Cloning and expression of mouse legumain, a lysosomal endopeptidase.
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ABSTRACT:
SUBMITTER: Chen JM
PROVIDER: S-EPMC1219758 | biostudies-other | 1998 Oct
REPOSITORIES: biostudies-other
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Project description:Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.
| S-EPMC3272831 | biostudies-literature
Project description:Legumain was recently discovered as a lysosomal endopeptidase in mammals [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098], having been known previously only from plants and invertebrates. It has been shown to play a key role in processing of the C fragment of tetanus toxin for presentation by the MHC class-II system [Manoury, Hewitt, Morrice, Dando, Barrett and Watts (1998) Nature (London) 396, 695-699]. We examine here the specificity of the enzyme from pig kidney by use of protein, oligopeptide and synthetic arylamide substrates, all determinations being made at pH 5.8. In proteins, only about one in ten of the asparaginyl bonds were hydrolysed, and these were mostly predicted to be located at turns on the protein surface. Bonds that were not cleaved in tetanus toxin were cleaved when presented in oligopeptides, sometimes faster than an equivalent oligopeptide based on a bond that was cleaved in the protein. Legumain cleaved the bait region of rat alpha1-macroglobulin and was 'trapped' by the macroglobulin, as most other endopeptidases are, but did not interact with human alpha2-macroglobulin, which contains no asparagine residue in its bait region. Glycosylation of asparagine totally prevented hydrolysis by legumain. Specificity for arylamide substrates was evaluated with reference to benzyloxycarbonyl-Ala-Ala-Asn-aminomethylcoumarin, and the preference for the P3-position amino acid was Ala>Tyr(tertiary butyl)>Val>Pro>Phe=Tyr>Leu=Gly. There was no hydrolysis of substrate analogues containing mono- or di-N-methylasparagines, l-2-amino-3-ureidopropionic acid or citrulline in the P1 position. We conclude that mammalian legumain appears to be totally restricted to the hydrolysis of asparaginyl bonds in substrates of all kinds. There seem to be no strong preferences for particular amino acids in other subsites, and yet there are still unidentified factors that prevent hydrolysis of many asparaginyl bonds in proteins.
| S-EPMC1220212 | biostudies-other
Project description:Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.
| S-EPMC2587490 | biostudies-literature
Project description:Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-? production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those ?1-thymosin and CTSL, which both drive intrinsically Th1 activity-but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-? production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn -/- mice also displayed a specific defect in IFN-? secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine ?1-thymosin activation in T cell-derived IFN-? production. These data suggest that AEP is an "upstream" activator of the CTSL-C3-IFN-? axis in human CD4+ T cells and hence an important supporter of human Th1 induction.
| S-EPMC6207624 | biostudies-literature
Project description:Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.
| S-EPMC3991597 | biostudies-literature
Project description:Astacin (EC 3.4.24.21) from the freshwater crayfish (Astacus astacus) is a prototype for the metzincin superfamily and for the astacin family of zinc peptidases, enzymes which are involved in hatching processes, embryonic patterning and tissue remodelling. Here we report on the cloning and overexpression in Escherichia coli of an astacin cDNA which was reverse-transcribed from crayfish midgut-gland mRNA. A cDNA construct based on this clone was generated which comprised the nucleotide sequence encoding mature astacin devoid of the signal and propeptide. This construct was cloned into the pET3a vector and used to transform E. coli BL21(DE3) cells. Recombinant astacin was purified from inclusion bodies and dissolved under reducing conditions. For folding, the protein was diluted into neutral buffer containing l-arginine, GSH and EDTA. Eventually, Zn(2+) was added by dialysis and the fraction of active enzyme was affinity-purified on immobilized Pro-Leu-Gly hydroxamate. As shown by superimposition of the corresponding three-dimensional structures, this inhibitor binds to a region of the active-site cleft that is conserved in most metzincins. Therefore this principle behind this affinity technique, originally introduced for fibroblast collagenase by Moore and Spilburg [Biochemistry (1986) 25, 5189-5195], is applicable throughout the metzincin superfamily of metalloproteases, despite their otherwise differing cleavage specificities. Recombinant astacin is active on gelatine zymograms and in a quenched fluorescence assay, yielding kinetic parameters comparable with those of wild-type astacin purified from crayfish stomach.
| S-EPMC1220708 | biostudies-literature
Project description:Understanding the regulation of lysosomal proteolytic/hidrolytic capacity has been recently advanced in a huge manner by the discovery of the lysosome-mTOR-TFEB pathway connecting nutrient sensing, autophagy and de novo lysosome generation. But, how lysosomes can adjust their proteolytic/hidrolytic capacity to meet varying conditions (more or less substrate) under normal fed conditions is not known. We have used AEP as a proxy to understand how lysosomes can compensate the lack of a key lysosomal protease to meet the requirements under physiological, fed conditions. To do this, we did compare cytoplasmic, membrane and nuclear fractions of WT and AEP MEFs that were expanded in SILAC media. AEP knock-out mice show pale kidney, with abnormal proliferation of the epithelial proximal tubular cells. In an attempt to understand the role of AEP in the kidney, we did compare kidney obtained from WT and AEP mice using kidneys from WT mice that were fed with SILAC food. Also, in an attempt to reproduce the changes observed in the absence of AEP in kidney and MEFs, we treated WT MEFs grown in SILAC media with MVO26630, specific inhibitor of AEP, for 12 and 48 hours.
2019-01-09 | PXD011473 | Pride
Project description:Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.
| S-EPMC6782589 | biostudies-literature
Project description:Background: Betatrophin, a novel secretory protein from liver and fatty tissues, is believed to be involved in lipid and glucose metabolism. However, its precise physiological role remains unclear. Here, we report the cloning, expression, and purification steps of mouse betatrophin in a prokaryotic system, followed by its structural analysis. Methods: Specific cloning primers were used to amplify the coding sequence of mouse liver betatrophin. The product was cloned into pET28 and expressed in E.coli BL21 (DE3) cells. The suitability of the refolding procedure was assessed by determining secondary structures of the initial and refolded proteins using circular dichroism spectroscopy. Results: The polymerase chain reaction resulted in a 549 bp nucleotide sequence, encoding a 183 amino acid polypeptide, with an apparent molecular weight of 21 kDa, which was expressed in an inclusion body. Following an optimization and refolding procedure, the recombinant protein was purified by anion exchange and metal affinity chromatography. CD spectra revealed that the refolded protein has suitable configuration. Conclusion: We believe that the produced betatrophin is suitable for further biochemical studies on glucose and lipid metabolism.
| S-EPMC6014755 | biostudies-literature
Project description:Lysosomal alpha-d-mannosidase from mouse tissues was separated into its constituent isoenzymes by DEAE-cellulose chromatography. Forms corresponding to the human isoenzymes B and A were present in testis, brain, spleen and kidney, whereas in epididymis and liver only the B form was present. Murine alpha-mannosidases A and B are glycoproteins and have pH optima, thermal stabilities and molecular masses similar to those of the human isoenzymes. A full-length cDNA (3.1 kb) containing the complete coding sequence for alpha-mannosidase was isolated from a mouse macrophage cDNA library. Comparison of the deduced amino acid sequences of human and mouse alpha-mannosidases showed that they had 75% identity and 83% similarity. Expression of this cDNA in COS cells showed that both the A and the B isoenzymes can arise from a single transcript. Northern blotting analysis showed a 10-fold range in the abundance of alpha-mannosidase mRNA in mouse tissues, with the highest levels found in epididymis, and the lowest in liver.
| S-EPMC1218761 | biostudies-other