Biochemical Analysis of the Plasmodium falciparum Erythrocyte-binding Antigen-175 (EBA175)-Glycophorin-A Interaction: IMPLICATIONS FOR VACCINE DESIGN*?

PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2–3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains (“region II,” PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2–3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a K_D ∼0.26 μm and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.     

Structures of TraI in solution

Cobalt and potassium are biologically important metal elements that are present in a large array of proteins. Cobalt is mostly found in vivo associated with a corrin ring, which represents the core of the vitamin B₁₂ molecule. Potassium is the most abundant metal in the cytosol, and it plays a crucial role in maintaining membrane potential as well as correct protein function. Here, we report a thorough analysis of the geometric properties of cobalt and potassium coordination spheres that was performed with high resolution on a representative set of structures from the Protein Data Bank and complemented by quantum mechanical calculations realized at the DFT level of theory (B3LYP/ SDD) on mononuclear model systems. The results allowed us to draw interesting conclusions on the structural characteristics of both Co and K centers, and to evaluate the importance of effects such as their association energies and intrinsic thermodynamic stabilities. Overall, the results obtained provide useful data for enhancing the atomic models normally applied in theoretical and computational studies of Co or K proteins performed at the quantum mechanical level, and for developing molecular mechanical parameters for treating Co or K coordination spheres in molecular mechanics or molecular dynamics studies.

An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP

A novel microflow technique is used to demonstrate that a weakened oxidant defense system found in diabetic erythrocytes leads to decreased levels of deformation-induced release of adenosine triphosphate (ATP) from erythrocytes. Addition of an oxidant to rabbit erythrocytes resulted in a 63% decrease in deformation-induced ATP release before eventually recovering to a value that was statistically equivalent to the initial value. Inhibition of glucose-6-phosphate dehydrogenase prevents recovery from the oxidant attack. Finally, results indicated that the ATP release from the erythrocytes of type II diabetics (91 nM +/- 10 nM) was less than half of that measured from the erythrocytes of healthy controls (190 +/- 10 nM). These data suggest that the antioxidant status of erythrocytes is a critical determinant in the ability of these cells to release ATP, a known nitric oxide stimulus.     

Overexpression of MMP Family Members Functions as Prognostic Biomarker for Breast Cancer Patients: A Systematic Review and Meta-Analysis

Background

Matrix metalloproteinases (MMPs) are regarded to be relevant to the prognosis of breast cancer. Numerous studies have confirmed the association between MMPs and tumor growth, invasion and metastasis in breast cancer. However, their prognostic values for survival in patients with breast cancer remain controversial. Hence, a meta-analysis was performed to clarify a more accurate estimation of the role of MMPs on prognosis of breast cancer patients.

Method

A systemic electronic search was conducted in PubMed, Embase and Web of science databases to identify eligible studies, which were associated with the relationship between MMPs and prognosis of breast cancer. The correlation in random-effect model was evaluated by using the hazard ratios (HRs) and 95% confidence intervals (CIs).

Results

A total of 28 studies covering 4944 patients were included for meta-analysis. A summary hazard ratio (HR) of all studies was calculated, as well as the sub-group HRs. The combined HRs calculated by either univariate or multivariate analysis both suggested that overexpression of MMPs had an unfavorable impact on overall survival (OS) (HR = 1.694, 95%CI: 1.347–2.129, P < 0.001; HR = 1.611, 95%CI: 1.419–1.830, P < 0.001, respectively). And the univariate analysis showed that patients with overexpression of MMPs had worse relapse-free survival (RFS) (HR = 1.969, 95%CI: 1.460–2.655, P < 0.001) in all eligible studies. In the sub-group analyses, HRs of MMP-9 positivity with poor OS were 1.794 (95%CI: 1.330–2.420, P < 0.001) and 1.709 (95%CI: 1.157–2.526, P = 0.007) which were separately evaluated by univariate and multivariate analysis. A small number of articles demonstrated that MMP-2 overexpression was not related with shorter OS (HR = 1.400, 95%CI: 0.610–3.029, P = 0.427). Four studies included in the OS analysis of MMPs expression in serum suggested that positive expression of serum MMPs may be an unfavorable factor (HR = 1.630, 95%CI: 1.065–2.494) for breast cancer patients. No publication bias was observed in the current meta-analysis.

Conclusions

Our findings suggested that MMPs overexpression (especially MMP-9, MMP-2, MMPs overexpression in serum) might indicate a higher risk of poor prognosis in breast cancer. Larger prospective studies are further needed to estimate the prognostic values of MMPs overexpression.     

Dimerization of the Glucan Phosphatase Laforin Requires the Participation of Cysteine 329

Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin’s oligomerization.     

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.     

Solution structure and small angle scattering analysis of TraI (381-569)

TraI, the F plasmid-encoded nickase, is a 1756 amino acid protein essential for conjugative transfer of plasmid DNA from one bacterium to another. Although crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. The central region (between residues 306 and 1520) is known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Here, we show that the ssDNA binding site is located between residues 381 and 858, and we also present the high-resolution solution structure of the N-terminus of this region (residues 381-569). This fragment folds into a four-strand parallel β sheet surrounded by α helices, and it resembles the structure of the N-terminus of helicases such as RecD and RecQ despite little sequence similarity. The structure supports the model that F TraI resulted from duplication of a RecD-like domain and subsequent specialization of domains into the more N-terminal ssDNA binding domain and the more C-terminal domain containing helicase motifs. In addition, we provide evidence that the nickase and ssDNA binding domains of TraI are held close together by an 80-residue linker sequence that connects the two domains. These results suggest a possible physical explanation for the apparent negative cooperativity between the nickase and ssDNA binding domain.     

A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world''s major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.Parasites of the Plasmodium genus are the etiological agents responsible for malaria, an infectious disease mostly occurring in developing countries with up to 40% of the world''s population described as being at risk of the disease. Among the Plasmodium species that can affect humans, Plasmodium falciparum is responsible for the highest mortality, causing around one million deaths annually, mostly in children under the age of five (1). The clinical symptoms of malaria occur during the cyclic asexual blood stage of the parasite lifecycle when merozoites, that have invaded and replicated within host erythrocytes, are released into the bloodstream before invading new red blood cells (2). Despite intensive efforts from the research community there is currently no licensed vaccine for malaria. The leading candidate RTS,S/AS01, which targets the pre-erythrocytic stage of the disease and was tested in phase III trials, conferred 30 to 50% protection from clinical malaria, depending on the age group studied (3, 4). This limited efficacy has led to calls for a more effective vaccine and many have suggested that a combinatorial vaccine that additionally targets the blood stage may increase efficacy.A vaccine targeting the proteins expressed on the surface of the blood stage of the parasite is conceptually attractive because merozoites are repeatedly and directly exposed to the human humoral immune system and naturally acquired antibodies against these proteins have been shown to confer at least partial immunity (5–8). Despite this, only a few antigens discovered before the completion of the parasite genome sequence have been assessed in detail (9) and clinical vaccine trials using antigens that target the blood stage have so far shown limited efficacy, mostly caused by antigenic diversity (10). The sequencing of the parasite genome (11) has identified all possible targets but the systematic screening of these new candidates to assess their potential as a vaccine is hampered by the inability to systematically express recombinant Plasmodium proteins in their native conformation (12–15). Likely explanations might be the high (∼80%) A:T content of the P. falciparum genome resulting in low codon usage compatibility in heterologous expression systems, the large size (> 50 kDa) of many proteins, the presence of long stretches of highly repetitive amino acids, and the difficulty in identifying clear structural domains within these proteins using standard prediction computer programs (11). Extracellular proteins, in particular, present an additional challenge because they often have signal peptides and transmembrane regions that can negatively impact expression (16–18) and contain structurally important disulfide bonds. However, unlike most other eukaryotic extracellular proteins, Plasmodium cell surface and secreted proteins are not modified by N-linked glycans because of the absence of the necessary enzymes (19).To express Plasmodium proteins for basic research and vaccine development, a diverse range of expression systems have been tried (12) ranging from bacteria (17, 18), yeast (13), Dictyostelium (20), and plants (21) to mammalian cells (22) and cell-free systems (23–25). To circumvent the problem of codon usage, bacterial (26) and yeast (27) strains with modified tRNA pools have been developed, or sequences of the gene of interest synthesized and codon-optimized to match that of the expression host (28, 29). Although Escherichia coli has been the most popular expression system because of its relative simplicity and cost effectiveness, large-scale production of soluble functional Plasmodium falciparum recombinant proteins remains challenging with success rates ranging from just 6 to 21% (17, 18) and is often hindered by the need for complex refolding procedures. Similarly, attempts have been made to compile large panels of parasite proteins using in vitro translation systems (23, 25, 30, 31). These systems, however, require reducing conditions and are therefore not generally suitable for the systematic expression of extracellular proteins that occupy an oxidizing environment and critically require the formation of disulfide bonds for proper function. As a result, functional analyses of extracellular parasite proteins have often been restricted to smaller subfragments of the proteins that can be expressed in a soluble form rather than the entire extracellular region. Although eukaryotic expression systems are able to add disulfide bonds, they also often inappropriately glycosylate parasite proteins, adding further complication (32). A generic method that would overcome these technical challenges to express, in a systematic way, panels of recombinant Plasmodium proteins that have retained their native function and conformation would therefore be a valuable resource for the molecular investigations of erythrocyte invasion and the development of a blood stage vaccine.To generate a resource of correctly folded recombinant merozoite proteins, we used a mammalian expression system and established the parameters necessary for high-level expression. Using this method, we compiled a panel of 42 proteins that corresponds to the repertoire of abundant cell surface and secreted merozoite proteins of the 3D7 strain of Plasmodium falciparum. Biochemical activity of these proteins was demonstrated by recapitulating known protein interactions and by showing conformation-sensitive immunoreactivity of the recombinant proteins using immune sera.