Aminopeptidase Play a Critical Role in the Accumulation of Free Amino Acids in Abalone(Haliotis discus hannai)During Cold Storage

Abalones reveal unique taste after processing,mainly because of their abundant free amino acids(FAAs)and nucleotides.FAAs are nutrition components that can contribute to the unique taste.However,which factor(s)is responsible for the accumulation of FAAs still need further studies.To analyze the production of FAAs,we studied the variation of FAAs during 7 days of storage at 4℃.The content of taste-active amino acids,including Asp,Glu,Ser,and Gly increased by 1.7-fold,2.0-fold,3.0-fold,and 8.4-fold,respectively.The relative activity of cathepsin L and aminopeptidase(AP)increased significantly during the cold storage period.To identify AP in abalone and its function in mediating the production of FAAs,an aminopeptidase with wide substrate specificity was then extracted and purified from abalone muscle to homogeneity.Purified AP with a molecular mass of 100 kDa exhibited its maximum activity at 30℃,pH 7.5,and was further confirmed by LC-MS.Bestatin specifically inhibited the activity of AP,and metalloproteinase inhibitors EDTA,EGTA and 1,10-phenanthroline also suppressed its activity to different degrees.Based on its highest activity to substrate Leu-MCA and its peptide sequences,the purified enzyme was identified as leucine aminopeptidase(LAP).Our present study indicated the essential role of AP for FAAs accumulation during cold storage of abalone.

Cloning and Sequencing of a Gene Encoding Aminopeptidase N in the Midgut of Helicoverpa armigera(Hubner)

A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108. 3 kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini is hydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accession number is AY181026.

Discovery of a Novel Aminopeptidase N Inhibitor Dino-Leucine Borate

Aminopeptidase N(APN/CD13), a Zn2+-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion of tumor cells. We found that boroleucine and dino-leucine borate exhibited a strong inhibitory effect on the enzyme activity of aminopeptidase N. The tested assay indicated that both compounds had an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay, migration test and matrigel-coated transwell assay showed that both boroleucine and dino-leucine borate inhibited the migration and invasion of breast cancer cells. Immunoblot analysis showed that both compounds down-regulated the expression of matrix metalloproteinase-2/9. In the capillary tube formation assay of human umbilical vein endothelial cells (HUVECs), dino-leucine borate showed better antiangiogenic activity than ubenimex even at a low concentration (10 μM). Moreover, compared with ubenimex, the anti-metastatic activity of dino-leucine borate in vivo was similar to or even better than that of ubenimex in the H22 pulmonary metastasis mouse model. In this paper, we found the novel APN inhibitors to markedly suppress the enzyme activity of APN and inhibit the migration and invasion of tumor cells in vitro and in vivo.

Single-Chain Expression and Crystallization of an Antigenic C-Terminus in Complex with the Regulatory Domain of ER Aminopeptidase 1

Human endoplasmic reticulum aminopeptidase 1 (ERAP1) is one of two ER luminal aminopeptidases that participate in the final processing of peptide precursors and generates the N-termini of the MHC class I-restricted epitopes. In order to investigate the interactions of its binding site with substrate peptides, X-ray crystallographic analyses have been carried out to study structures of ERAP1 regulatory (ERAP1_R) domain in complex with antigenic peptides. Single-chain bimodular constructs with various antigenic peptides linked to the C-terminal end of ERAP1_R domain are designed to facilitate crystallization process of these complexes. These recombinant proteins have been purified and crystalized, and x-ray diffraction data of one crystal have been processed to a resolution of 2.8 . The crystal belongs to the space group P21, with unit cell parameters a =64.2, b = 66.8, c = 66.3 , β = 110.2°. A Refmac-refined omit map reveals a clear density for the antigenic peptide’s carboxylate-end that is in contact with the ERAP1 regulatory domain of neighboring molecule. Thus the single-chain bimodular constructs have provided an expedited approach to study sequence-specific interactions between the ERAP1 regulatory domain and antigen peptide’s C-terminal ends.

Leukotriene-A4-Hydrolase and Basic Aminopeptidase Activities Are Related with Collagen-Induced Arthritis in a Compartment-Dependent Manner

Objective: Previous study demonstrated the involvement of basic aminopeptidase (APB) activity in the development of collagen-induced arthritis (CIA). Two zinc dependent metalloenzymes (EC 3.4.11.6 and EC 3.3.2.6) are known to exhibit concomitantly APB and leukotriene-A4-hydrolase (LT-A4-H) activities. Influence of the interrelationship between both activities on arthritic processes, however, is presently uncertain. This study aimed to compare these activities in CIA. Methods: CIA was induced in rats and arthritis was assessed macroscopically. Ultracentrifugation was used to separate soluble (S) and solubilized membrane-bound (M) fractions from peripheral blood mononuclear cells (PBMCs) and synovial tissue (ST). Enzyme immunoassay was used to measure LT-A4-H activity, and Real Time Polymerase Chain Reaction was used for evaluating EC 3.4.11.6 and EC 3.3.2.6 gene expressions. Results: The existence of genes for EC 3.3.2.6 and EC 3.4.11.6 was demonstrated in the ST. Compared with control, LT-A4-H activity increased in synovial fluid (SF) and in S-PBMCs of CIA-arthritic and CIA-resistant and in M-ST of CIA-resistant, while it decreased in M-PBMCs of CIA-arthritic and CIA-resistant. In all these locations APB activity remained unchanged or inversely correlated with LT-A4-H activity. Conclusions: LT-A4-H and APB activities in joint-related samples are associated, for the first time, with EC 3.3.2.6 and EC 3.4.11.6 genes, exhibiting a compartment-dependent differential modulation of their specificity, efficiency and/or affinity or an inverse concurrent pattern. Changes in LT-A4-H activity have implications for development or resistance to arthritis in CIA model with a potential to be a diagnostic tool.

Statins markedly potentiate aminopeptidase inhibitor activity against(drug-resistant)human acute myeloid leukemia cells

Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells.Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation.Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents;and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation;this was experimentally confirmed by impaired Rheb prenylation by simvastatin.Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.

Cloning of aminopeptidase N promoter and its activity in hematopoietic cell and different tumor cell lines

Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXPl-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expression in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may provide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radiotherapy.

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins

Bacillus thuringiensis(Bt)insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops.Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action.Previous studies suggested aminopeptidase N1(APN1)as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches.In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout.Three APN genes(HaAPN1,HaAPN2 and HaAPN5)were individually knocked out in a susceptible strain(SCD)of H.armigera to establish three homozygous knockout strains.Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout.Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain.This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H.armigera.

Expression levels of aminopeptidase-N genes in the lightbrown apple moth, Epiphyas postvittana

Five aminopeptidase-N genes （EpAPN1-5） of the tortricid moth Epiphyas postvittana have been isolated from a midgut expressed sequence tag （EST） library. Relative RNA expression of these genes was measured by quantitative reverse transcription polymerase chain reaction using actin as a reference gene. Measurements were made on various tissues of fifth instar larvae, over all stages of the life cycle and under differing dietary conditions： different protein sources and levels, and in presence of trypsin and metalloprotease inhibitors. Gene expression for all five genes was the greatest in midgut tissue, but was also detected in the hindgut, fat body and Malpighian tubules. EpAPN4 was consistently the highest expressed, with EpAPN3 at about half that level; EpAPN5 was the least expressed. During larval stages expression was high, generally increasing over the instars, after an early peak as neonates or first instars. Expression in other life stages was much lower. Males and females showed differing expression in the pupal and adult stages： female expression was higher in the pupa, this reversed in the adult. Gene expression levels and ratios both changed with diet. A natural apple leaf diet depressed levels. Diets with the most impaired amino acid balance induced the greatest change; generally EpAPN1 increased by the greatest proportion. The addition of proteinase inhibitor also increased gene expression, and it was noteworthy that the trypsin inhibitor addition, which has been shown to double aminopeptidase activity, also doubled levels of gene expression.

Anti-tumor targeted drug delivery systems mediated by aminopeptidase N/CD13

Aminopeptidase N(APN)/CD13 is a transmembrane glycoprotein,which is overexpressed on tumor neovascular endothelial cells and most tumor cells,where it plays an important role in tumor angiogenesis.Peptides containing the Asn-Gly-Arg(NGR)motif can specifically recognize APN/CD13 allowing them to act as tumor-homing peptides for the targeted delivery of anti-tumor drugs to tumor neovascular endothelial cells and tumor cells.This article reviews the literature and recent developments related to APN/CD13,its role in tumor growth and some antitumor drug delivery systems containing NGR peptides designed to target APN/CD13.

Gene cloning and expression of aminopeptidase N and cadherin from midgut of the rice stem borer, Chilo suppressalis

The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed in the laboratory with good resistance to this pest and other Lepidopteran insects, which will provide a possible alternative tool for this pest control. The full-length cDNAs encoding an aminopeptidase N （CsAPN） and a cadherin （CsCad） were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here （CsCad） showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region （CR7）, which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin CrylAb. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.

Real-time identification of gut microbiota with aminopeptidase N using an activable NIR fluorescent probe

A NIR fluorescent probe(DDAA) derived from fluorophore DDAO with alanine as the recognition group was developed for sensing aminopeptidase N(APN) in gut microbiota.Using DDAA as the real-time guidance tool for the fluorescence imaging of intestinal microorganism,target bacteria and saccharomycete possessing active APN were identified successfully from human feces.

Clone, Purification and Characterization of Thermostable Aminopeptidase ST1737 from Sulfolobus tokodaii

The aminopeptidase gene from thermophilic archaea Sulfolobustokodaii was cloned and expressed in Escherichia coli BL21 codon-plus（DE3）. To overexpress the aminopeptidase, the vector pET32a was constructed, in which the target gene was fused with the genes of histidine-tag and thioredoxin（Trx）. The expressed protein was purified using Ni^2＋-column affinity chromatography and ion exchange chromatography and cleft with enterokinase（EK） to obtain the purified aminopeptidase（ST1737）. The biochemical and enzymic properties of the expressed ST1737 were characterized. The results show that its optimal pH and temperature are 8 and 80 ℃, respectively. The half-life of ST1737（0.2 mg/mL） is about 85 h at 90 ℃, indicating that the enzyme exhibits an excellent thermostability. The activity of ST1737 could still maintain over 85% after its treatment at 25 ℃ in different buffers with a pH range of from 6.0 to 10.5 for 24 h, demonstrating that ST1737 is stable in neutral or slight alkali environment. The enzyme shows a high activity for the substrates such as unmodified peptide Asp-Ala, while the pNPC8 shows an optimal esterase substrate specificity. These results indicate that the enzyme is a bifunctional enzyme, and different from the aminopeptidase reported before.

A bestatin-based fluorescent probe for aminopeptidase N cell imaging

Aminopeptidase N （APN） is an important drug target and biomarker for various tumors. The current work characterizes a novel APN-targeted fluorescent probe （Bes-Green, 2） that manifests comparable inhibitory activity with Bestatin. This probe has capacity of tightly binding to the APN for imaging endogenous APN in living human ovarian clear cell carcinoma cells （ES-2） and has potential application in biological study of cellular APN.

Synthesis and biological evaluation of novel 1,2,3-benzotriazin-4-one derivatives as leukotriene A4 hydrolase aminopeptidase inhibitors

A series of novel 1,2,3-benzotriazin-4-one derivatives were designed,synthesized and their inhibitory activities against leulcotriene A4 hydrolase aminopeptidase in vitro were evaluated.Many compounds showed moderate to good activities at the concentration of 10 μmol/L.Among them,compound Ⅳ-16 exhibited the highest inhibitory activity up to 80.6% with an IC50 of 1.30 ± 0.20 μmol/L The compound Ⅳ-16 was also tested the proliferation inhibitory activities in THP1 human AML cell line and its binding model with LTA_4H enzyme by molecular docking was studied.It indicated that 1,2,3-benzotriazin-4-one was a promising scaffold for further study.The relationship between structure and inhibitory activity was also preliminarily discussed.

沉默APN对胶质瘤细胞U251增殖、侵袭和凋亡的影响

目的探究aminopeptidase N(APN)对胶质瘤细胞U251增殖能力、迁移侵袭能力和凋亡等生物学行为的影响及可能的作用机制。方法通过RNA干扰技术沉默胶质瘤细胞U251中APN的表达后,运用RT-qPCR的方法检测APN mRNA表达,采用Western blot检测APN蛋白表达水平,CCK-8检测各组U251细胞的增殖活性;采用流式细胞仪检测细胞凋亡和细胞周期;采用细胞划痕实验检测U251细胞的迁移能力,Transwell实验检测细胞侵袭能力;采用Western blot检测STAT3通路蛋白t-STAT3、p-STAT3、Bcl-2和MMP-2的蛋白表达水平。结果与空白对照组和阴性对照组相比,沉默U251中APN的表达后APN在基因和蛋白表达水平下降(P<0.01),沉默APN后U251的增殖活性明显下降(P<0.05),细胞周期停滞在G0/G1期,并且凋亡率明显增加(P<0.01);另外沉默APN的表达后U251细胞的迁移能力和侵袭能力明显下降(P<0.01);沉默APN后STAT3磷酸化水平下降,引起下游蛋白Bcl-2和MMP-2等蛋白表达下降(P<0.05)。结论沉默APN可通过STAT3信号途径抑制胶质瘤细胞U251增殖、迁移和侵袭,诱导凋亡。

Early Morphological and Physiological Events Occurring During Germination of Maize Seeds

The early morphological and physiological events occurring during maize （Zea mays cv. Nongda 108） seed imbibition and germination were studied. Water uptake of seeds exhibited a triphasic pattern with a marked increase during the initial phase of imbibition, and then a slow increase, followed by a second substantial increase. Imbibition time for 10 and 50% of seed germination was about 26 and 46 h at 30℃, respectively. The relative conductivity of maize seeds dramatically decreased during the initial phase of imbibition, followed by a substantial increase. Respiratory rate of seeds gradually increased with imbibition. Length of root cap cells decreased during the initial phase and then increased; those of meristematic zone cells increased during the initial phase and then decreased; and those of elongation zone cells and of the whole elongation zone of the radicle gradually increased during germination. The contents of soluble sugars and starch in embryos gradually decreased as the activities of α- and β-amylase strikingly increased with imbibition. In the meantime, protein contents of embryos gradually decreased and free amino acid content increased. The activities of aminopeptidase and endopeptidase increased until 12 h of imbibition and then decreased. It is concluded that germination of maize seeds is mainly completed by extension of cells in the elongation zone of the radicle, and that mobilization of stored reserves in the embryo during the initial phase of imbibition is also an early event during seed germination.

Effect of adrenalectomy and hydrocortisone on ventral prostate of rats

Aim: To study the effects of adrenalectomy and hydrocortisone on the ventral prostate of SD rats. Methods: Inadrenalectomised (ADX) and ADX + hydrocortisone (1, 2, or 4 mg) treated rats, the prostatic histology and thecholesterol, protein, zinc, and copper levels and the enzymic profile (acid phosphatase, alkaline phosphatase, aryl sul-phatase, lactic dehydrogenase, and leucine aminopeptidase) in the prostatic tissue were determined; the serum hormon-al profile (testosterone, FSH and LH) was also assayed. Results; Adrenalectomy caused a progressive degenerationin prostatic structure that was not reversed by hydrocortisone treatment. The serum testosterone were significantly lowerin ADX than in sham operated rats and lower in ADX + hydrocortisone than in ADX-C rats (P < 0.01). The serumFSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham op-erated rats and higher in ADX + hydrocortisone than in ADX-C rats (P<0.05-0.01). The prostatic zinc levels weresignificantly higher in sham operated than in ADX, and higher in ADX-C than in ADX + hydrocortisone rats (P < 0.05-0.01). The prostatic copper level was significantly lower in sham operated than in ADX, and lower in ADX-C thanin the ADX + hydrocortisone rats (P <0.01). Conclusion; In rats, adrenalectomy leads to pathological and func-tional changes of the prostate. Hydrocortisone treatment at the doses employed did not reverse these changes. (Asian JAndrol 2001 Dec; 3: 289 - 300)

Arrhythmogenic properties of dismantling cadherin-mediated adhesion in murine hearts

Objective： To evaluate the arrhythmogenic effects of dismantling cadherin-mediated adhesion by recombinant mouse aminopeptidase N （rmAPN） in murine hearts. Methods： rmAPN was incubated with cultured neonatal rat cardiomyocytes as well as being infused in adult mice. The cell-cell connections were immunolabelled and observed by laser confocal microscopy. Disruption of the N-terminal of N-cadherin （N-cad） was detected by western blot and quantitative immunofluorescence. The risk of inducible ventricular tachyarrhythmia was evaluated in mice by an electrophysiological study. Results： Disrupted cell-cell contact was observed in cultured neonatal rat cardiomyocytes in response to 30-40 ng/μL rmAPN. Loss of the N-terminal in N-cad and altered distribution of connexin 43 （Cx43） were observed in hearts from rmAPN-infused mice. In addition, a reduction of phosphorylated Cx43 was also detected concomitant with redistribution of Cx43. Electrophysiological studies of rmAPN-infused mice showed prolonged QRS duration and increased inducibility of ventricular tachycardias. Conclusion： Disruption of N-cad by rmAPN contributes to gap junction remodeling and may elicit arrhythmogenic effects. The disorder of adherent junctions by proteolytic enzymes may play an important role in arrhythmogenic mechanisms in correlated diseases.

Effect of adrenalectomy on rat epididymidis

AIM: To investigate the effect of adrenalectomy (ADX) on the epididymidis of Sprague-Dawley rats. METHODS: The histological, biochemical (cholesterol protein, zinc, copper, alkaline and acid phosphatase aryl sulphatase, lactic dehydrogenase and leucine amino peptidase) and hormonal (FSH, LH and testosterone) changes of caput and cauda epididymis in ADX rats were observed. RESULTS: Organ wet weight, histological studies and morphometric measurements indicated a cellular degeneration in caput and cauda epididymis of ADX rats. Serum testosterone level was significantly lower in ADX than in sham-operated rats, while the serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham-operated rats. Epididymal zinc level increased whereas copper level decreased in ADX rats compared to the sham-operated. CONCLUSION: Adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone.