Role of Escherichia coli endopeptidases and dd-carboxypeptidases in infection and regulation of innate immune response.

The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and dd-carboxypeptidases from Escherichia coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with dd-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of dd-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and dd-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.

Trypanosoma cruzi serinecarboxipeptidase is a sulfated glycoprotein and a minor antigen in human Chagas disease infection.

In this work, the presence of sulfated N-glycans was studied in a high-mannose-type glycoprotein of Trypanosoma cruzi with serinecarboxipeptidase (TcSCP) activity. The immune cross-reactivity between purified SCP and Cruzipain (Cz) was evidenced using rabbit sera specific for both glycoproteins. Taking advantage that SCP co-purifies with Cz from Concanavalin-A affinity columns, the Cz-SCP mixture was desulfated, ascribing the cross-reactivity to the presence of sulfate groups in both molecules. Therefore, knowing that Cz is a sulfated glycoprotein, with antigenic sulfated epitopes (sulfotopes), SCP was excised from SDS-PAGE and the N-glycosydic chains were analyzed by UV-MALDI-TOF-MS, confirming the presence of short-sulfated high-mannose-type oligosaccharidic chains. Besides, the presence of sulfotopes was analyzed in lysates of the different parasite stages demonstrating that a band with apparent molecular weight similar to SCP was highly recognized in trypomastigotes. In addition, SCP was confronted with sera of infected people with different degrees of cardiac dysfunction. Although most sera recognized it in different groups, no statistical association was found between sera antibodies specific for SCP and the severity of the disease. In summary, our findings demonstrate (1) the presence of sulfate groups in the N-glycosidic short chains of native TcSCP, (2) the existence of immune cross-reactivity between Cz and SCP, purified from epimastigotes, (3) the presence of common sulfotopes between both parasite glycoproteins, and (4) the enhanced presence of sulfotopes in trypomastigotes, probably involved in parasite-host relationship and/or infection. Interestingly, we show for the first time that SCP is a minor antigen recognized by most of chronic Chagas disease patient's sera.

Construction of human MASP-2-CCP1/2SP, CCP2SP, SP plasmid DNA nanolipoplexes and the effects on tuberculosis in BCG-infected mice.

The lectin pathway, one of the complement cascade systems, provides the primary line of defense against invading pathogens. The serine protease of MASP-2 plays an essential role in complement activation of the lectin pathway. The C-terminal segment of MASP-2 is comprised of the CCP1-CCP2-SP domains, and is the crucial catalytic segment. However, what is the effect of CCP1-CCP2-SP domains in controlling chronic infection is unknown. In order to evaluate the potential impact of CCP1-CCP2-SP domains on tuberculosis, we constructed the human MASP-2 CCP1/2SP, CCP2SP and SP recombinant plasmids, and delivered these plasmids by DNA-DOTAP:cholesterol cationic nanolipoplexes to BCG-infected mice. After 21 days post DNA-DOTAP:chol nanolipoplexes application, we analyzed bacteria loads of pulmonary, pathology of granuloma, lymphocyte subpopulations. The C3a, C4a and MASP-2 levels in serum were measured with enzyme-linked immunosorbent assays. Compared to the control group that received GFP DNA-DOTAP:chol nanolipoplexes, MASP-2 CCP1/2SP DNA-DOTAP:chol nanolipoplexes treated group showed significantly enlarged pulmonary granulomas lesion (P < 0.05) and did not reduce bacteria loads in the lung tissue (P < 0.05). Furthermore, the levels of C3a in serum were decreased (P < 0.05), the number and percentage of PD1+ and Tim3+ cells subgroups were increased in BCG-infected mice after treated with MASP-2 CCP1/2SP DNA-DOTAP:chol nanolipoplexes (P < 0.05). But, there was no statistical difference in the serum C4a and MASP-2 level among DNA nanolipoplexes treated groups (P > 0.05). These findings provided experimental evidence that MASP-2 CCP1/2SP DNA nanolipoplexes shown the negative efficacy in controlling Mycobacterium tuberculosis infection, and displayed a potential role of down-regulating T-cell-mediated immunity in tuberculosis.

Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): molecular characterization, genomic arrangement and immune responses upon pathogenic stresses.

Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.

Peptidases trimming MHC class I ligands.

Peptides presented by MHC class I molecules are typically produced through antigen degradation by the proteasome followed by trimming by exopeptidases. According to recent results, these include both aminopeptidases and carboxypeptidases in the cytosol and the endoplasmic reticulum. While cytosolic peptidases have a net neutral or destructive effect on MHC ligands, endoplasmic reticulum aminopeptidases are required for efficient class I loading and have a strong effect on the repertoire of peptide/MHC complexes. Cells lacking these enzymes can be eliminated both by NK cells and by CD8+ T cells recognizing complexes formed between an MHC class Ib molecule and a conserved peptide. Cross-presented peptides derived from internalized antigens can be processed by insulin-regulated aminopeptidase, the only endosomal trimming peptidase.

Screening for inhibitors of plant protease D1 using novel monoclonal antibodies directed against its carboxyl terminal.

Carboxyl terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of D1 protein, which is predicted to be an excellent target for a general broad-spectrum herbicide. In this study, the CtpA gene from spinach cDNA was cloned and overexpressed and the recombinant CtpA fusion protein (rCtpA) was used as antigen to immunize BALB/c mice for the production of monoclonal antibody (MAb). Western blot and ELISA results indicated that both rCtpA and the PDZ domain protein of CtpA had specific binding abilities to MAbs, while the specificity and sensitivity of rCtpA were much higher than that of the PDZ domain. These results suggest that parts of the antigen determinant of CtpA were located in the PDZ domain. The MAbs and related results obtained in this study proved the feasibility of high-throughput screening of lead compounds for protease as inhibitors and mechanism analysis of CtpA enzyme.

[Cloning, expression, purification of spinach carboxyl-terminal processing protease of D1 protein with hydrolysis activity and preparation of polyclonal antibody].

Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8 degrees C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg x min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.

Prolylcarboxypeptidase: a cardioprotective enzyme.

Prolylcarboxypeptidase (PRCP) is involved in regulating the blood flow through active tissues in order to preserve the internal environment. The expression of PRCP in tissues is determined by a number of pharmacological stimuli such as glucocorticoids and a combination of dexamethasone plus the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate. PRCP is an enzyme which is associated with preeclampsia, rheumatoid arthritis, and tonsillitis. The interplay between inward cellular signalling required for induced and basal transcription, and PRCP expression have not been mechanistically characterized. Molecules modulated by PRCP include angiotensin II (Ang II), angiotensin III (Ang III), alpha-MSH, and prekallikrein (PK), demonstrating its cardiovascular protective role. In addition to regulating vascular tone, PRCP may modulate proliferation, cell migration, and angiogenesis through regulating angiotensin molecules--and bradykinin--induced endothelium activation. The anti-hypertensive and proinflammatory properties of PRCP implicate that this enzyme may well be an accessible target for anti-inflammatory therapy.

Preparation, characterization and epitope mapping of monoclonal antibodies specific to human mast cell carboxypeptidase.

Human mast cell carboxypeptidase (hMC-CP) is a unique product of mast cells. Unlike tryptase and chymase, its potential function and expression in diseased conditions remain largely unknown. To develop an assay for hMC-CP, the recombinant fusion protein of hMC-CP and purified native skin hMC-CP was prepared, and two novel monoclonal antibodies against hMC-CP named CCP1 (IgG1 isotype) and CCP2 (IgM isotype) were raised in the present study. Epitope analysis shows that CCP1 and CCP2 antibodies recognize epitopes located in the region of amino acids 112-202 of hMC-CP, and hydrophilicity analysis implies that epitopes might be located in the amino acid residues 123-134 and 165-177. Furthermore, using a competition enzyme-linked immunosorbent assay, it was shown that the epitope recognized by CCP1 is close to that recognized by CCP2 or the two antibodies partially share the same epitope. Flow cytometry analysis shows that basophilic leukemia cell line KU812 reacts with both CCP1 and CCP2 antibodies, suggesting that this cell line expresses hMC-CP. In conclusion, although the two antibodies possess different isotypes, they may partially share the same epitope. These two antibodies will be valuable tools for the development of an assay to detect the levels of hMC-CP in the biological fluids in man.

Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues.

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses.

Molecular characterization of the severe acute respiratory syndrome coronavirus has revealed genetic diversity among isolates. The spike (S) glycoprotein, the major target for vaccine and immune therapy, shows up to 17 substitutions in its 1,255-aa sequence; however, the biologic significance of these changes is unknown. Here, the functional effects of S mutations have been determined by analyzing their affinity for a viral receptor, human angiotensin-converting enzyme 2 (hACE-2), and their sensitivity to Ab neutralization with viral pseudotypes. Although minor differences among eight strains transmitted during human outbreaks in early 2003 were found, substantial functional changes were detected in S derived from a case in late 2003 from Guangdong province [S(GD03T0013)] and from two palm civets, S(SZ3) and S(SZ16). S(GD03T0013) depended less on the hACE-2 receptor and was markedly resistant to Ab inhibition. Unexpectedly, Abs that neutralized most human S glycoproteins enhanced entry mediated by the civet virus S glycoproteins. The mechanism of enhancement involved the interaction of Abs with conformational epitopes in the hACE-2-binding domain. Finally, improved immunogens and mAbs that minimize this complication have been defined. These data show that the entry of severe acute respiratory syndrome coronaviruses can be enhanced by Abs, and they underscore the need to address the evolving diversity of this newly emerged virus for vaccines and immune therapies.

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.

Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.

Recognition of prostate tumor cells by cytotoxic T lymphocytes specific for prostate-specific membrane antigen.

The development of immunotherapy for cancer, such as synthetic peptide-based vaccines, relies heavily on the identification of appropriate epitopes capable of eliciting antitumor T-cell responses. We have used a combination of computer-based algorithms to predict peptide sequences from prostate-specific membrane antigen (PSMA) capable of stimulating in vitro CTLs restricted by the HLA-A2 MHC molecule. Four of the five peptides that were predicted by these algorithms were capable of inducing antigen-specific CTLs that killed target cells that were pulsed exogenously with the corresponding peptides. However, only one of the four peptides, PSMA(27), induced CTLs that were effective at recognizing prostate tumor cells expressing the HLA-A2 and PSMA molecules. These results underline the importance of demonstrating antitumor reactivity of peptide-induced CTLs for the selection of epitopes destined to become immunotherapeutic for prostate cancer.

The utility of monoclonal antibodies in the imaging of prostate cancer.

Monoclonal antibodies (mAbs) to prostate-specific antigens, such as PSMA, have great potential as diagnostic and therapeutic tools in the management of advanced prostate cancer. PSMA is a very attractive target for mAb-based imaging. It is expressed by virtually all prostate cancers and its expression is further increased in poorly differentiated, metastatic, and hormone-refractory carcinomas. The ProstaScint scan (Cytogen, Princeton, NJ), based on the mAb 7E11-C5.3, is currently approved for the imaging of prostate cancer in soft tissue but is not approved for imaging bone metastases. It appears superior to conventional imaging studies for soft-tissue disease but has limitations attributed to its intracellular binding site on PSMA. Overcoming this limitation, new mAbs to the extracellular domain of PSMA have been developed. The radioisotopes, (111)Indium, (90)Yttrium, and (177)Lutetium have been conjugated to one such mAb, J591. Radioimmunoscintigraphy with this immunoconjugate has demonstrated excellent tumor targeting of prostate cancer sites not only in soft tissue but also in bone.

Anti-tumor effects of toxins targeted to the prostate specific membrane antigen.

BACKGROUND: There is presently no effective therapy for relapsing, metastatic, androgen-independent prostate cancer. Immunotherapy with monoclonal antibody-vehicled toxins (Immunotoxins, ITs) may be a promising novel treatment option for the management of prostate cancer in these cases. METHODS: Three anti-prostate specific membrane antigen (anti-PSMA) monoclonals (J591, PEQ226.5, and PM2P079.1) were cross-linked to ricin A-chain (RTA; native or recombinant), and their cytotoxic effects were investigated in monolayer and three-dimensional (3-D) cell cultures of prostate carcinoma cells (LNCaP). RESULTS: The various Immunotoxins showed effects in the nanomolar range (IC(50s) of 1.6-99 ng/ml) against PSMA+ cells (IC(50) being the concentration inhibiting 50% cell proliferation or protein synthesis). PSMA(-) cell lines were 62- to 277-fold less sensitive to anti-PSMA ITs, evidencing an appreciable therapeutic window. Treatment with J591-smpt-nRTA (0.35-31.7ng/ml) resulted in complete eradication of 3-D tumor micromasses or in 1.46- to 0.35-log reduction of target cells number, depending on the dose. CONCLUSION: Anti-PSMA ITs appear to be promising for use in the eradication of small prostate tumor cell aggregates present in tissues and in the bone marrow.

Ins and outs of clinical trials with peptide-based vaccines.

Peptides are the smallest antigenic components that are recognized by T cells when presented in MHC molecules on the cell surface. After the identification of peptides from tumor associated and tumor specific antigens, the exploration of the use of peptides in immunotherapy of cancer was instigated. From initial exploration of peptide-mediated induction of immune responses in mice, the peptide based vaccines have evolved to clinical testing in cancer patients. Many different clinical trials have been performed to address the ability of peptide-based vaccines to induce both clinical and immunological responses in patients. This review will provide an overview of the results of the majority of the clinical trials with peptide-based vaccines directed against various antigens in patients with solid tumors.

[Cloning and expression of extracellular domain of prostate specific membrane antigen in Escherichia coli and preparation of polyclonal antibody].

Human Prostate Specific Membrane Antigen(PSMA) cDNA was amplified using total RNA extracted from prostate carcinoma tissue by RT-PCR. The cDNA fragment of extracellular domain of PSMA(edPSMA) gene was amplified by PCR and cloned into expression vector pMAL-c2x. Sequence analysis of both PSMA and edPSMA revealed identity to the GenBank reported. The edPSMA was expressed in E. coli as part of a fusion protein with MBP as the induction of IPTG. Western blot analysis showed the recombinant protein could react with PSMA monocloned antibodies 4G5. MBP-edPSMA fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE(120 kD). BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody. The polyclonal antibody, which had a title of 1:12,800, were indicated the specificity to prostate tissue.

Technology evaluation: MLN-591, Cornell University/BZL Biologics/ImmunoGen/Millennium.

MLN-591 is a monoclonal antibody specific for prostate-specific membrane antigen that is being developed by Cornell University, BZL Biologics, ImmunoGen Inc and Millennium Pharmaceuticals Inc for the potential treatment of prostate cancer. MLN-591 was in phase II trials by May 2002.