Using intracellular SCGB1A1-sorted, formalin-fixed club cells for successful transcriptomic analysis.

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1+) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells.

Targeting human secretory phospholipase A2 with designed peptide inhibitors for inflammatory therapy.

Phospholipase A2 (PLA2) is potentially an important target for anti-inflammatory therapeutics. Here, we described a systematic scheme that integrated protein docking and peptide redocking, molecular dynamics simulation, and binding affinity analysis to rationally design PLA2 inhibitory peptides based on a solved PLA2 crystal structure. The scheme employed protein docking to sample the interaction modes of PLA2 with its natural inhibitor Clara cell protein, from which a number of peptide fragments, including a pentapeptide LLLGS, were cut off and redocked to serve as the lead entities of PLA2 inhibitory peptides. In addition, a systematic mutation energy map that characterized the binding free energy changes ΔG upon mutations of each position of the putative pentapeptide to 20 amino acids was also profiled, which was subsequently used to guide peptide structure optimization. In order to solidify the computational findings, we performed kinetic and inhibition studies of few designed peptides against human secretory PLA2. Consequently, eight peptides were successfully identified to have potent inhibition potency, in which the LLAYK and AVFRS were found to suppress enzymatic activity significantly (Ki = 0.75 ± 0.06 and 4.2 ± 0.3 µM, respectively). A further structure examination revealed that the designed peptides can form intensive nonpolar networks of van der Waals contacts and hydrophobic interactions at their complex interfaces with PLA2, conferring considerable stability and affinity for the formed complex systems.

Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli.

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.

Selective targeted delivery of the TNF-alpha receptor p75 and uteroglobin to the vasculature of inflamed tissues: a preliminary report.

BACKGROUND: Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. We hypothesized that the specific targeting of TNF-alpha antagonists to inflamed tissues could increase drug efficacy and reduce side effects. RESULTS: Using uteroglobin (UG), a potent anti-inflammatory protein, as a scaffold, we prepared a bispecific tetravalent molecule consisting of the extracellular ligand-binding portion of the human TNF-alpha receptor P75 (TNFRII) and the scFv L19. L19 binds to the ED-B containing fibronectin isoform (B-FN), which is expressed only during angiogenesis processes and during tissue remodeling. B-FN has also been demonstrated in the pannus in rheumatoid arthritis. L19-UG-TNFRII is a stable, soluble homodimeric protein that maintains the activities of both moieties: the immuno-reactivity of L19 and the capability of TNFRII to inhibit TNF-alpha. In vivo bio-distribution studies demonstrated that the molecule selectively accumulated on B-FN containing tissues, showing a very fast clearance from the blood but a very long residence time on B-FN containing tissues. Despite the very fast clearance from the blood, this fusion protein was able to significantly improve the severe symptomatology of arthritis in collagen antibody-induced arthritis (CAIA) mouse model. CONCLUSIONS: The recombinant protein described here, able to selectively deliver the TNF-alpha antagonist TNFRII to inflamed tissues, could yield important contributions for the therapy of degenerative inflammatory diseases.

Identification and characterization of Ch806 mimotopes.

The chimeric antibody 806 (Ch806) is a promising antitumor agent that recognizes both the epidermal growth factor receptor variant III (EGFRvIII) and the overexpressed epidermal growth factor receptor (EGFR) in cancer tissues but does not recognize the wild type EGFR in normal tissues. However, passive antibody immunization could not produce effective antitumor titers unless the immunization was administered repeatedly over long periods. To overcome this limitation, we generated epitope mimics that bind to Ch806 and tested whether the peptide mimics could induce the production of similar antibodies when actively immunizing mice with the peptides. We used the PH.D-12 phage display peptide library to identify peptides that bind to the monoclonal antibody (mAb) 12H23, which also recognizes similar epitopes of Ch806. Two mimotopes (WHTEILKSYPHE and LPAFFVTNQTQD) were shown to mimic the mAb 12H23 and Ch806 epitope using immunoassays. The mimotopes were conjugated to immunogenic carrier proteins and used to intraperitoneally immunize BALB/c mice. Interestingly, sera from the mice immunized with the isolated mimotopes not only recognize the recombinant or synthetic 806 eptitope, but can also recognize EGFR that is overexpressed in A431 cells and EGFRvIII expressed in Huh7-EGFRvIII cells, whereas sera from mice immunized with the control peptide-KLH (keyhole limpet hemocyanin) and carrier KLH alone failed to show a similar reactivity. Furthermore, in an antibody-dependent cellular cytotoxicity assay (ADCC), the mimotope-induced antibodies specifically lysed human Huh-7-EGFRvIII cells. Our data indicate that the isolated mimotopes reported here may potentially be used as new alternative agents for treating cancer with EGFRvIII expression or EGFR overexpression.

Airway regeneration: the role of the Clara cell secretory protein and the cells that express it.

Clara cell secretory protein (CCSP) is one of the most abundant proteins in the airway surface fluid, and has many putative functions. Recent advances in the field of stem cells and lung regeneration have identified potentially new roles of CCSP and CCSP-expressing cell populations in airway maintenance, repair and regeneration. This review focuses on the airway regenerative potential of CCSP and the cells that express this protein. The use of this protein or CCSP-expressing cells as an indication of biologic processes that contribute to lung injury or repair is highlighted.

Hypochlorous acid reacts with the N-terminal methionines of proteins to give dehydromethionine, a potential biomarker for neutrophil-induced oxidative stress.

Electrophilic halogenating agents, including hypohalous acids and haloamines, oxidize free methionine and the N-terminal methionines of peptides and proteins (e.g., Met-1 of anti-inflammatory peptide 1 and ubiquitin) to produce dehydromethionine (a five-membered isothiazolidinium heterocycle). Amide derivatives of methionine are oxidized to the corresponding sulfoxide derivatives under the same reaction conditions (e.g., Met-3 of anti-inflammatory peptide 1). Other biological oxidants, including hydrogen peroxide and peroxynitrite, also produce only the corresponding sulfoxides. Hypothiocyanite does not react with methionine residues. We suggest that dehydromethionine may be a useful biomarker for the myeloperoxidase-induced oxidative stress associated with many inflammatory diseases.

Use of uteroglobin for the engineering of polyvalent, polyspecific fusion proteins.

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-alpha, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.

A multi-objective evolutionary algorithm for protein structure prediction with immune operators.

Genetic algorithms (GA) are often well suited for optimisation problems involving several conflicting objectives. It is more suitable to model the protein structure prediction problem as a multi-objective optimisation problem since the potential energy functions used in the literature to evaluate the conformation of a protein are based on the calculations of two different interaction energies: local (bond atoms) and non-local (non-bond atoms) and experiments have shown that those types of interactions are in conflict, by using the potential energy function, Chemistry at Harvard Macromolecular Mechanics. In this paper, we have modified the immune inspired Pareto archived evolutionary strategy (I-PAES) algorithm and denoted it as MI-PAES. It can effectively exploit some prior knowledge about the hydrophobic interactions, which is one of the most important driving forces in protein folding to make vaccines. The proposed MI-PAES is comparable with other evolutionary algorithms proposed in literature, both in terms of best solution found and the computational time and often results in much better search ability than that of the canonical GA.

Interaction of antiflammin-1 with uteroglobin-binding protein induces phosphorylation of ERK1/2 in NIH 3T3 cells.

Previously, it has been suggested that uteroglobin (UG)-binding protein functions as a putative receptor of UG; however, the specific epitope of UG that interacts with this receptor has not yet been identified. The downstream events of UG-binding protein signaling remain unclear. Here we report that antiflammin-1 (AF-1, a bioactive C-terminal peptide of UG) specifically binds to UG-binding protein and has a cellular signaling consequence. We reduced the level of endogenous UG-binding protein expression in murine fibroblast cell line NIH 3T3 by RNA interference and found that knockdown of UG-binding protein inhibited AF-1-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Meanwhile, the interaction between AF-1 and UG-binding protein was confirmed by flow cytometry-based binding assays and co-localization of AF-1 and enhanced green fluorescent protein (EGFP)-tagged UG-binding protein. The present study provides evidence for the first time for AF-1 binding with UG-binding protein, and preliminarily characterized UG-binding protein as a point downstream of AF-1 in mediating ERK phosphorylation.

Structural characterization of the tetrameric form of the major cat allergen Fel d 1.

Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.

Effects of antiflammins on transglutaminase and phospholipase A2 activation by transglutaminase.

Two anti-inflammatory peptides, named antiflammins (AFs), corresponding to a region with high amino acid similarity between lipocortin-1 and uteroglobin were tested for their ability to inhibit transglutaminase (TG) and low-molecular-mass phospholipase A2 (PLA2). Porcine pancreatic PLA2 activity and guinea pig hepatic TG activity were determined by arachidonyl release from arachidonyl-phosphatidylcholine and by the incorporation of putrescine into succinylated casein, respectively. AFs inhibited TG activity but did not affect PLA2 activity. Moreover, porcine pancreatic PLA2 was activated by TG and AFs decreased porcine pancreatic PLA2 activation induced by TG. Taken together, our results support the hypothesis that the anti-inflammatory effects of AFs are, at least in part, due to the action of AFs on TG activity.

Coupe du roi bisection of proteins. Spontaneous tetramerization of two peptides that span the sequence of the rabbit uteroglobin monomer.

The study of dividing objects into isometric segments has yielded novel approaches to the synthesis of high-symmetry organic compounds. Reported herein is the first application of this concept to a protein, rabbit uteroglobin (UG). Bisection of UG into two identical homochiral segments led to the design of the heterodimeric 70mer peptide alpha(1,2)-S-S-alpha(3,4) that spans the sequence of the native UG monomer. The ability of this compound to form a globular 140mer tetramer consisting of two noncovalently bound heterodimers was assessed by ultracentrifugation at sedimentation equilibrium and by fluorescent spectroscopy. On the other hand, the monomeric peptides alpha(1,2)-SH and alpha(3,4)-SH were shown to selectively form the alpha(1,2)-S-S-alpha(3,4) heterodimer via spontaneous air oxidation in phosphate buffer at neutral pH.

The safety, pharmacokinetics, and anti-inflammatory effects of intratracheal recombinant human Clara cell protein in premature infants with respiratory distress syndrome.

Clara cell 10-kD protein (CC10) is a potent anti-inflammatory protein that is normally abundant in the respiratory tract. CC10 is deficient and oxidized in premature infants with poor clinical outcome (death or the development of bronchopulmonary dysplasia). The safety, pharmacokinetics, and anti-inflammatory activity of recombinant human CC10 (rhCC10) were evaluated in a randomized, placebo-controlled, double-blinded, multicenter trial in premature infants with respiratory distress syndrome. A total of 22 infants (mean birth weight: 932 g; gestational age: 26.9 wk) received one intratracheal dose of placebo (n = 7) or 1.5 mg/kg (n = 8) or 5 mg/kg (n = 7) rhCC10 within 4 h of surfactant treatment. Pharmacokinetic analyses demonstrated that the serum half-life was 11.6 (1.5 mg/kg group) and 9.9 h (5 mg/kg group). Excess circulating CC10 was eliminated via the urine within 48 h. rhCC10-treated infants showed significant reductions in total cell count (p < 0.0002), neutrophil counts (p < 0.001), and total protein concentrations (p < 0.01) and tended to have decreased IL-6 (p < 0.07) in tracheal aspirate fluid collected over the first 3 d of life. Infants in all three groups showed comparable growth. At 36 wk postmenstrual age, five of seven infants were still hospitalized and two of seven infants were receiving oxygen in the placebo group compared with two of seven hospitalized and one of seven receiving oxygen in the 1.5-mg/kg group and four of six hospitalized and three of six receiving oxygen in the 5-mg/kg group. A single intratracheal dose of rhCC10 was well tolerated and had significant anti-inflammatory effects in the lung. Multiple doses of rhCC10 will be investigated for efficacy in reducing pulmonary inflammation and ameliorating bronchopulmonary dysplasia in future studies.

Recombinant bovine uteroglobin at 1.6 A resolution: a preliminary X-ray crystallographic analysis.

Uteroglobin (UG) is a conserved protein which is induced by progesterone and secreted by the epithelia of various mammalian reproductive and respiratory organs. Recombinant bovine uteroglobin (recbUG), consisting of 80 amino acids with a C-terminal His6 tag, was overexpressed in Escherichia coli and purified. The protein was crystallized in two geometric forms, rhomboid and cuneate (wedge-shaped), by the hanging-drop vapour-diffusion method at 295 K. The rhomboid crystals diffracted to a maximum resolution of 1.6 A using synchrotron radiation. These crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 81.42, b = 82.82, c = 45.26 A, and contain four monomers per asymmetric unit. The cuneate crystals diffracted to 2.35 A resolution using a rotating-anode generator. These crystals belong to space group C222(1), with unit-cell parameters a = 43.39, b = 93.94, c = 77.30 A, and contain two molecules per asymmetric unit.

Mammaglobin a in breast cancer: existence of multiple molecular forms.

Existing serum-based markers for breast cancer all lack organ specificity. Mammaglobin A (MGA) is a 93 amino acid protein expressed almost exclusively in breast tissue. The aim of our study was to investigate the different forms of MGA protein in fibroadenomas and breast carcinomas. MGA protein was measured by Western blotting in 132 breast cancers, 29 fibroadenomas and 14 nonbreast tissues. MGA protein in breast tissue was found to exist in 2 main forms. These forms migrated with approximate molecular masses of 18 and 25 kDa. Both forms of MGA were detected more frequently in breast carcinomas compared to fibroadenomas. The high molecular weight form of MGA but not the low molecular weight form was found more frequently in hormone receptor-positive than in receptor-negative cancers. Furthermore, an inverse relationship was found between the high molecular weight form of MGA and both tumour grade and proliferation index. No significant correlation was found between the MGA proteins and either tumor size or nodal status. Our results show that MGA protein exists in 2 main forms in breast tissue. As the high molecular weight form correlated positively with hormone receptors and negatively with tumor grade and proliferation rate, its presence is likely to be associated with a favourable prognosis for breast cancer. As expression of MGA is almost breast specific, it is a promising marker for breast cancer. Its most immediate use is likely to be in detecting micrometastases from breast cancer.

cDNA sequence, 5'-flanking region, and promoter activity of the Neotomodon alstoni alstoni Clara cell secretory protein gene.

To better understand the phylogenetic divergence and the species-specific characteristics of the Clara cell secretory protein (CCSP), we cloned the cDNA encoding the neotomodon CCSP (nCCSP) and analyzed its tissue-specific expression. The full-length cDNA is 451bp long and predicts an amino acid sequence of 93 residues. Northern blot analysis from different neotomodon tissues demonstrated that the mRNA of CCSP appears to be solely expressed in the lung. To study the transcriptional regulation of the CCSP gene, we cloned the 5'-flanking region of the nCCSP gene and compared its features with those previously reported for the hamster gene. The neotomodon and hamster genes share 89% sequence homology in their promoter region as well as a number of conserved cis-acting elements. However, in H441 cells the expression of a reporter gene driven by the nCCSP promoter was about 4-fold greater than its hamster counterpart. Functional analysis of progressive 5'-deletion mutants identified a region involved in the higher transcriptional activity of the neotomodon promoter.

Mammaglobin: a candidate diagnostic marker for breast cancer.

Mammaglobin, known for its mammary tissue specificity, has been discussed as a promising diagnostic marker in breast cancer for almost 10 years. In particular, the application of mammaglobin RT-PCR to detect disseminated breast cancer cells has been reported. More than 25 publications evaluate the detection of mammaglobin mRNA in lymph node, blood, and bone marrow specimens of breast cancer patients. Recently, structural details about the mammaglobin complex have been discovered, and these findings can be implemented to optimize detection of the secreted protein. This review summarizes the findings of almost 50 published studies and the current knowledge about the diagnostic utility of mammaglobin.

Clara cell secretory protein: determination of serum levels by an enzyme immunoassay and its importance as an indicator of bronchial asthma in children.

Clara cell secretory protein (CC16) is a 16kDa protein secreted by Clara cells in the lining fluid of bronchiolar and bronchial epithelium. CC16 presents several biologic properties, and has been shown to have immunomodulatory and anti-inflammatory activity. It may play a role in controlling inflammation in the airway. There is some evidence that the CC16 level is primarily lower in adult individuals with bronchial asthma, thus contributing to its pathophysiology. This study was designed to examine CC16 serum levels of children, healthy and with asthma. An enzyme solid phase immunoassay utilizing monoclonal antibody to CC16 was the analytical method to determine the protein concentration in blood sera. The method showed excellent linearity, high sensitivity (detection limit: <50 ng/l) and precision. It was found that asthmatic children appear significantly lower levels (P < 0.001) of CC16 in serum as compared to healthy ones. It is, therefore, concluded that CC16 may be a useful diagnostic index of bronchial asthma in the early child-age.