Vesicular localization and characterization of a novel post-proline-cleaving aminodipeptidase, quiescent cell proline dipeptidase

A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell proline dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N:-glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.     

A novel apoptotic pathway in quiescent lymphocytes identified by inhibition of a post-proline cleaving aminodipeptidase: a candidate target protease, quiescent cell proline dipeptidase.

The vast majority of lymphocytes in vivo persist in a quiescent state. These resting lymphocytes are maintained through a cellular program that suppresses apoptosis. We show here that quiescent PBMC, but not activated PBMC or transformed lymphocytes, die in the presence of highly specific post-proline aminodipeptidase inhibitors. This form of death has the hallmarks of apoptosis, such as phosphatidylserine externalization and loss of mitochondrial transmembrane potential. However, it differs from apoptosis induced by gamma irradiation in the same cells or by Fas ligation in transformed lymphocytes in terms of caspase involvement. In addition, the aminodipeptidase inhibitor-induced cell death, but not gamma-irradiation-mediated apoptosis, can be prevented by inhibition of the proteasome complex. The target of these inhibitors is not CD26/DPPIV, but probably a novel serine protease, quiescent cell proline dipeptidase, that we have recently isolated and cloned. These studies will yield a better understanding of the requirements and the mechanisms that mediate quiescent lymphocyte homeostasis in vivo.     

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase

Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a Km value of 2.2 mM and a Vmax value of 0.045 micromol(-1) ml(-1) min(-1). Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca2+ and Mg2+ at 2-12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining.     

Construction and properties of an intracellular serine protease mutant of Bacillus subtilis.

An intracellular serine protease (ISP-1) mutant of Bacillus subtilis was created by introducing a frameshift into the coding region of the cloned gene. Intracellular protease activity in the mutant was very low, yet sporulation in both nutrient broth and minimal medium was normal. The rate of bulk protein turnover in the mutant was slightly slower than that in the wild-type strain. These results suggest that the gene for ISP-1 is not essential and that ISP-1 is not the major enzyme involved in protein turnover during sporulation.     

A new alkaline serine protease from alkalophilic Bacillus sp.: Cloning,sequencing, and characterization of an intracellular protease

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D37921.     

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca²⁺ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca²⁺ ions and gramicidin S; its pH and temperature optima were 9.0 and 37°C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.     

叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析

为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征,为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列,使用生物信息学软件进行序列分析以及系统进化分析,采用实时荧光定量PCR (Real time quantitativate PCR,RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明,EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp,分别编码125个氨基酸和306个氨基酸,预测均为亲水蛋白质,理论分子量分别为13.48 kDa和33.82 kDa,等电点分别为6.68和5.80,分别有30个和23个氨基酸残基的信号肽序列,EfSPI20有跨膜结构域,EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高,氨基酸序列一致性达58%;EfSP1与稻绿蝽Nezara viridula SP同源性最高,氨基酸序列一致性达66%;系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达,推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关;EfSP1基因在卵期、卵巢和肠道中高表达,推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。     

Purification and cloning of an extracellular serine protease from the nematode-trapping fungus Monacrosporium cystosporium

An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and 56 degrees (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant (Km) and Vmax for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were 1.67x10-4 M and 0.6071 OD410 per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might play a role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mc1 showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mc1 possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.     

Characterization of an intracellular serine protease from sporulating cells of Bacillus brevis.

Sporulating cells of Bacillus brevis ATCC 9999 produced a high level of an intracellular serine protease when grown in nutrient medium. The protease activity in the crude extracts of this strain appeared at hour 5 (t5) after the end of exponential growth and increased gradually during sporulation, reaching a maximum at t12 to t13. The enzyme isolated in a partially purified state showed a pH optimum between 7.3 and 9.0 and had an apparent molecular weight of about 60,000. The activity was completely inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, EDTA, and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The protease possessed a high activity for azocoll and low activities for azocasein and 14C-labeled hemoglobin. It cleaved the cyclic decapeptide gramicidin S specifically at the peptide linkage between valine and ornithine and hydrolyzed the oxidized insulin B-chain mainly at peptide bonds 4-5 (Glu-His), 6-7 (Leu-CysSO3H), and 15-16 (Leu-Tyr). No catalysis of bond cleavage by the enzyme on a variety of small peptides or esters was detected. Unlike other Bacillus species, B. brevis ATCC 9999 grown in nutrient medium excreted no extracellular proteases.     

Expression, purification, and functional analysis of the human serine protease HtrA2

HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.     

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP intracellular serine protease - ISP-A-Bsu A-50 and ISP-B-Bsu A-50 molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively - SDS sodium dodecyl sulfate - PMSF phenylmethyl sulfonylfluoride - pNA p-nitroanilide - Buffer A 50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl₂ adjusted to pH 8.5 with HCl     

Molecular cloning,expression, and anti-tumor activity of a novel serine protease from Arenicola cristata

Arenicola cristata, a marine annelid, is a wellknown and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease ofA. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA （cDNA） of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the pro posed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was 26 kDa after overexpression in E. coli. The recombinant pro tein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma （ESCC） in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activa tion of caspase9 as well as downregulation of Mcl1 and Bcl2. Taken together, our findings indicated that the recom binant serine protease ofA. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.