Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Burkholderia cenocepacia-induced delay of acidification and phagolysosomal fusion in cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages

The Burkholderia cepacia complex (Bcc) is a group of opportunistic bacteria chronically infecting the airways of patients with cystic fibrosis (CF). Several laboratories have shown that Bcc members, in particular B. cenocepacia, survive within a membrane-bound vacuole inside phagocytic and epithelial cells. We have previously demonstrated that intracellular B. cenocepacia causes a delay in phagosomal maturation, as revealed by impaired acidification and slow accumulation of the late phagolysosomal marker LAMP-1. In this study, we demonstrate that uninfected cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages or normal macrophages treated with a CFTR-specific drug inhibitor display normal acidification. However, after ingestion of B. cenocepacia, acidification and phagolysosomal fusion of the bacteria-containing vacuoles occur in a lower percentage of CFTR-negative macrophages than CFTR-positive cells, suggesting that loss of CFTR function contributes to enhance bacterial intracellular survival. The CFTR-associated phagosomal maturation defect was absent in macrophages exposed to heat-inactivated B. cenocepacia and macrophages infected with a non-CF pathogen such as Salmonella enterica, an intracellular pathogen that once internalized rapidly traffics to acidic compartments that acquire lysosomal markers. These results suggest that not only a defective CFTR but also viable B. cenocepacia are required for the altered trafficking phenotype. We conclude that CFTR may play a role in the mechanism of clearance of the intracellular infection, as we have shown before that B. cenocepacia cells localized to the lysosome lose cell envelope integrity. Therefore, the prolonged maturation arrest of the vacuoles containing B. cenocepacia within cftr(-/-) macrophages could be a contributing factor in the persistence of the bacteria within CF patients.

The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion

The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.

Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein)--haptoEGFPs--which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein-haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.