Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta.

Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates and plays a critical role in protein metabolism and growth. In the present study, we investigated the full-length cDNA sequence of the LAP3 gene in Sinonovacula constricta (ScLAP3) using expressed sequence tags and rapid amplification of cDNA ends. The full-length ScLAP3 cDNA was 2885 bp, with a 1560 bp open reading frame encoding 519 amino acids. Sequence analysis revealed that ScLAP3 shared 70.9% identity with LAP3 from the blood clam Tegillarca granosa and 62.0-68.0% with other species. ScLAP3 was expressed in all six tested tissues, with significantly higher expression levels in the foot compared with mantle, adductor muscle, liver, gills, and siphon tissues in adults (P < 0.01). In the eight developmental stages, ScLAP3 expression gradually increased, with significantly higher levels in D-shaped larvae compared with other developmental stages (P < 0.01), suggesting that it may be involved in the formation of certain organs during early development. Association analysis identified three shared single nucleotide polymorphisms (SNPs), c.1073A > G, c.1139C > T and c.1154A > G in exons of ScLAP3 gene from 177 individuals of two groups, one selective strain and one wild population, which had significant effects on growth traits of S. constricta. The results provided candidate genetic markers to assist selective breeding of razor clams toward improved growth.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis.

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention.

Diversity evaluation based on morphological, physiological and isozyme variation in genetic resources of garlic (Allium sativum L.) collected worldwide.

The aim of this study was to obtain primary information about the global diversity of garlic (Allium sativum L.) by evaluating morphological, physiological and isozyme variation. A total of 107 garlic accessions collected worldwide were grown in Yamaguchi, Japan. Five morphological traits (bulb weight, bulb diameter, number of cloves per bulb, number of bulbils and scape length) and one physiological trait (bolting period) of the collected garlic showed wide variation. Meanwhile, a total of 140 garlic accessions, including the 107 mentioned above, were characterized by leucine aminopeptidase (LAP) and phosphoglucoisomerase (PGI) isozyme analyses; they clearly showed polymorphisms in putative isozyme loci (Lap-1, Lap-2 and Pgi-1). Allelic frequencies were estimated in each group of accessions categorized by their geographical origin, and the observed (Ho) and expected (He) heterozygosities were calculated. The allelic frequencies differed between groups. A principal component analysis based on morpho-physiological data indicated a grouping of the garlic accessions into Central Asian and Northern Mediterranean groups as well as others. We discuss the roles of artificial and natural selection that may have caused differentiation in these traits, on the assumption that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations that accumulated during expansion, and have evolved along with human-preferred traits over a long history of cultivation.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 µM, respectively, while the Ki was 2210 ± 358 µM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 µM] than B. gibsoni [IC50 = 460.8 ± 114.45 µM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica.

M17 leucine aminopeptidase (LAP) is one of a family of metalloexopeptidases, of which short peptide fragments are cleaved from the N-terminals. In this study, the full length of cDNA encoding Fasciola gigantica LAP (FgLAP) was cloned from adult parasites. The amino acid sequences of FgLAP showed a high degree of identity (98%) with that from Fasciola hepatica and a low degree of identities (11% and 9%) with those from cattle and human. Phylogenetic analysis revealed that the FgLAP was closely related and grouped with F. hepatica LAP (FhLAP). Northern analysis showed that FgLAP transcriptional products have 1800 base pairs. Analysis by RNA in situ hybridization indicated that LAP gene was expressed in the cecal epithelial cells of adult parasites. A polyclonal antibody to a recombinant FgLAP (rFgLAP) detected the native LAP protein in various developmental stages of the parasite. In a functional test, this rFgLAP displayed aminolytic activity using a fluorogenic Leu-MCA substrate, and was significantly inhibited by bestatin. Its maximum activity was at pH 8.0 and enhanced by Mn(2+) ions. Localization of LAP proteins by immunohistochemistry and immunofluorescence techniques indicated that the enzyme was distributed in the apical cytoplasm of cecal epithelial cells. Because of its important metabolic role and fairly exposed position, FgLAP is a potential drug target and a possible vaccine candidate against fasciolosis.

Up-regulation of leucine aminopeptidase-A in cadmium-treated tomato roots.

The effects of cadmium (Cd) on aminopeptidase (AP) activities and Leucine-AP (LAP) expression were investigated in the roots of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 10 days in the presence of 0.3-300 µM Cd and compared to control plants grown in the absence of Cd. AP activities were measured using six different p-nitroanilide (p-NA) substrates. Leu, Met, Arg, Pro and Lys hydrolyzing activities increased in roots of Cd-treated plants, while Phe-pNA cleavage was not enhanced after Cd treatments. The use of peptidase inhibitors showed that most of the Leu-pNA hydrolyzing activity was related to one or several metallo-APs. Changes in Lap transcripts, protein and activities were measured in the roots of 0 and 30-µM Cd-treated plants. LapA transcript levels increased in Cd-treated roots, whereas LapN RNAs levels were not modified. To assess amount of Leu-pNA hydrolyzing activity associated with the hexameric LAPs, LAP activity was measured following immunoprecipitation with a LAP polyclonal antiserum. LAP activity increased in Cd-treated roots. There was a corresponding increase in LAP-A protein levels detected in 2D-immunoblots. The role of LAP-A in the proteolytic response to Cd stress is discussed.

Identification and characterization of Paragonimus westermani leucine aminopeptidase.

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.

Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis.

Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host's homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn(2+), and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development.

Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis.

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the Km value 0.19 +/- 0.011 mM and Vmax value 157.2 +/- 3.17 nmol/min/mgprotein) and a methionyl substrate (with the Km value 0.12+/-0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mgprotein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host's blood digestion process.

Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP.

The family M1 of Zn-dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA-library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn-binding motif and displays high homology to the other M1 family members except for its N-terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS-7 and BHK cells. Northern-blot analysis revealed ubiquitous expression of a major 3. 1-kb transcript. For enzymatic studies, the complete protein was expressed in Sf 9 insect cells. When aminoacyl beta-naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin-insensitive leucyl-specific aminopeptidase is a signal-sequence-bearing member of family M1 and may be another example of the small subset of substrate-specific peptidases.

Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase.

In the current study, we report the cloning and initial characterization of a novel human cytosolic aminopeptidase named adipocyte-derived leucine aminopeptidase (A-LAP). The sequence encodes a 941-amino acid protein with significant homology (43%) to placental leucine aminopeptidase (P-LAP)/oxytocinase. The predicted A-LAP contains the HEXXH(X)18E consensus sequence, which is characteristic of the M1 family of zinc-metallopeptidases. Although the deduced sequence contains a hydrophobic region near the N-terminus, the enzyme localized mainly in cytoplasm when expressed in COS-7 cells. Northern blot analysis revealed that A-LAP was expressed in all the tissues tested, some of which expressed at least three forms of mRNA, suggesting that the regulation of the gene expression is complex. When aminopeptidase activity of A-LAP was measured with various synthetic substrates, the enzyme revealed a preference for leucine, establishing that A-LAP is a novel leucine aminopeptidase with restricted substrate specificity. The identification of A-LAP, which reveals strong homology to P-LAP, might lead to the definition of a new subfamily of zinc-containing aminopeptidases belonging to the M1 family of metallopeptidases.

A -308 deletion of the tomato LAP promoters is able to direct flower-specific and MeJA-induced expression in transgenic plants.

Tomato and potato leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding and the wound signal molecules, ABA and jasmonic acid. Here, we report the isolation of two LAP genes, LAP17.1A and LAP17.2, from tomato. Functional analysis in transgenic tomato and potato plants show that fusions of the corresponding 5' non-coding regions to the gusA gene are constitutively expressed in flowers and induced in leaves upon wounding or by treatment with methyl jasmonate (MeJA). Comparison of the 5' non-coding regions of the two genes revealed a region from -317 to -3 relative to the ATG, which is strongly conserved in both promoters. This 0.3 kb proximal promoter fragment is sufficient to direct flower-specific and MeJA-inducible GUS activity in transgenic potato plants, and thus contains a MeJA-responsive element that mediates induction by MeJA. Dimeric TGACG motifs or G-box elements similar to those found in other MeJA-inducible genes are not observed in this region, which suggests that a different DNA sequence is involved in MeJA induction of the LAP genes.

Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ-specific signal transduction pathways.

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound-inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root-specific pin2 homologue accumulated in the JA-treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA-induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA-induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA-mediated gene induction has enabled identification of a JA-independent pathway for ABA induction of pin2 and the other wound-inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound-inducible genes. Our data suggest the presence of a complex, organ-specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.

Localization and post-translational processing of the wound-induced leucine aminopeptidase proteins of tomato.

Leucine aminopeptidase (LAP) is induced by wounding and bacterial pathogen infection in tomato. DNA blot analysis of XbaI-digested lambdalap genomic clones demonstrated that LapA1 and LapA2 cDNAs were encoded by two different LapA genes in the tomato genome. The coding and untranslated regions of LapA1 and LapA2 mRNAs shared more than 93% identity. The deduced amino acid sequences of LapA cDNA clones and in vitro translation of LapA1 mRNA indicated that LAP-A was synthesized as a 60-kDa precursor protein. The processing of a 60-kDa preLAP-A into the mature 55-kDa LAP-A was demonstrated in vivo by expression of the full-length LapA1 cDNA in insect cells. Sequencing of a single LAP-A form isolated from a two-dimensional polyacrylamide gel indicated that LAP-A proteins had two different N termini that were separated by two residues. The LAP-A presequence had features similar to chloroplast transit peptides. Comparison of LAP-A levels in chloroplast and total protein extracts from methyl jasmonate-treated leaves indicated that a small proportion of the LAP-A proteins was detected in the plastids. Inspection of the LAP-A presequence indicated the presence of a dibasic protease (Kex2/furin) processing site motif 6-8 residues upstream from the LAP-A N termini. Its potential role in LAP-A precursor biogenesis is discussed.

Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family.

The serum level of placental leucine aminopeptidase (P-LAP) increases during pregnancy. P-LAP degrades several peptide hormones such as oxytocin and vasopresin, suggesting a role in maintaining homeostasis during pregnancy. In the study reported here, we have isolated a cDNA clone with 4084 base pairs encoding P-LAP from a human placental cDNA library. The amino acid sequence deduced from the cDNA contained all of the sequences of the peptide fragments obtained by digestion of the purified protein with trypsin. The predicted P-LAP contains the HEXXH consensus sequence of zinc metallopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase N and aminopeptidase A. The deduced sequence also contains a hydrophobic region near the N terminus, suggesting that the enzyme is a type II integral membrane protein. Northern blot analysis revealed that P-LAP was expressed in several tissues, some of which expressed two forms of mRNAs. These results suggest that the enzyme is synthesized as an integral membrane protein and is released into blood under some physiological conditions.

Nature and regulation of pistil-expressed genes in tomato.

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.