Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.

Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.

A measles virus vaccine strain derivative as a novel oncolytic agent against breast cancer.

Breast cancer is the most common malignancy and the second leading cause of female cancer mortality in the United States. There is an urgent need for development of novel therapeutic approaches. In this study, we investigated the antitumor potential of a novel viral agent, an attenuated strain of measles virus deriving from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA) against breast cancer. CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of breast cancer cell lines including MDA-MB-231, MCF7 and SkBr3 at different multiplicities of infection (MOIs) from 0.1 to 10 resulted in significant cytopathic effect consisting of extensive syncytia formation and massive cell death at 72-96 h from infection. All breast cancer lines overexpressed the measles virus receptor CD46 and supported robust viral replication, which correlated with CEA production. TUNEL assays indicated an apoptotic mechanism of syncytial death. The efficacy of this approach in vivo was examined in a subcutaneous Balb C/nude mouse model of MDA-MB-231 cells. Intravenous administration of MV-CEA at a total dose of 1.2 x 10(7) TCID50 resulted in statistically significant tumor growth delay ( p=0.005) and prolongation of survival ( p=0.001). In summary, MV-CEA has potent antitumor activity against breast cancer lines and xenografts. Monitoring marker peptide levels in the serum could serve as a low-risk method of detecting viral gene expression during treatment and could allow dose optimization and individualization of treatment. Trackable measles virus derivatives merit further exploration in breast cancer treatment.

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments.

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE- and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by alpha(1)-antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val(15)-aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.

Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.

BACKGROUND: Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. METHODS: Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. RESULTS: The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP inhibitors 1,10 phenanthroline and N-hydroxy-5,5-dimethylpiperazine-2-carboxamide completely abolished AdM40-induced fusion, while the non-specific serine protease inhibitor soybean trypsin inhibitor did not affect it, thus demonstrating specificity of the observed effect. Intratumoral treatment of BalbC/nude mice bearing subcutaneous U87 glioma xenografts with AdM40 at a total dose of 1.2 x 10(10) plaque-forming units (pfu) resulted in statistically significant tumor regression as compared with control animals either treated with AdN40 (p = 0.01) or untreated animals (p = 0.01). Treatment with AdM40 also resulted in survival improvement as compared with AdN40-treated animals (p = 0.006) or untreated animals (p = 0.001). Histopathologic examination of treated tumors demonstrated extensive syncytia formation. CONCLUSIONS: Our data indicate that AdM40, a replication-defective adenovirus expressing the GALV fusogenic glycoprotein, attached to a blocking ligand via an MMP-cleavable linker, can target the cytotoxicity of GALV in MMP-overexpressing glioma lines and xenografts, and maintain significant antitumor activity both in vitro and in vivo. Given the high frequency of MMP overexpression in gliomas, AdM40 represents a potentially promising agent in the gene therapy of these tumors.