Rat thimet oligopeptidase: large-scale expression in Escherichia coli and characterization of the recombinant enzyme.
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ABSTRACT:
SUBMITTER: McKie N
PROVIDER: S-EPMC1135820 | biostudies-other | 1995 Jul
REPOSITORIES: biostudies-other
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Project description:The consequences of sleep deprivation on memory, cognition, nociception, stress, and endocrine function are related to the balance of neuropeptides, with peptidases being particularly essential. Thimet oligopeptidase (THOP1) is a metallopeptidase implicated in the metabolism of many sleep-related peptides, including angiotensin I, gonadotropin releasing hormone (GnRH), neurotensin, and opioid peptides. In the present study, we evaluated the effect of sleep deprivation and sleep recovery in male rats on THOP1 expression and specific activity in the central nervous system. In the striatum and hypothalamus, THOP1 activity decreased following sleep deprivation and a recovery period. Meanwhile, THOP1 activity and immunoexpression increased in the hippocampal dentate gyrus during the sleep recovery period. Changes in THOP1 expression after sleep deprivation and during sleep recovery can potentially alter the processing of neuropeptides. In particular, processing of opioid peptides may be related to the known increase in pain sensitivity in this model. These results suggest that THOP1 may be an important player in the effects of sleep deprivation.
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Project description:Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.
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Project description:We have purified human thimet oligopeptidase to homogeneity from erythrocytes, and compared it with the enzyme from rat testis and chicken liver. An antiserum raised against rat thimet oligopeptidase also recognized the human and chicken enzymes, suggesting that the structure of the enzyme has been strongly conserved in evolution. Consistent with this, the properties of the human enzyme were very similar to those for the other species. Thus human thimet oligopeptidase also is a thiol-dependent metallo-oligopeptidase with M(r) about 75,000. Specificity for cleavage of a number of peptides was indistinguishable from that of the rat enzyme, but Ki values for the four potent reversible inhibitors tested were lower. In discussing the results, we consider the determinants of the complex substrate specificity of thimet oligopeptidase. We question whether substrates containing more than 17 amino acid residues are cleaved, as has been suggested. We also point out that the favourable location of a proline residue and a free C-terminus in the substrate may be as important as the hydrophobic residues in the P2, P1 and P3' positions that have been emphasized in the past.
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Project description:Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).
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Project description:A systematic analysis of the peptide sequences and lengths of several homologues of bioactive peptides and of a number of quenched-fluorescence (qf) opioid- and bradykinin-related peptides was performed to determine the main features leading the oligopeptides to hydrolysis by the recombinant rat testis thimet oligopeptidase (EC 3.4.24.15). The results indicate that a minimum substrate length of six amino acids is required and that among the oligopeptides six to thirteen amino acid residues long, their susceptibility as substrates is highly variable. Thimet oligopeptidase was able to hydrolyse, with similar catalytic efficiency, peptide bonds having hydrophobic or hydrophilic amino acids as well as proline in the P1 position of peptides, ranging from a minimum of six to a maximum of approximately thirteen amino acid residues. An intriguing observation was the shift of the cleavage site, at a Leu-Arg bond in qf dynorphin-(2-8) [qf-Dyn2-8; Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl)ethylenediamine], to Arg-Arg in qf-Dyn2-8Q, in which Gln was substituted for Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluorescence bradykinin analogues containing Gln at the C-terminal position, namely Abz-RPPGFSPFR-EDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-Ser bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQ-EDDnp are cleaved at the Pro-Phe bond.
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Project description:Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure-function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.
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Project description:Paenibacillus polymyxa A-8, which secretes ?-mannosidase, was isolated from the soil sample under a pine tree located in the "Laoban" mountain region of Sichuan, China. The ?-mannosidase gene (MANB) was isolated from P. polymyxa A-8, using primers according to the complete genome. The MANB (2,550 bp) encoding 849 amino acid residues was expressed in Escherichia coli. The specific activities of ?-mannosidase produced by P. polymyxa A-8 and E. coli pET30a-MANB were 12 nkat/mg and 635 nkat/mg respectively. SDS-PAGE analysis indicated that the molecular mass of the recombinant MANB was approximately 96 kDa. The recombinant MANB was active between pH 7.0-8.5 with the maximum activity at pH 7.0. It had good pH stability and adaptability. The MANB had the optimal temperature of 35°C and was relatively stable at 35-40°C. In addition, the MANB activity was enhanced by K+, Ca2+, Mn2+, and Mg2+ and inhibited by Zn2+, Cu2+, and Hg2+.
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Project description:The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galalpha1,4Galbeta1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori beta-l,4-galactosyltransferase and a Neisseria meningitidis alpha-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.
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