A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Abstract

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

“…Our findings rationalize the elevated levels of serum lipase found in patients undergoing DPP4 inhibitor based therapies 28, 29 , although these reports are in disagreement with other findings 30, 31 . While it is logical and expected to find scaffolds that are congruent to trypsin and DPP4 active sites in lipases based on the current results and our previous findings 22 , we also show the presence of the serine catalytic triad in close proximity to the active site residues of proteins which have a completely different enzymatic mechanism (for example, in glutaminyl cyclase which is a transferase 32 ). This corroborates the current belief that convergent evolution occurs more frequently than previously believed 33 .…”

“…Thus, in addition to the trypsin motif used previously (Asp102, Ser195 and His57 - PDBid 1A0J) 22 (Motif1), we choose another motif from a DPP4 enzyme (Asp708, Ser630 and His740 - PDBid:1N1M) (Motif2) ( Table 1). Apart from the catalytic triad, we chose another non-polar residue in order to increase the specificity of the matches (Ala56 in Motif1 and Val711 in Motif2).…”

“…DPP4 (EC 3.4.14.5), a serine protease that is expressed in many tissues (kidney, liver, lung, intestinal membranes, lymphocytes and endothelial cells), cleaves peptides with Pro or Ala residues in the second amino terminal position. Previously, we have experimentally demonstrated the existence of the serine protease domain in PI-PLC from Bacillus cereus - both by virtue of its proteolytic activity, and the inhibition of its native activity on phospholipids in the presence of serine protease inhibitors 22 . Furthermore, the specificity of the proteolytic activity indicated that it was a prolyl peptidase - thus, leading us to believe that DPP4 inhibitors should have a similar inhibitory effect on the PI-PLC enzyme.…”

“…CLASP analysis indicated that the phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus has spatial and electrostatic congruence with a serine protease motif 22 . This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC 50 = 0.018 mM).…”

“…Subsequently, we used a motif derived from a DPP4 protein 25 , in addition to the trypsin motif used previously 22 , to query a comprehensive and non-redundant (50% sequence identity) list of ~5000 human proteins with known structures using CLASP, intending to identify other proteins that might be inhibited by the gliptins. From the set of proteins with significant congruent matches with these two motifs, we identified a pancreatic lipase 26 and a gastric lipase 27 , keeping the context of lipases, acute pancreatitis and GLP-1 based therapies in mind.…”