Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.     

Novel and highly sensitive fluorescent assay for leucine aminopeptidases

l-Leucine aminopeptidases (LAPs) are implicated in the progress of many pathological disorders and play some regulatory roles in tumor cell proliferation, invasion, and/or angiogenesis. Thus, LAPs not only could become new diagnostic or prognostic biomarkers but also may have potential as novel molecular targets for the treatment of several cancers. Highly sensitive assays are critical for early detection of changes in LAP activity and for screening potent LAP inhibitors. In this study, we developed a novel and highly sensitive fluorescent assay for LAPs based on substituted aminopyridines as fluorescent reporters. This assay was at least 100- and 20-fold more sensitive than commercial colorimetric and fluorescent LAP substrates, respectively. We also showed that this assay was a useful tool for monitoring LAP activities in extracts from cancer cell lines, as well as for the high-throughput screening of inhibitors, which could lead to new cancer treatments.     

Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves

The constitutive and wound-inducible leucine aminopeptidases (LAP-N and LAP-A, respectively) of tomato encode 60-kDa proteins with 5-kDa presequences that resemble chloroplast-targeting peptides. Cell fractionation studies and immunoblot analyses of chloroplast and total proteins have suggested a dual location of the mature LAP-A proteins in the cytosol and the plastids. In this study, the subcellular localization of tomato LAPs was further investigated using in vitro chloroplast-targeting assays and immunocytochemical techniques at the light and TEM levels. In vitro-translated LAP-A1 and LAP-N preproteins were readily transported into pea chloroplasts and processed into mature proteins of 55 kDa indicating the presence of a functional chloroplast-targeting signal in the LAP-A1 and LAP-N protein precursors. In addition, a LAP polyclonal and a LAP-A-specific antisera were used to immunolocalize LAP proteins in leaves from healthy, wounded and methyl jasmonate (MeJA)-treated plants. Low levels of LAPs and/or LAP-like proteins were detected in leaves from unwounded plants. The LAP polyclonal antiserum, which detected LAP-A, LAP-N and LAP-like proteins, and the LAP-A specific antibodies, which detected only LAP-A, showed that LAP levels increased in leaf sections after wounding and MeJA treatments. LAP-A proteins were primarily detected within the chloroplasts of spongy and palisade mesophyll cells. The localization of LAP-A was distinct from the location of early wound-response proteins that are important in the biosynthesis of jasmonic acid or systemin and more similar to the late wound-response proteins with primary roles in defense. The importance of these findings relative to the potential roles of LAP-A in defense is discussed.     

Plant leucine aminopeptidases moonlight as molecular chaperones to alleviate stress-induced damage

Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress.     

Small-molecule probe using dual signals to monitor leucine aminopeptidase activity

Leucine aminopeptidases (LAPs) are widely distributed in organisms from bacteria to humans, and play crucial roles in cell maintenance and cell growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this Letter, we report a small-molecule probe which exhibits colorimetric and fluorogenic changes according to LAP activity.     

Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae were isolated which have decreased ability to hydrolyze leucine beta-naphthylamide, a chromogenic substrate for amino-peptidases. The mutations were shown by starch gel electrophoresis to affect one of four different aminopeptidases. Mutations affecting a given enzyme belong to a single complementation group. The four genes were symbolized lap1, lap2, lap3, and lap4, and the corresponding enzymes LAPI, LAPII, LAPIII, and LAPIV. Both lap1 and lap4 were mapped to the left arm of chromosome XI, and lap3 was mapped to the left arm of chromosome XIV. Strains which possessed only one of the four leucine aminopeptidases (LAPs) were constructed. Crude extracts from these strains were used to study the properties of the individual enzymes. Dialysis against EDTA greatly reduced the activity of all the LAPs except for LAPIII. Of the cations tested, Co2+ was the most effective in restoring activity. LAPIV was the only LAP reactivated by Zn2+. LAPI was purified 331-fold and LAPII was purified 126-fold from cell homogenates. Both of the purified enzymes had strong activity on dipeptides and tripeptides. The activity levels of the LAPs are strongly dependent on growth stage in batch culture, with the highest levels in early-stationary phase. Strains lacking all four LAPs have slightly lower growth rates than wild-type strains. The ability of leucine auxotrophs to grow on dipeptides and tripeptides containing leucine is not impaired in strains lacking all four LAPs.     

Specificity of the wound-induced leucine aminopeptidase (LAP-A) of tomato activity on dipeptide and tripeptide substrates.

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) that preferentially hydrolyzes amino acid-p-nitroanilide and -beta-naphthylamide substrates with N-terminal Leu, Met and Arg residues. To determine the substrate specificity of LAP-A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined. For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel. Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed. Substrates with variable N-terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1') position. The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N-terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur-containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides. Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted. To systematically evaluate the impact of the P1' residue on cleavage of dipeptides, three series of dipeptides (Leu-Xaa, Gly-Xaa, and Arg-Xaa) were evaluated. The P1' residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue. P1' Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP-A, porcine LAP, and E. coli PepA markedly. Analysis six Arg-Gly-Xaa tripeptides showed that more diversity was tolerated in the P2' position. P2' Arg inhibited tripeptide cleavage by all three enzymes, while P2' Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs.     

A Complex Array of Proteins Related to the Multimeric Leucine Aminopeptidase of Tomato

Leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding, pathogen infection, and insect infestation (V. Pautot, F.M. Holzer, B. Reisch, L.L. Walling [1993] Proc Natl Acad Sci USA 90: 9906-9910). Polyclonal antibodies to a glutathione S-transferase-LAP fusion protein and affinity-purified antibodies recognizing LAP antigenic determinants detected four classes of polypeptides in tomato (Lycopersicon esculentum) leaves. All four classes had multiple polypeptides in two-dimensional polyacrylamide gel electrophoresis immunoblots. Although antigenically related to the wound-induced tomato LAP proteins, the 77- and 66-kD LAP-like proteins accumulated in both healthy and wounded leaves. Two classes of 55-kD polypeptides with distinctive isoelectric points were designated as plant LAPs; only the acidic LAP proteins accumulated to high levels after mechanical wounding or Pseudomonas syringae pv tomato infection of tomato leaves. The temporal accumulation of LAP mRNAs was correlated with the increase in acidic LAP protein subunits. A slow-migrating LAP activity was detected using a native gel assay after wounding. The molecular mass of the native wound-induced LAP enzyme was 353 kD. The 55-kD acidic LAP proteins were associated with induced LAP activity, whereas the neutral LAPs and the LAP-like proteins were not associated with this exopeptidase. A second, fast-migrating aminopeptidase was detected in both healthy and wounded tomato leaves. Cell fractionation experiments revealed that wound-induced LAP is a soluble enzyme.     

Salmonella enterica serovar typhimurium peptidase B is a leucyl aminopeptidase with specificity for acidic amino acids

Peptidase B (PepB) of Salmonella enterica serovar Typhimurium is one of three broad-specificity aminopeptidases found in this organism. We have sequenced the pepB gene and found that it encodes a 427-amino-acid (46.36-kDa) protein, which can be unambiguously assigned to the leucyl aminopeptidase (LAP) structural family. PepB has been overexpressed and purified. The active enzyme shows many similarities to other members of the LAP family: it is a heat-stable (70 degrees C; 20 min) hexameric ( approximately 270-kDa) metallopeptidase with a pH optimum of 8.5 to 9.5. A detailed study of the substrate specificity of the purified protein shows that it differs from other members of the family in its ability to hydrolyze peptides with N-terminal acidic residues. The preferred substrates for PepB are peptides with N-terminal Asp or Glu residues. Comparison of the amino acid sequence of PepB with those of other LAPs leads to the conclusion that PepB is the prototype of a new LAP subfamily with representatives in several other eubacterial species and to the prediction that the members of this family share the ability to hydrolyze peptides with N-terminal acidic residues. Site-directed mutagenesis has been used to show that this specificity appears to be determined by a single Lys residue present in a sequence motif conserved in all members of the subfamily.     

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta

Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.     

Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.     

In vitro binding of synthetic acylated lipid-associating peptides to high-density lipoproteins: effect of hydrophobicity

To measure the effect of hydrophobicity on the binding of model apoproteins to lipoproteins, we synthesized a 15 amino acid lipid-associating peptide (LAP) with acyl chains of various lengths (0-18 carbons) bound to the N-terminal amino acid through a peptide bond. The acylated LAPs preferentially bound to high-density lipoprotein (HDL) and were activators of lecithin:cholesterol acyltransferase. Circular dichroic spectra indicated that the LAP association with phospholipid was accompanied by increased alpha-helical structure. The LAPs self-associated in solution as judged from tryptophan fluorescence analysis. These characteristics, which are comparable to those of apolipoprotein A-I, were strongly dependent upon the acyl chain length of the LAPs. The equilibrium constants (Keq) for the association of LAPs to reassembled HDL were measured by equilibrium dialysis at several temperatures. At 37 degrees C, Keq increased by 3 orders of magnitude as the number of carbon units was increased from 0 to 16; there was a log-linear relationship between Keq and the acyl chain length. The free energy of association (delta Ga) decreased by a constant value for each methylene unit added to the acyl chain (0.35 kcal mol-1), clearly demonstrating a strict hydrophobic effect. This change of delta Ga was enthalpy rather than entropy driven. Our data show that, with all other parameters including putative alpha-helicity, sequence, and molecular weight being constant, the binding of a lipid-associating peptide to lipoprotein is governed by its hydrophobicity.     

Comparison of metal‐bound and unbound structures of aminopeptidase B proteins from Escherichia coli and Yersinia pestis

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal‐dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn²⁺ and Mn²⁺, while the second structure has two Zn²⁺ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N‐terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C‐terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.     

The Plasmodium LAP complex affects crystalloid biogenesis and oocyst cell division

Malaria parasite oocysts located on the mosquito midgut generate sporozoites by a process called sporogony. Plasmodium berghei parasites express six LCCL lectin domain adhesive-like proteins (LAPs), which operate as a complex and share a localisation in the crystalloid – an organelle found in the ookinete and young oocyst. Depletion of LAPs prevents crystalloid formation, increases oocyst growth, and blocks sporogony. Here, we describe a LAP4 mutant that has abnormal crystalloid biogenesis and produces oocysts that display reduced growth and premature sporogony. These findings provide evidence for a role of the LAP complex in regulating oocyst cell division via the crystalloid.     

Peptidases in Drosophila melanogaster. I. Characterization of dipeptidase and leucine aminopeptidase activities

Four major peptidases of Drosophila melanogaster have been described and distinguished by their electrophoretic mobilities, molecular weights, net electrical charges, and substrate specificities. The previously described leucine aminopeptidase, LAP D, consists of at least two isozymes, designated here LAP P and LAP G. In pupae most LAP activity results from LAP P (pupal); in larvae and adults, in contrast, most LAP activity results from LAP G (gut). These two LAPs may be separated by electrophoresis in the presence of the nonionic detergent Triton X-100. A specific assay for LAP P, which exploits the large difference between the net electrical charge of LAP P and that of LAP G, is described. The activity levels of two dipeptidases, Dip A and Dip B, were high in all the postembryonic stages examined. Specific assays for Dip A and Dip B were used to show that for each of these isozymes, the activity in an adult is proportional to gene dosage.     

Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships

Background  

The neprilysin (M13) family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2), which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles.     

The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.     

Structural basis for dimerization of LAP2alpha, a component of the nuclear lamina

Lamina-associated polypeptides (LAPs) are important components of the nuclear lamina, the dense network of filaments that supports the nuclear envelope and also extends into the nucleoplasm. The main protein constituents of the nuclear lamina are the constitutively expressed B-type lamins and the developmentally regulated A- and C-type lamins. LAP2alpha is the only non-membrane-associated member of the LAP family. It preferentially binds lamin A/C, has been implicated in cell-cycle regulation and chromatin organization, and has also been found to be a component of retroviral preintegration complexes. As an approach to understanding the role of LAP2alpha in cellular pathways, we have determined the crystal structure of the C-terminal domain of LAP2alpha, residues 459-693. The C-terminal domain is dimeric and possesses an extensive four-stranded, antiparallel coiled coil. The surface involved in binding lamin A/C is proposed based on results from alanine-scanning mutagenesis and a solid-phase overlay binding assay.