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.

In vivo fluorescence imaging of exogenous enzyme activity in the gastrointestinal tract

“…The activity of FM, MX, and SC PEPs was first characterized in vitro and their low stability in artificial GI fluids (Fig. S1A) was confirmed (21). Moreover, PEPs could not reduce the abundance of the peptide QLQPFPQPQLPYPQ PQPF from whole wheat gluten under conditions simulating in vitro GI digestion (Fig.…”

“…With this tool in hand, the enzymatic activity of PEPs was examined in vivo in female rats (see Movie S1 for a live illustration of the in vivo assay). FM and MX PEP were selected for the in vivo experiments because of their comparable in vitro cleavage pattern against gliadin peptides with similar chain length (21). The animals were gavaged with PEPs, followed by the labeled peptide 5 min later.…”

“…Unlike human GI enzymes, PEPs can efficiently hydrolyze proline-rich gluten peptides (9) and they have been shown to alleviate gluten immunotoxicity under simulated intestinal conditions (19,20). Earlier studies conducted with PEPs from Flavobacterium meningosepticum (FM) and Myxococcus xanthus (MX) revealed moderate enzyme stability in vitro under artificial GI conditions (21). Aspergillus niger (AN) PEP was recently investigated in an artificial compartmental system mimicking the human GI tract (TIM model).…”

Designing a Microbial Prolyl Peptidase Delivery System for the Treatment of Celiac Disease

“…Recently a new treatment strategy based upon the possibility of enzyme therapy using prolyl endopeptidases derived from bacteria or fungi has been proposed (Shan et al 2004, Stepniak et al 2006. These enzymes break down the immunodominant peptides by hydrolyzing proline-Xaa bonds.…”

“…Prolyl endopeptidases from Aspergillus niger and Myxococcus xanthus have been described previously (Stepniak et al, 2006;Shan et al, 2004). Four peptidases from L.…”