Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue

Abstract

Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

Figure 1. Resistance of the FM and the MX prolyl endopeptidases to inactivation by gastric and pancreatic enzymes

Pancreatic enzyme stability was evaluated by treating 5 units/ml of the FM and the MX enzymes with 1 mg/ml trypsin, 1 mg/ml chymotrypsin, 0.2 mg/ml elastase and 0.2 mg/ml carboxypeptidase A (40 mM phosphate, pH 6.5). Pepsin stability (gastric) was tested by treating FM and MX (5 units/ml) with 1 mg/ml pepsin (pH 2, 20 mM HCl).

Figure 2. Site specificity of PQPQLPYPQPQLP hydrolysis by individual prolyl endopeptidasess

UV HPLC (215 nm) traces are shown for each reaction mixture. Initial cleavage fragments (100 μM PQPQLPYPQPQLP, 0.1 μM enzyme, t=5 min) were identified by tandem MS. The starting material PQPQLPYPQPQLP and the cleavage fragments (A, PQPQLPYP; B, YPQPQLP; C, PQPQLP; D, QPQLP) are indicated in the traces. AU, arbitrary units of absorbance.

Figure 3. Hydrolysis of the 33-mer peptide by FM, MX and SC prolyl endopeptidases

(A) Time dependence of hydrolysis in the presence of 10 μM substrate and 0.1 μM enzyme. The substrate appears as a doublet at a retention time of approx. 18 min, due to the presence of equal quantities of the 32-mer from which the N-terminal leucine residue is deleted; presence of this contaminant does not affect analysis. From the residual peak areas, the rates of substrate (33-mer+32-mer) disappearance were calculated as 2.3 μM/min (FM), 0.43 μM/min (MX) and 0.07 μM/min (SC). (B) Initial cleavage fragments observed due to hydrolysis by the FM enzyme (t=1 min) and the MX enzyme (t=5 min). (C) Summary of initial cleavage fragments from FM and MX prolyl endopeptidases catalysed hydrolysis of the 33-mer substrate. MW, molecular mass.

Figure 4. Competitive proteolysis of PQPQLPYPQPQLP (50 μM) and the 33-mer peptide (10 μM) in the presence of 30 mg/ml pepsin-treated gluten

This complex mixture of substrates was treated under physiological conditions with a mixture of pancreatic enzymes (trypsin, chymotrypsin, carboxypeptidase and elastase), BBM enzymes (derived from rat small intestine) and either (A) FM or (B) MX. For details, see the text.

Figure 5. Proteolysis of the 33-mer peptide (5 μM) co-perfused with individual prolyl endopeptidases (0.1 μM) in the small intestinal lumen of an anesthetized rat

Each enzyme-substrate mixture was introduced via a catheter into a 15–20 cm segment of the upper jejunum. Samples were collected at the other end of the segment, and analysed by UV HPLC (at a wavelength of 215 nm). The control without any prolyl endopeptidases is shown in the top trace. AU, arbitrary units.