Facile purification of active recombinant mouse cytosolic carboxypeptidase 6 from Escherichia coli.

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification - polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg2+/K+, the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved.

In-Depth Venome of the Brazilian Rattlesnake Crotalus durissus terrificus: An Integrative Approach Combining Its Venom Gland Transcriptome and Venom Proteome.

Snake venoms are complex mixtures mainly composed of proteins and small peptides. Crotoxin is one of the most studied components from Crotalus venoms, but many other components are less known due to their low abundance. The venome of Crotalus durissus terrificus, the most lethal Brazilian snake, was investigated by combining its venom gland transcriptome and proteome to create a holistic database of venom compounds unraveling novel toxins. We constructed a cDNA library from C. d. terrificus venom gland using the Illumina platform and investigated its venom proteome through high resolution liquid chromotography-tandem mass spectrometry. After integrating data from both data sets, more than 30 venom components classes were identified by the transcriptomic analysis and 15 of them were detected in the venom proteome. However, few of them (PLA2, SVMP, SVSP, and VEGF) were relatively abundant. Furthermore, only seven expressed transcripts contributed to ∼82% and ∼73% of the abundance in the transcriptome and proteome, respectively. Additionally, novel venom proteins are reported, and we highlight the importance of using different databases to perform the data integration and discuss the structure of the venom components-related transcripts identified. Concluding, this research paves the way for novel investigations and discovery of future pharmacological agents or targets in the antivenom therapy.

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.

Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

SUPPRESSOR OF LLP1 1-mediated C-terminal processing is critical for CLE19 peptide activity.

Cell-to-cell communication is essential for the coordinated development of multicellular organisms. Members of the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED (CLE) family, a group of small secretory peptides, are involved in these processes in plants. Although post-translational modifications are considered to be indispensable for their activity, the detailed mechanisms governing these modifications are not well understood. Here, we report that SUPPRESSOR OF LLP1 1 (SOL1), a putative Zn²âº carboxypeptidase previously isolated as a suppressor of the CLE19 over-expression phenotype, functions in C-terminal processing of the CLE19 proprotein to produce the functional CLE19 peptide. Newly isolated sol1 mutants are resistant to CLE19 over-expression, consistent with the previous report (Casamitjana-Martinez, E., Hofhuis, H.F., Xu, J., Liu, C.M., Heidstra, R. and Scheres, B. (2003) Curr. Biol. 13, 1435-1441). As expected, our experiment using synthetic CLE19 peptide revealed that the sol1 mutation does not compromise CLE signal transduction pathways per se. SOL1 possesses enzymatic activity to remove the C-terminal arginine residue of CLE19 proprotein in vitro, and SOL1-dependent cleavage of the C-terminal arginine residue is necessary for CLE19 activity in vivo. Additionally, the endosomal localization of SOL1 suggests that this processing occurs in endosomes in the secretory pathway. Thus, our data indicate the importance of C-terminal processing of CLE proproteins to ensure CLE activities.

Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10.

Bacillus licheniformis HS10 is a good biocontrol agent against Pseudoperonospora cubensis which caused cucumber downy disease. To identify and characterize the antifungal proteins produced by B.licheniformis HS10, the proteins from HS10 were isolated by using 30-60% ammonium sulfate precipitation, and purified with column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. And the SDS-PAGE and MALDI-TOF/TOF-MS analysis results demonstrated that the antifungal protein was a monomer with molecular weight of about 55 kDa, identified as carboxypeptidase. Our experiments also showed that the antifungal protein from B. licheniformis HS10 had significantly inhibition on eight different kinds of plant pathogenic fungi, and it was stable with good biological activity at as high as 100°C for 30 min and in pH value ranged from 6 to 10. The biological activity was negatively affected by protease K and 10mM metal cations except Ca(2+).

Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

UNLABELLED: An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates.

BACKGROUND: Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated. RESULTS: Screening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser309-Lys334) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and ß- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase. CONCLUSIONS: Our work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

Plasma prolylcarboxypeptidase (angiotensinase C) is increased in obesity and diabetes mellitus and related to cardiovascular dysfunction.

BACKGROUND: Prolylcarboxypeptidase (PRCP) (angiotensinase C) has 3 major targets, angiotensin II, prekallikrein, and α-melanocyte stimulating hormone(1-13). The truncation of the latter leads to loss in appetite regulation and obesity in experimental animals. The objectives of this study were to purify PRCP from a native source, establish a sensitive immunoassay for PRCP, and relate plasma PRCP concentrations to signs and symptoms of obesity, diabetes mellitus, and cardiovascular dysfunction. METHODS: Purification of PRCP from human neutrophils and establishment of a sensitive ELISA was carried out with the use of samples from study participants. Three cohorts were studied: healthy individuals (n = 40); a chest pain cohort (Fast Assessment of Thoracic Pain by Neural Networks) (n = 165); and a community-based cohort [Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS)] (n = 1004). RESULTS: PRCP was purified to homogeneity. Mean (SD) plasma concentrations in healthy individuals were 12.9 (3.2) µg/L and were increased in patients with chest pain and in patients with obesity and/or diabetes mellitus (P < 0.0001). In the PIVUS cohort the concentrations were related to several measures of arterial plaque formation, thickness of arterial intima media and posterior wall of the heart (P = 0.04-0.000005); the Framingham score (r = 0.14, P < 0.0001); and concentrations of C-reactive protein (r = 0.16, P < 0.0001) and N-terminal pro B-type natriuretic peptide (r = -0.13, P < 0.0001). CONCLUSIONS: Plasma concentrations of PRCP may be used to reflect metabolic conditions in individuals with obesity and diabetes mellitus. The associations of PRCP concentrations with signs of cardiovascular dysfunction and cardiovascular abnormalities suggest a pivotal role of the enzyme in disease.

Carboxypeptidase activity common to viridans group streptococci cleaves angiotensin I to angiotensin II: an activity homologous to angiotensin-converting enzyme (ACE).

We have found that Streptococcus gordonii FSS2, an infective endocarditis (IE) isolate, expresses a dipeptidyl-carboxypeptidase with activity homologous to angiotensin-converting enzyme (ACE). The carboxypeptidase activity was purified to homogeneity as a complex/aggregate from a bacterial surface extract and was also active as a 165 kDa monomer. The specific activity for the carboxypeptidase activity was eightfold higher than that for recombinant human ACE. Selected ACE inhibitors, captopril, lisinopril and enalapril, did not inhibit the ACE activity. The carboxypeptidase also hydrolysed the Aα and Bß-chains of human fibrinogen, which resulted in impaired fibrin formation by thrombin. The gene encoding ACE carboxypeptidase activity was sequenced and the inferred polypeptide product showed 99 % amino acid homology to SGO_0566, sgc, 'challisin' of S. gordonii CL1 Challis, and had no significant amino acid sequence homology to human ACE. Homologues of challisin ACE activity were commonly detected among the viridans group streptococci most often associated with IE.

Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH.

Enzymatic properties of the recombinant serine-type carboxypeptidase OcpC, which is unique to Aspergillus oryzae.

Gene AO090103000153 is unique to Aspergillus oryzae RIB40 and A. flavus NRRL3357, and is speculated to encode a serine-type carboxypeptidase. In this study, we purified and characterized a heterologously expressed gene product of AO090103000153. 5'-Rapid amplification of cDNA ends indicated that the translation start site of the gene is located 1,586 bp downstream of the translation start site predicted by the genome sequencing project. The gene, starting from the revised translation start codon, termed ocpC, was transcribed constantly in A. oryzae RIB40. Purified recombinant OcpC exhibited the enzymatic properties of a serine-type carboxypeptidase. This protease was stable at temperatures below 45°C and a low pH, and had broad substrate specificity for N-acylpeptides, but it exhibited significantly lower specific activity and a lower k(cat) value for substrates than previously reported serine-type carboxypeptidases from A. oryzae.

A multifunctional human Argonaute2-specific monoclonal antibody.

Small regulatory RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs) guide regulation of gene expression in many different organisms. The Argonaute (Ago) protein family constitutes the cellular binding partners of such small RNAs and regulates gene expression on the levels of transcription, mRNA stability, or translation. Due to the lack of highly specific and potent monoclonal antibodies directed against the different Ago proteins, biochemical analyses such as Ago complex purification and characterization rely on overexpression of tagged Ago proteins. Here, we report the generation and functional characterization of a highly specific monoclonal anti-Ago2 antibody termed anti-Ago2(11A9). We show that anti-Ago2(11A9) is specific for human Ago2 and detects Ago2 in Western blots as well as in immunoprecipitation experiments. We further demonstrate that Ago2 can be efficiently eluted from our antibody by a competing peptide. Finally, we show that anti-Ago2(11A9) recognizes Ago2 in immunofluorescence experiments, and we find that Ago2 not only localizes to cytoplasmic processing bodies (P-bodies) and the diffuse cytoplasm but also to the nucleus. With the anti-Ago2(11A9) antibody we have generated a potent tool that is useful for many biochemical or cell biological applications.

Expression, purification and crystallization of human prolylcarboxypeptidase.

Prolylcarboxypeptidase (PrCP) is a lysosomal serine carboxypeptidase that cleaves a variety of C-terminal amino acids adjacent to proline and has been implicated in diseases such as hypertension and obesity. Here, the robust production, purification and crystallization of glycosylated human PrCP from stably transformed CHO cells is described. Purified PrCP yielded crystals belonging to space group R32, with unit-cell parameters a = b = 181.14, c = 240.13 A, that diffracted to better than 2.8 A resolution.

Carboxypeptidase from Streptomyces bikiniensis: primary structure, isolation, and properties.

A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0-7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).

Molecular cloning of ocpO encoding carboxypeptidase O of Aspergillus oryzae IAM2640.

Carboxypeptidase O from Aspergillus oryzae IAM2640 is a serine-type carboxypeptidase. In this study, we cloned and sequenced cDNA and genomic DNA carrying ocpO encoding carboxypeptidase O. The results showed that the length of ocpO was 1,816 bp, and the open reading frame encoded a putative preproenzyme composed of 472 amino acid residues of the mature carboxypeptidase O and an additional N-terminal sequence of 50 amino acid residues. A BLASTN search revealed that a gene, AO090020000351, in A. oryzae RIB40, which is strain used in genome-wide sequencing, is a homolog of ocpO. The difference between AO090020000351 and ocpO was only one nucleotide. The difference caused substitution of Ala for Pro at the 277th position of the enzyme; therefore the protein encoded by AO090020000351 was overproduced and purified. The purified protein showed enzymatic properties similar to carboxypeptidase O, indicating that carboxypeptidase O and protease encoded by AO090020000351 are same enzyme.

Functional annotation of two new carboxypeptidases from the amidohydrolase superfamily of enzymes.

Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea ( gi|44371129 ). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5) M(-1) s(-1), respectively, and N-formyl-l-Tyr with k(cat) and k(cat)/K(m) values of 33 s(-1) and 3.9 x 10(5) M(-1) s(-1), respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with k(cat) and k(cat)/K(m) values of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A with l-methionine bound in the active site. The alpha-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The alpha-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane.

We have investigated the localization of Kex1p, a type I transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. Indirect immunofluorescence demonstrated the presence of Kex1p in a punctate organelle resembling the yeast Golgi apparatus as identified by Kex2p and Sec7p (Franzusoff, A., K. Redding, J. Crosby, R. S. Fuller, and R. Schekman. 1991. J. Cell Biol. 112:27-37). Glycosylation studies of Kex1p were consistent with a Golgi location, as Kex1p was progressively N-glycosylated in an MNN1-dependent manner. To address the basis of Kex1p targeting to the Golgi apparatus, we examined the cellular location of a series of carboxy-terminal truncations of the protein. The results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the Golgi apparatus. Deletions of the retention region or overproduction of wild-type Kex1p led to mislocalization of Kex1p to the vacuolar membrane. This unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane.

Isolation and characterization of carboxypeptidase III from germinating triticale grains.

Carboxypeptidase III from germinating triticale grains was purified 434.2-fold with a six-step procedure including: homogenization, ammonium sulfate precipitation, cation-exchange chromatography on CM-cellulose, gel filtration chromatography on Sephadex G-150, cation-exchange chromatography on SP8HR column (HPLC), and affinity chromatography on CABSSepharose 4B. Triticale carboxypeptidase III is a monomer with a molecular weight of 45 kDa, which optimally hydrolyzes peptides at temperature 30-50 degrees C and pH 4.6. N-CBZ-Ala-Phe, N-CBZ-Ala-Leu, and N-CBZ-Ala-Met are hydrolyzed at the highest rates. Amino acids with aromatic or large aliphatic side chains are preferred in position P1', whereas the presence of these types of groups in position P1 of the substrate results in a lower rate of hydrolysis. Peptides containing glutamic acid in positions P1 are poor substrates for the enzyme. This phenomenon suggests the hydrophobic substrate- binding sites S1 and S1'. The active site contains serine since diisopropylfluorophosphate and phenylmethanesulfonyl fluoride reduce the activity by 89.9% and 81.5%, respectively. Moreover, the activity of triticale carboxypeptidase III is reduced by mercury ions and organomercurial compounds, which suggests the presence of a sulfhydryl group adjacent to the active site of the enzyme. Identification of purified enzyme by mass spectrometry method demonstrated that the enzyme is a homolog of barley carboxypeptidase III.

Trichophyton rubrum secreted and membrane-associated carboxypeptidases.

Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides.