Exploring proteoforms of the IgG2 monoclonal antibody panitumumab using microchip capillary electrophoresis-mass spectrometry.

The IgG2 type monoclonal antibody panitumumab is an anti-epidermal growth factor receptor (EGFR) drug used for the treatment of EGFR-expressing, chemotherapy resistant, metastatic colorectal carcinoma. In this study, panitumumab drug product was first analysed using size exclusion chromatography coupled to mass spectrometry for rapid identity testing. The experimental data led to the identification of two panitumumab isoforms with several prominent forms remaining unidentified, despite apparently low sample complexity. Microchip capillary electrophoresis-mass spectrometry (CE-MS) was subsequently utilised for a more detailed characterization. It was observed that panitumumab is subject to partial N-terminal pyroglutamate formation. Incomplete conversion is uncharacteristic for N-terminally exposed glutamines and in case of panitumumab gives rise to forms which show successive mass offsets of 17 Da, respectively. If not separated before mass spectrometric analysis, e.g. by capillary electrophoresis, such near isobaric species coalesce into single MS peaks, which subsequently hampers or prevents their assignment. With a total of 42 panitumumab isoforms assigned by CE-MS, these observations highlight a potential pitfall of commonly applied rapid identity testing workflows and demonstrate that even low complexity biopharmaceuticals can require separation strategies which offer high separation selectivity for species close in mass.

Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.

Despite the global medical needs associated with Staphylococcus aureus infections, no licensed vaccines are currently available. We identified and characterized a protein annotated as an epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. In addition, we determined the structure of the recombinant protein (rEpiP) by X-ray crystallography. The crystal structure revealed that rEpiP was cleaved somewhere between residues 95 and 100, and we found that the cleavage occurs through an autocatalytic intramolecular mechanism. The protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine whether the protein acts as a serine protease, we mutated the hypothesized catalytic serine 393 residue to alanine, generating rEpiP-S393A. The crystal structure of this mutant protein showed that the polypeptide chain was not cleaved and was not interacting stably with the active site. Indeed, rEpiP-S393A was shown to be impaired in its protease activity. Mice vaccinated with rEpiP were protected from S. aureus infection (34% survival, P=0.0054). Moreover, the protective efficacy generated by rEpiP and rEpiP-S393A was comparable, implying that the noncleaving mutant could be used for vaccination purposes.

Streptococcus pyogenes SpyCEP: a chemokine-inactivating protease with unique structural and biochemical features.

SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids. The same type of organization is present in the enzyme obtained from S. pyogenes. Furthermore, N-terminal SpyCEP is not involved in the folding of the mature enzyme. The 2 protease fragments were separately expressed in E. coli as soluble polypeptides that, when combined, reconstituted a fully active enzyme complex. Therefore, SpyCEP appears to possess a completely new structural architecture that has not been described so far for other microbial proteases.

Proteomics characterization of outer membrane vesicles from the extraintestinal pathogenic Escherichia coli DeltatolR IHE3034 mutant.

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.

Proteomic analysis of the airway surface liquid: modulation by proinflammatory cytokines.

The airway surface is covered by a fluid, the airway surface liquid, interposed between the mucous layer and the epithelium. The airway surface liquid contains proteins, secreted by different cell types, that may have pro-/anti-inflammatory or bactericidal functions or have a role in the mucociliary clearance. We have used a proteomics approach to identify the proteins secreted by an isolated in vitro model of human airway epithelium, at resting and under proinflammatory conditions, as a strategy to define the factors involved in epithelial barrier function. To this aim, we have analyzed the airway surface liquid from human bronchial epithelial cells grown as polarized monolayers in the presence and absence of inflammatory stimuli such as IL-4, IL-1beta, TNF-alpha, and IFN-gamma. Two-dimensional electrophoresis followed by mass spectrometry analysis has allowed the identification of approximately 175 secreted protein spots, among which are immune-related proteins, structural proteins, an actin severer, some protease inhibitors, and a metalloproteinase. Comparisons between treated and untreated conditions have shown that the expression of several proteins was significantly modified by the different cytokines. Our results indicate that the surface epithelium is an active player in the epithelial barrier function and that inflammatory conditions may modulate protein secretion.

Gelsolin secretion in interleukin-4-treated bronchial epithelia and in asthmatic airways.

RATIONALE: The airway surface liquid, the thin layer of liquid covering the airways, is essential for mucociliary clearance and as a barrier against microbial and other noxious agents. Proteins secreted into the airway surface liquid by epithelial and nonepithelial cells may be important in innate immunity and to improve the fluidity of mucous secretions. OBJECTIVES: We aimed to identify proteins specifically secreted into the airway surface liquid by human bronchial epithelial cells, in resting conditions and after treatment with interleukin 4 (IL-4), a cytokine released in asthma. METHODS AND MAIN RESULTS: By using a proteomics approach, we found that one of the most abundant proteins was gelsolin, which breaks down actin filaments. Gelsolin mRNA and protein secretion were increased threefold in the airway surface liquid of epithelia treated with IL-4. These results were confirmed at the functional level by measuring actin depolymerization using a fluorescence assay. Gelsolin protein was also upregulated in the airways of subjects with asthma. CONCLUSIONS: Our findings indicate that gelsolin is released by epithelial cells into the airways and that its secretion is increased by IL-4 in vitro. In addition, we found that the concentration of both IL-4 and gelsolin were raised in the bronchoalveolar lavage of patients with asthma. These results suggest that gelsolin might improve the fluidity of airway surface liquid in asthma by breaking down filamentous actin that may be released in large amounts by dying cells during inflammation.

Discovering high mobility group A molecular partners in tumour cells.

DNA-based activities rely on an extremely coordinated sequence of events performed by several chromatin-associated proteins which act in concert. High Mobility Group A (HMGA) proteins are non-histone architectural nuclear factors that participate in the regulation of specific genes but they are also believed to have a more general role in chromatin dynamics. The peculiarity of these proteins is their flexibility, both in terms of DNA-binding and in protein-protein interactions. Since these proteins act as core elements in the assembly of multiprotein complexes called enhanceosomes, and have already displayed the ability to interact with several different proteins, we started a proteomic approach for the systematic identification of their molecular partners. By a combination of affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry we have identified about twenty putative HMGA interactors which could be roughly assigned to three different classes: mRNA processing proteins, chromatin remodelling related factors and structural proteins. Direct HMGA interaction with some of these proteins was confirmed by glutathione-S-transferase pull-down assays and the HMGA domain involved was mapped. Blot-overlay experiments reveal that members of the HMGA family share most of their molecular partners but, interestingly, it seems that there are some cell-type specific partners. Taken together, these experimental data indicate that HMGA proteins are highly connected nodes in the chromatin protein network. Since these proteins are strongly implicated with cancer development, the identification of molecules able to perturb the HMGA molecular network could be a possible tool to interfere with their oncogenic activity.

Induction of Mycobacterium avium proteins upon infection of human macrophages.

Induction of Mycobacterium avium proteins labelled with [35S]methionine and mRNAs upon infection of the human macrophage cell line THP-1 was investigated by two-dimensional gel electrophoresis-mass spectrometry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. M. avium overexpressed proteins within the macrophages that are involved in fatty acids metabolism (FadE2, FixA), cell wall synthesis (KasA), and protein synthesis (EF-tu). The correlation of differential protein and mRNA expression varied between good and no correlation. Overall, these four proteins may be involved in the adaptation and survival of M. avium within human macrophages.

Translationally controlled tumor protein (TCTP) in the human prostate and prostate cancer cells: expression, distribution, and calcium binding activity.

BACKGROUND: The translationally controlled tumor protein (TCTP) is an abundantly expressed protein found in a wide range of organisms from both the animal and plant kingdom. Initially described as a growth-related protein, knowledge of the biological actions of TCTP has been recently extended to include calcium binding, regulation of apoptosis, and microtubules stabilization. This report describes expression, distribution, and characterization of TCTP in human prostatic tissues and cell lines. METHODS: Samples were analyzed by Western blot, RT-PCR, immunohistochemistry, and confocal microscopy. Calcium binding activity of the recombinant human prostatic protein was evaluated on a calcium overlay assay. A public SAGE database was analyzed to determine TCTP expression levels in normal and cancer tissues. RESULTS: TCTP protein and mRNA were detected in all the specimens and cell lines analyzed. The protein was mainly expressed by the secretory luminal epithelial and basal layer cells. A significant amount of protein was present in the prostatic fluids. Subcellular distribution studies in prostate epithelial cells detected the protein in the cytoplasm in interphase and colocalized with tubulin during mitosis. The calcium binding capacity of prostatic TCTP was shown in vitro. Finally, SAGE data indicated TCTP as the calcium binding protein with the highest expression levels among those examined. CONCLUSIONS: The results of the present study demonstrate, for the first time, the expression of TCTP in the human prostate and in prostate cancer cells, and suggest the involvement of the protein in key-processes such as apoptosis, cellular differentiation, and in the control of sperm functions.

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed.

Normal human dermal fibroblasts: proteomic analysis of cell layer and culture medium.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

Characterization of membrane nongenomic receptors for progesterone in human spermatozoa.

Rapid, nongenomic actions of steroid hormones have been characterized only recently. They may be mediated by interaction with a poorly characterized membrane receptor, by classic receptor located to the plasma membrane, or by interaction of the classic receptor with other signaling effectors. Among these, rapid effects of progesterone on human spermatozoa have been shown to be mediated by interaction with one or more membrane receptors. Two proteins, respectively of 57 and 28 kDa, representing the possible surface progesterone receptors in human spermatozoa, have been identified by our group employing an antibody (c-262) directed against the progesterone binding domain of the genomic receptor. The two proteins have been immunoprecipitated using c-262, isolated by 2D gel electrophoresis and analyzed by Maldi-Tof. Preliminary results of the analysis in data bank of the obtained masses suggest that the two proteins represent previously unidentified ones since they do not match with any protein in the database. We have also performed RT-PCR analysis with RNA extracted from human spermatozoa, utilizing various oligoprimers in different regions of the human progesterone genomic receptor. Results indicate the presence of transcripts for the complete genomic receptor. However, several previously published studies in the literature indicate the absence of expression of the genomic receptor in human spermatozoa. In this light posttranscriptional/posttraductional modifications of the receptor can be hypothesized. Interestingly, with primers amplifying in the DNA-binding domain of the progesterone receptor gene, we detected a higher molecular weight transcript when compared to the placenta. Further studies are needed to determine whether the sequences of the transcripts obtained by RT-PCR analysis of human sperm RNA match exactly with the human genomic receptor gene and to define the sequence of the higher molecular weight transcript detected in the DNA-binding region.