Presence Of Maltose Binding Protein Research Articles

Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK2, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 μM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium toward the transition state of MalFGK2. We tested this hypothesis by using site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy, which showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open, and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted toward the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.

Modular protein switches derived from antibody mimetic proteins.

Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms.

Abstract P6-09-04: M2 macrophages induced by mammary carcinoma are switched to M1 macrophages by Escherichia coli maltose-binding protein

Abstract Background: The maltose-binding protein (MBP) is a component of the maltose transport system in the Escherichia coli. It is used to increase the stability and solubility of proteins in bacterial protein expression systems and is increasingly being used to facilitate the production and delivery of subunit vaccine. Tumor-associated macrophages (TAMs) are alternatively activated macrophages (M2) within the tumor microenvironment, which directly promoting tumor cell growth, extracellular matrix remodeling, and angiogenesis. Our previous studies revealed that MBP could nonspecifically induce Th1 cells activation and classically activate macrophages (M1). In the present study, we investigated the effect of MBP on re-polarization of mammary carcinoma induced M2 macrophage to pro-inflammatory M1 macrophages, in order to develop a novel antitumor immune adjuvant. Methods: We mimicked a tumor microenvironment by in vitro coculturing RAW264.7 macrophages with conditioned media (CM) from two different kinds of mammary carcinoma cell lines EMT6 and 4T1. In the absence or presence of MBP, the mammary carcinoma CM-induced macrophages were tested for their polarization types. The production of IL-12 and IL-10 were assessed by ELISA, the mRNA expression of iNOS and Arg-1 were detected by RT-PCR, and the expression of CD206 and CD16/32 were determined by flow cytometry and immunofluorescence staining. All of these important factors were taken as criteria for M1 and M2 polarization. The expression levels of TLR2 and TLR4 on the surface of RAW264.7 cells were determined by flow cytometry. By the addition of TLR2 or TLR4 blocking antibody, the ratio of IL-12 and IL-10 production was used to distinguish M1 and M2 macrophages in the absence or presence of MBP. For the investigation of the signal transduction pathways, the expression level of MyD88 and the phosphorylation levels of IκB-α, NF-κB, PI3K, Akt, MAPKs, JNK were analyzed by western blotting. Results: The mammary carcinoma CM induced the RAW264.7 macrophage to polarize to M2 macrophages. In the mammary carcinoma CM-induced macrophages, the expression level of CD206 was increased, the ratio of IL-12/IL-10 production was decreased, and the gene expression of Arg-1 was increased. In the presence of MBP, M2 polarization was attenuated as shown by the increase of CD16/32 and iNOS, the decrease of CD206 and Arg-1, an altered ratio of IL-12/IL-10, and accompanied with the up-regulation of TLR2 and TLR4 expressions. The effect of MBP on mammary carcinoma CM-induced macrophages was blocked by anti-TLR2 antibody, but not anti-TLR4 antibody or TLR4 inhibitor polymyxin B. Further studies confirmed that the effects of MBP on switching mammary carcinoma induced M2 macrophages to M1 depended on the activation of NF-κB and MAPKs. Conclusion: Our results provide new insights into the modulation of TAM polarization. MBP can be used as a novel antitumor adjuvant, which may stimulate a tumor-rejecting environment by switching M2 macrophages to classical pro-inflammatory M1 macrophages through TLR2 signaling and a combination of transcription factors including NK-κB and MAPKs. Accordingly, MBP may have the therapeutic potential in mammary carcinoma. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-04.

Both Lobes of Maltose Binding Protein are Engaged in Stabilization of the Semi-Open State of the Malk Dimer in the Maltose ABC Transporter

ATP-binding cassette (ABC) transporters involved in uptake in bacteria are capable of accumulating substrates against a concentration gradient at the expense of ATP. In most systems, high-affinity binding protein located in the periplasmic space or outside the cell scavenges solutes for delivery to and through the transmembrane components. One such system is the maltose transporter in Escherichia coli, which is composed of a periplasmic maltose binding protein (MBP), two transmembrane domains, MalF and MalG, and a nucleotide-binding homodimer, MalK2. Interaction of the ligand-bound MBP with the MalFGK2 transporter in the presence of MgATP triggers conformational changes that result in reorientation of transmembrane helices to receive maltose from MBP and closure of the nucleotide-binding interface in MalK2 to hydrolyze ATP. Following ATP hydrolysis, the MalK dimer opens and transmembrane helices reorient, bringing maltose into the cell. Recently, evidence of a semi-open state of MalK2 was found when the transporter was allowed to hydrolyze ATP in the presence of MBP. We have used site-directed spin labeling and electron paramagnetic resonance spectroscopy to further characterize this “post-hydrolysis” state. By placing EPR probes in different locations along the interface where MBP is known to interact with MalFGK2, we found that whereas only the N-lobe is bound to the transporter in the resting state conformation, both the N- and C-terminal lobes of MBP are engaged with the transporter under conditions where the semi-open state of MalK2 is stabilized. Interpreted in light of how MBP may stimulate the ATPase activity of the transporter, it appears that both lobes of MBP are involved in dictating the conformation of the MalK dimer on the opposite site of the membrane.

Preserved epitope-specific T cell activation by recombinant Bet v 1-MBP fusion proteins.

Allergen-specific T cells play an important role in the allergic immune response, and are thought to be the principal target in specific immunotherapy. The aim of the present study was to evaluate if fusion proteins of allergens with bacterial proteins can be used to activate and bias allergen-specific T cells, and to characterize T cell epitopes. The complete gene of Bet v 1, the major birch pollen allergen, was amplified by PCR from birch pollen mRNA, and cloned in pKK223-3. The complete gene or truncated sequences were transferred to pMAL-c and expressed in E. coli as fusion proteins with maltose binding protein (MBP). The complete fusion protein, and the truncated fusion proteins were used for studies with Bet v 1-specific T cells. Bet v 1-specific T cells reacted similarly with purified and crude Bet v 1-MBP proteins. Therefore, crude preparations were used to study the epitope-specificity of 11 Bet v 1-specific T cell clones. Six distinct T cell epitopes were determined in this way. Interestingly, the T cell epitope of three T cell clones, that did not react with synthetic peptides in a previous study, was identified. In addition, the presence of MBP as a fusion partner to Bet v 1 was shown to influence TH2/TH1 cytokine production in T cell lines, but not in established T cell clones. Using crude preparations of recombinant fusion proteins of Bet v 1 with MBP, multiple T cell epitopes were identified in Bet v 1. As T cell activation is preserved in this system, the generation of recombinant allergens with TH1-inducing proteins as fusion partners might be considered as a T-cell targeted approach for specific immunotherapy.

Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system.

Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK2), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK2 complex and that MBP plays a crucial role in triggering the transport process.

Interaction between ATP, oleandomycin and the OleB ATP-binding cassette transporter of Streptomyces antibioticus involved in oleandomycin secretion.

The OleB protein of Streptomyces antibioticus, oleandomycin (OM) producer, constitutes an ATP-binding cassette transporter containing two nucleotide-binding domains and is involved in OM resistance and its secretion in this producer strain. We have characterized some properties of the first nucleotide-binding domain of OleB using an overexpressed fusion protein (MBP-OleB') between a maltose-binding protein (MBP) and the first half of OleB (OleB'). Extrinsic fluorescence of the base-modified fluorescent nucleotide analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) and 2'(3')-o-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate was determined in the presence of MBP and the fusion protein MBP-OleB', and it was found that epsilon ATP binds to MBP-OleB' with a stoichiometry of 0.9. Measurements of the intrinsic fluorescence of the MBP-OleB' fusion protein indicated that ATP induces a decrease in the accessibility of the MBP-OleB' tryptophans to acrylamide, an indication of a folding effect. This conclusion was confirmed by the fact that ATP also induces considerable stabilization against guanidine chloride denaturation of MBP-OleB'. Two effects were found to be associated with the presence of Mg2+ ions: (1) an increase in the quenching of MBP-OleB' intrinsic fluorescence by ATP; and (2) an increase in the accessibility of MBP-OleB' tryptophans to acrylamide. Significant changes in the intrinsic fluorescence of the fusion protein were also observed in the presence of OM, demonstrating the existence of interaction between the transporter and the antibiotic in the absence of any hydrophobic membrane component.

The Maltose Transport System of Escherichia coli Displays Positive Cooperativity in ATP Hydrolysis

Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.

Cloning of ODAP Degrading Gene and Its Expression as Fusion Protein in Escherichia coli

A gene responsible for the degradation of s-N-Oxalyl diaminopropionic acid (ODAP) was fused to the maIE gene, which codes for maltose binding protein, by cloning into an expression vector pMAL c2. The gene has been expressed as fusion protein of mol wt approximately 62 kD. It has been purified by affinity chromatography. The fusion protein has been cleaved by an endoprotease factor Xa and the presence of maltose binding protein and the product of the cloned gene confirmed. SDS-PAGE has shown that the product of the ODAP degrading gene is a single polypeptide of mol wt of about 20.7 kD.

Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli.

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hydrolysis of ATP was directly coupled to maltose uptake. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.