inhibitors: from passive resistance to active destruction

Abstract

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.

Figure 1

Organization of the PRSS3 gene on chromosome 9p11.2. The GCGAGT sequence indicates the suboptimal splicing site, which probably facilitates alternative splicing of exon-1 and exon-1A. The location of the TATA-box containing promoter upstream of exon-1 is indicated. The promoter elements upstream of exon-1A have not been identified yet. Exons are not drawn to scale.

Figure 2

Inhibition of human mesotrypsin by soybean trypsin inhibitor (Kunitz). Left panel. Tight-binding inhibition of cationic trypsin (PRSS1), anionic trypsin (PRSS2), mesotrypsin (PRSS3) and R198G-mesotrypsin [13]. Trypsins were incubated with the indicated concentrations of soybean trypsin inhibitor at 22 °C in 100 μL of 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂ and 1.5 mg/mL bovine serum albumin for 10 min. Residual trypsin activity was then determined after addition of 100 μL N-CBZ-Gly-Pro-Arg-p-nitroanilide to 0.1 mM final concentration. Trypsin concentrations were 25 nM with the exception of wild-type mesotrypsin, which was used in 15 nM concentration. Right panel. Competitive inhibition of mesotrypsin. The K_M values for N-CBZ-Gly-Pro-Arg-p-nitroanilide were determined in the presence of the indicated concentrations of soybean trypsin inhibitor at 22 °C, in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂ and 1.5 mg/mL bovine serum albumin. The mesotrypsin concentration was 1 nM. The K_M values were plotted against the inhibitor concentration and the inhibitory constant (K_i) was determined from the x-axis intercept of the linear fit.

Figure 3

Digestion of trypsin inhibitors by mesotrypsin. Inhibitors and mesotrypsin were incubated at the indicated concentrations at 37 °C in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂ and 1 mg/mL bovine serum albumin, and residual inhibitory activity was determined against bovine trypsin. Alternatively, samples were precipitated with trichloroacetic acid and analyzed by reducing SDS-PAGE and Coomassie-blue staining. Left panel. Hydrolysis of the reactive site of soybean trypsin inhibitor (Kunitz). Right panel. Degradation of human pancreatic secretory trypsin inhibitor (Kazal) by mesotrypsin. Note that pancreatic secretory trypsin inhibitor stains poorly with Coomassie blue, and the relative band intensities do not reflect the actual concentrations of mesotrypsin and inhibitor. Figure modified from [13].

Figure 4

Activation of human trypsinogens by cathepsin B. Human cationic trypsinogen (PRSS1), anionic trypsinogen (PRSS2) and mesotrypsinogen (PRSS3) were activated at 2 μM concentration with human cathepsin B (90 μg/mL) at 37 °C in 0.1 M Na-acetate buffer (pH 4.0) in the presence of 1 mM dithiothreitol, 2 mg/mL bovine serum albumin, 1 mM K-EDTA, and 300 μM benzamidine. Aliquots (2.5 μL) were withdrawn at indicated times and trypsin activity was measured on the synthetic substrate N-CBZ-Gly-Pro-Arg-p-nitroanilide. Trypsin activity was expressed as percentage of potential maximal activity. Figure adapted from [13].