Dependent Peptidase Research Articles

Structure and functional analysis of theLegionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

Characterization of gene expression profiles and functional analysis of peptidoglycan recognition protein 2 from rock bream (Oplegnathus fasciatus)

Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6–53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection.

The Mechanism of Peptide Hydrolysis Catalysed by Dipeptidyl Peptidase III from Bacteroides thetaiotaomicron

Dipeptidyl peptidase III (DPP III) is a zinc- dependent peptidase that cleaves dipeptides off of N-termini of its substrates. Previous studies on human DPP III reveal a reaction mechanism similar to that of thermolysin. Since the active site is conserved within the DPP III family, it is not surprising that the mechanism determined for Bacteroides thetaiotaomicron DPP III (BtDPP III) closely resembles that of hDPP III. However, the hydrogen bond network within the model differs slightly from that in the human ortholog, which results in two proposed pathways. The calculated Gibbs activation energy of 90.1 kJ mol–1 is larger than the one calculated from kinetic data for the preferred substrate Arg2-2-naphthylamide at room temperature (69 kJ mol–1), suggesting the importance of treating the whole DPP III enzyme in the calculations.

Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

Understanding the Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN)

Aminopeptidase N (PepN) from E. coli is an M1 class, Zn(II)‐dependent peptidase with broad specificity. PepN was mechanistically characterized in an effort to understand the role of tyrosine 381. pH Dependence kinetic studies revealed a log kcat versus pH and kcat/KM versus pH curves with inflection points at a pH of 9.0. Solvent isotope studies suggest at least one rate‐limiting proton transfer in the mechanism. In an effort to characterize the functional roles of binding region components, several active‐site amino acids were mutated. A tyrosine to alanine mutated PepN (Y381A) was successfully over‐expressed, purified, and characterized. Y381A‐PepN maintained Zn(II) binding efficiency but exhibited greater than 900‐fold loss of activity (kcat) when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product. This work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Miami University.

LPS activates ADAM9 dependent shedding of ACE from endothelial cells

Angiotensin-I converting enzyme (ACE) is a zinc dependent peptidase with a major role in regulating vasoactive peptide metabolism. ACE, a transmembrane protein, undergoes proteolysis, or shedding, by an as yet unidentified proteinase to release a catalytically active soluble form of the enzyme. Physiologically, soluble ACE in plasma is derived primarily from endothelial cells. We demonstrate that ACE shedding from confluent endothelial cells is increased in response to bacterial lipopolysaccharide, but not phorbol esters. Characterisation of lipopolysaccharide stimulated shedding showed that there is a lag phase before soluble ACE can be detected which is sensitive to inhibitors of translation, NF-κB, TNFα and TNFR-I/II. The shedding phase is less sensitive to these inhibitors, but is ablated by BB-94, a Matrix Metalloproteinase (MMP)/A Disintegrin and Metalloproteinase (ADAM) inhibitor. Tissue Inhibitor of Metalloproteinase (TIMP) profiling suggested a requirement for ADAM9 in lipopolysaccharide induced ACE shedding, which was confirmed by depletion with siRNA. Transient transfection of ADAM9 and ACE cDNAs into HEK293 cells demonstrated that ADAM9 requires both membrane anchorage and its catalytic domain to shed ACE.

DapE can function as an aspartyl peptidase in the presence of Mn2+.

Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn(2+). Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn(2+)-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His(6)). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn(2+)-dependent aspartyl peptidase activity relative to the MPD dapE(+) strain. In addition, purified DapE-His(6) exhibited Mn(2+)-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn(2+) in the high-affinity site and Mn(2+) in the low-affinity site.

Partial Purification and Characterization of Gelatinase and Metal Dependent Peptidase from Rabbit Uterus and Their Synergistic Action on Gelatin In Vitro

Two metal dependent proteases were investigated in rabbit uterus using a synthetic substrate, 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gin-D-Arg (Dnp-peptide). One was extracted by homogenization in 50 mM Tris-HCl/0.25% Triton X-100/100 mM CaCl2, pH 7.4, from rabbit uterus, and the other from the insoluble fraction by heating at 60 degrees C for 4 min in 50 mM Tris-HCl/100 mM CaCl2, pH 7.4. Both enzymes were partially purified by gel filtration, ion-exchange chromatography and chromatofocusing, and further characterized. The soluble enzyme was a metal dependent peptidase, and hydrolysed 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg as well as Dnp-peptide. Its molecular weight was about 7.0 X 10(4), and the cleavage sites for Dnp-peptide were Gln-Gly and possibly Gly-Ile in the ratio of 3:1. On the other hand, the enzyme extracted from the insoluble fraction was present as a latent form, and was found to be activated by 4-aminophenylmercuric acetate but not by trypsin. The activated enzyme hydrolysed gelatin, in addition to Dnp-peptide, indicating that the enzyme is a gelatinase. The molecular weight was about 7.4 X 10(4) for the active form, and the cleavage site for Dnp-peptide was only the Gly-Ile bond. The rabbit uterine metal dependent peptidase obtained here had negligible activity on gelatin, but once it had been cleaved by the above gelatinase, the presence of metal dependent peptidase accelerated the action of gelatinase. Thus, the actions of both enzymes on gelatin were found to be synergistic.

γ-Glutamyltransferase bound to prostatic subcellular organelles and in free form in human seminal plasma

Most of the gamma-glutamyltransferase occurring in semen was found to be bound to the membranes of prostatic organelles, as is the case with Mg++-Ca++-dependent ATPase, and Zn++-dependent peptidase hydrolysing succinyl (alanine)3-paranitroanilide. Organelle-bound gamma-glutamyltransferase was released in a water-soluble form by papain. Charge shift electrophoresis revealed the presence in seminal fluid of a small additional amount of gamma-glutamyltransferase that is unbound and water-soluble, but also of prostatic origin. After papain treatment there was no sign of the organelle-bound Zn++-dependent peptidase activity, nor of the Mg++-Ca++ dependent ATPase activity, both having been inactivated by the papain. Both organelle-bound gamma-glutamyltransferase and Zn++-dependent peptidase could be rendered soluble in active form with the non-ionic detergent Triton X-100.

A zinc-dependent peptidase in prostatic organelles present in seminal plasma

The electrophoretic seminal plasma protein pattern changes obviously within a few hours if the plasma is not frozen after the ejaculation. Mainly cathodal proteins disappear. Addition of serine protease inhibitors has no apparent affect, but o-phenanthroline markedly retards the conversions seen on electrophoresis. The subcellular organelles in seminal plasma are shown to contain a zinc ion dependent peptidase with optimal activity at pH 7.8. Estimations of enzyme activity may be performed using the elastase substrates n-tert butyloxycarbonyl- l-alanine-paranitrophenylester (Boc(Ala)) and succinyl(alanine) 3-paranitroanilide (Suc(Ala) 3pNA). The enzyme is inactivated by addition of o-phenanthroline and is completely reactivated by the addition of zinc ions.

Peptidases in Escherichia coli K-12 Capable of Cleaving Lysine Homopeptides

Abstract The peptidases present in Escherichia coli K-12 that can cleave lysine oligopeptides were characterized to establish a system in which one could examine questions relating to peptidase specificity, function, and regulation. As an aid to this study, a mutant with diminished peptidase activity was isolated with the use of the reduced ability to cleave trilysine as a screening procedure. In this simplified system, the remaining enzymatic capacity to cleave lysine homopeptides was then characterized. On the basis of substrate specificities and cofactor requirements as well as resolution of the enzymes by ion exchange and Sephadex chromatography; it was shown that the mutant contains the following lysine peptidases: (a) a Co++-dependent peptidase capable of splitting dilysine, (b) an EDTA-sensitive peptidase specific for trilysine, and (c) an endopeptidase that can cleave tetralysine but has no activity toward di- or trilysine. The parental K-12 strain contains, in addition to these three activities, a metal-independent peptidase capable of cleaving all three lysine peptides.