Effects of Environmental pH on Membrane Proteins in Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector, Ixodes scapularis, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent B. burgdorferi B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6. 0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of B. burgdorferi to the tick or mammalian microcosm.     

pH-dependent elution profiles of selected proteins in HPLC having a stationary phase modified with pH-sensitive sulfonamide polymers

An approach to controlling protein interactions with silica (glass) beads grafted with pH-sensitive copolymers of a sulfonamide and N,N-dimethylacrylamide is proposed. The bead surface was modified with poly(N.N-dimethyl acrylamide-co-methacryloyl sulfadimethoxine), which exhibits hydrophobic/hydrophilic switching around pH 7.0. Phase transition in the aqueous solution, the wettability change of the modified surface and surface tension of the polymer-grafted glass surface was characterized by changes in turbidity and the dynamic contact angle as a function of pH. The discontinuous phase transition in the copolymer solution shifted to a higher pH region as the composition of sulfadimethoxine monomer (SDM) was increased. However, the transition pH in continuous wettability changes on the modified surface was not affected by polymer composition due to differences in the dynamic motion. The ionized SDM of the copolymers enhanced wettability. Therefore, the advancing contact angle of the surface decreased by 45 degrees above pH 7.0, whereas wettability at low pH values was low due to the hydrophobic surface. Elution behavior of the selected four model proteins with different pI (bovine serum albumin (pI 5.0), insulin (pI 5.3), fibrinogen (pI 6.1) and myoglobin (pI 7.1)) was investigated by using pH-sensitive copolymer-modified glass beads at four pH values (5.0, 6.0, 7.0 and 8.0). Retention time of the proteins in pH-sensitive aqueous chromatography was easily regulated by eluent pH that controls the proportion of electrostatic and hydrophobic interactions between the stationary phase and the protein. At pH 5.0, hydrophobic interaction on the deionized polymer-grafted surface is an important force in separating the proteins. However, identification of the proteins at pH 7.0 was effectively facilitated due to the electrostatic interaction between the negatively charged polymer support and positive charges on protein.     

Comonomer composition distribution of P(3HB-co-3HP)s produced by Alcaligenes latus at several pH conditions

A series of poly(3-hydroxybutyric acid-co-3-hydroxypropionic acid) (P(3HB-co-3HP)) samples was biosynthesized by fermentation with Alcaligenes latus using sucrose/3-hydroxypropionic acid as carbon source at different pH conditions from 6.0 to 8.0. The copolymers were compositionally fractionated using the mixed solvent chloroform/heptane. It was found that A. latus produces P(3HB-co-3HP)s with different 3HP content under the same conditions except for the pH value of the medium. The higher the pH value of the fermentation medium is, the less 3HP units are introduced into the copolyester. The comonomer composition distribution for the copolyesters also depends on the pH value of medium. The copolyester sample produced at pH 6.0 has a much broader composition distribution than those produced at pH values between 6.5 and 8.0. The copolyester with the narrowest distribution was obtained at pH 7.0. It was also found that the distribution becomes broader with increasing fermentation time.     

pH-Dependent urea sensitivity of southern bean mosaic virus

The behavior of southern bean mosaic virus (SBMV) virions in urea was markedly pH dependent and revealed several novel features. In the absence of urea (5 degrees , 1 hr) SBMV was stable as a 115 S entity between pH 2.5 and 9.5, yielded a slowly sedimenting (ca 60-65 S) entity at pH 10.5, and was degraded into a 35-40 S product at pH 11.0. In 4.0 M urea, SBMV was precipitated irreversibly at pH 2.5 or 10.0, yielded variously sedimenting aggregates at pH 3.0, was transformed progressively into a 80 S entity between pH 7.0 and 9.5, or remained structurally intact at pH 3.5 to 6.5. In 8.O M urea, SBMV was precipitated at pH 3.5 and below or pH 7.5 and above, showed a tendency for the particle:particle interaction at pH 6.5 and 7.0, but remained stable between pH 4.0 and 6.0. Under conditions where SBMV was somewhat destabilized with urea, increase in the temperature or ionic concentration caused virion precipitation without any detectable degradation. SBMV was dissociated with 4.0 or 8.0 M urea at pH 5.5 but only in the presence of 1.0 M NaCl and at the elevated temperatures (>50 degrees ). These results on the rather unusual SBMV behavior in urea, as compared to other viruses, are discussed in relation to the available information on the SBMV stabilizing interactions.     

Clustering and anchoring mechanisms of molecular constituents of postsynaptic scaffolds in dendritic spines

Recent technological progress has yielded great amounts of information about the molecular constituents of postsynaptic scaffolds in the dendritic spine. Actin filaments are major cytoskeletal elements in the dendritic spine, and they functionally interact with neurotransmitter receptors via regulatory actin-binding proteins. Drebrin A and alpha-actinin-2 are two major actin-binding proteins in dendritic spines. In adult brains, they are characteristically concentrated in spines, but not in dendritic shafts or cell bodies. Thus, they are part of a unique postsynaptic scaffold consisting of actin filaments, PSD protein family, and neurotransmitter receptors. Localization of NMDA receptors, actin filaments, and actin-binding proteins in spines changes in parallel with development, and in response to synaptic activity. This raises the possibility that clustering and anchoring of these characteristic molecular constituents at postsynaptic scaffolds play important roles in spine function. This article focuses on the clustering and anchoring mechanisms of NMDA receptors and actin filaments, and the involvement of actin-binding proteins, in dendritic spines, and the way in which characteristic postsynaptic scaffolds are built up.     

Signal transduction cascades underlying de novo protein synthesis required for neuronal morphogenesis in differentiating neurons

Differentiating neurons must acquire many unique morphological and functional characteristics in creating the precise neural circuits of the mature nervous system. The phenomenon of 'neuronal differentiation' includes a special set of simple, separate processes, that is, neuritogenesis, neurite outgrowth, pathfinding, targeting and synaptogenesis. All of these processes are critically dependent on the reorganization of actin cytoskeleton by many actin-binding proteins that function downstream of Rho-family GTPases. Furthermore, de novo synthesis of key proteins are critically involved in the reorganization of actin cytoskeleton during neuronal differentiation. In this article, we review recent progresses in the general mechanisms that control actin dynamics by various actin-binding proteins in differentiating neurons, including a series of recent studies from our laboratory on de novo synthesis of several key proteins that are essential for actin reorganization induced by second messengers. We demonstrated that dual regulation of cyclic AMP and Ca2+ determines cofilin (an actin-binding protein) phosphorylation states and LIM kinase 1 (a cofilin kinase) expression level during neuritogenesis.     

The influences of L(+)-lactate and pH on contractile performance in rabbit glycerinated skeletal muscle

To know whether L(+)-lactate directly induces the decrease in muscle contractile performance, several parameters of cross-bridge function were measured at various concentrations of lactate and pH in glycerinated rabbit psoas and soleus muscles at three different temperatures (5, 20, 28 degrees C). At all pHs studied (pH 7.0, 6.5, 6.0, and 5.5), isometric tension, unloaded velocity of shortening, and stiffness of a fiber during active and resting state in the presence of 50 mM lactate were not virtually different from those in the absence of lactate, but pH had remarkable effects on these parameters. The active stiffness decreased only slightly, and the small resting stiffness appeared at low pH; they were not affected by the presence of lactate. The present results indicate that the lactate anions may not have marked influence on the interaction between actin and myosin, and the concomitant change in pH with the production of lactate may remarkably affect it, as far as they were examined under the existing conditions of the experimental solutions.     

Conservation of antigen components from two recombinant hybrid proteins protective against malaria.

Recently, we have shown that two hybrid proteins carrying partial sequences of the blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum confer protective immunity on Aotus monkeys against an experimental parasite infection (B. Knapp, E. Hundt, B. Enders, and H. A. Küpper, Infect. Immun. 60:2397-2401, 1992). The malarial components of the hybrid proteins consist of amino acid residues 630 to 892 of SERP, amino acid residues 146 to 260 of MSAI, and the 189 C-terminal residues of HRPII. We have studied the diversity of these protein regions in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from two different areas where malaria is endemic. The gene regions of SERP and MSAI coding for the corresponding sequences of the protective hybrid proteins and the exon II region of the HRPII gene were amplified by polymerase chain reaction and sequenced. All three regions were found to be highly conserved. In the 262-amino-acid fragment of SERP, one single conservative amino acid substitution was found. The exon II region of HRPII showed only a slight variability in number and arrangement of the repeat units. The 115-amino-acid fragment of MSAI which is located within an N-terminal region known to be conserved among different parasite strains was shown to be the most variable among the vaccine components: amino acid substitutions were found in 14 different positions of this MSAI region when both laboratory strains and field isolates were compared.     

Moesin is involved in the cytoskeletal remodelling of rat decidual cells

The extensive actin cytoskeletal remodelling of the uterine stroma during early pregnancy involves changes in actin-binding proteins. This study provides the first detailed localisation of the actin-binding protein, moesin, in rat uterine endometrium during this period. Western blot analysis and immunofluorescence microscopy showed that the amount of moesin in the uterus peaked at the time of implantation, corresponding to the presence of intensely immunolabelling decidual cells. Furthermore, moesin increased in active membrane/cytoskeleton bound protein at the time of implantation, concomitantly decreasing in cytosolic protein. The increase of moesin at decidualisation corresponds with the appearance of alpha smooth muscle actin (alphaSMA) suggesting that decidual cells have contractile abilities that may aid in containing an invasive trophoblast. The results of this study suggest that moesin is important in developing a specialised cytoskeleton and increased adhesiveness of decidual cells, possibly functioning to bridge adhesion molecules to the underlying cytoskeleton.     

Role of 3'-phosphoinositides in oncogenic KRAS-induced modulation of shape and motility of airway epithelial cells

The authors' previous study demonstrated that oncogenic KRAS modulates the shape and motility of airway epithelial cells. To explore detailed mechanism mediating these events, the possible involvement of phosphatidylinositides (PIP) was investigated. The intracellular localization of PIP was visualized with a pleckstrin homology domain-enhanced green fluorescent protein (EGFP) construct. PIP accumulated at the leading edges of polarizing epithelial cells, while they co-localized with cortical actin at cell–cell contacts, suggesting that PIP play important roles in the cytoskeletal organization. Transduction of oncogenic KRAS induced multiple pseudopodia and disrupted cortical actin, enhancing motility. A mitogen activated protein kinase kinase (MEK) inhibitor reduced the accumulation of PIP at membranes and development of pseudopodia, and restored stable cortical actin, reducing the motility. A phosphoinositide 3-kinase (PI3K) inhibitor also reduced accumulation of PIP at membranes, formation of pseudopodia and motility, but its effect on cortical actin was indistinct. The KRAS V12/S35 mutant, activating only the MEK pathway, induced multiple pseudopodia and disrupted the cortical actin. The KRAS V12/C40 mutant, activating only the PI3K pathway, also induced pseudopodia, but its effect on cortical actin was obscure. Taken together, oncogenic KRAS could cause the accumulation of PIP via the PI3K and MEK pathways and modulate the cell shape and migration.     

The serology of the bromoviruses. I. Serological interrelationships of the bromoviruses

The serological relationships between brome mosaic (BMV), cowpea chlorotic mottle (CCMV), and broad bean mottle (BBMV) viruses, and their coat proteins, were studied by gel precipitin, "rocket immunoelectrophoresis" (RIE), and enzyme-linked immunosorbent assay (ELISA) techniques. Gel precipitin and RIE tests indicated that capsid swelling altered the antigenicity of both BMV and CCMV, as although the coat proteins were serologically related at both pH 6.0 and pH 7.0, the viruses appeared to be related only when swollen at pH 7.0. Fixation of the viruses with 2% formaldehyde at pH 6.0 appeared to remove the relationship. BBMV was not related by precipitin tests to either BMV or CCMV but was related to both by direct and indirect ELISA at both pH 6.0 and pH 7.4, though less closely than BMV and CCMV were related to each other. Indirect ELISA showed the three coat proteins to be more closely related than the parent viruses. Formalinized BMV and CCMV appeared less related than the native viruses at pH 6.0. Sandwich ELISA proved too strain specific to show any group relationships. The implications of these results and a serological map proposed for the bromoviruses are discussed.     

Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C.

We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.     

Identification of pH-specific protein expression responses by Campylobacter jejuni strain NCTC 11168

In this study a data dependent acquisition label-free based proteomics approach was used to identify pH-dependent proteins that respond in a growth phase independent manner in Campylobacter jejuni reference strain NCTC 11168. NCTC 11168 was grown within its pH physiological normal growth range (pH 5.8, 7.0 and 8.0, μ = ∼0.5 h⁻¹) and exposed to pH 4.0 shock for 2 h. It was discovered that gluconate 2-dehydrogenase GdhAB, NssR-regulated globins Cgb and Ctb, cupin domain protein Cj0761, cytochrome c protein CccC (Cj0037c), and phosphate-binding transporter protein PstB all show acidic pH dependent abundance increases but are not activated by sub-lethal acid shock. Glutamate synthase (GLtBD) and the MfrABC and NapAGL respiratory complexes were induced in cells grown at pH 8.0. The response to pH stress by C. jejuni is to bolster microaerobic respiration and at pH 8.0 this is assisted by accumulation of glutamate the conversion of which could bolster fumarate respiration. The pH dependent proteins linked to growth in C. jejuni NCTC 11168 aids cellular energy conservation maximising growth rate and thus competitiveness and fitness.     

The specificity of papaya mosaic virus assembly.

Papaya mosaic virus protein forms sinuous tubular particles with homologous RNA or that from a related virus at pH 8.0, whereas it makes thin extended particles with RNAs from unrelated viruses under the same conditions. The coat protein forms particles with discontinuities with both homologous and heterologous RNAs from pH 6.0 to 7.5. It will also encapsidate DNA under similar conditions. The protein encapsidates poly(A) and poly(C), but not poly(U) or poly(I) at pH 8.0, nor will it form tubular particles at pH 8.0 with homologous RNA in which cytosine has been transformed to uracil.     

The influence of pH on the growth and stability of transmissible gastroenteritis virusin vitro

The influence of pH on the growth of transmissible gastroenteritis virus (TGEV) in adult pig thyroid cell culture, and on the stability of the virus was studied. At pH 7.2 and 100 fold higher than those at pH 8.0. The adsorption, penetration and uncoating steps of the viral replicative cycle were shown to be unaffected by pH variation. Synthesis of TGEV RNA during the first 12 hours post infection was found to be unaffected by pH variation between the range 6.5-8.0. After 12 hours breakdown of this RNA appeared to occur in cultures held at pH 7.2 and 8.0 but not at pH 6.5. When incubated at 37 degrees C for 24 hours the virus infectivity was found to be least affected by pH 6.5 but when kept at 4 degrees C for the same length of time, the virus infectivity remained constant between pH 5.0 and pH 8.0.     

Recombinant Yersinia YopT Leads to Uncoupling of RhoA-Effector Interaction

Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis deliver different Yop (Yersinia outer proteins) effector proteins into mammalian cells by a type III secretion mechanism. Recently, it was shown that Yersinia producing YopT leads to disruption of the actin cytoskeleton of HeLa cells (M. Iriarte and G. R. Cornelis, Mol. Microbiol. 29:915-929, 1998). To analyze the molecular mechanism of YopT, we cloned and expressed YopT as a glutathione S-transferase fusion protein. Recombinant YopT caused rounding up of embryonic bovine lung cells and redistribution of the actin cytoskeleton rapidly after microinjection. The Escherichia coli cytotoxic necrotizing factor (CNF1), which constitutively activates Rho proteins, was not able to inhibit or revert YopT-induced cell rounding. YopT caused release of RhoA from embryonic bovine lung membranes and released recombinant isoprenylated RhoA from artificial PE or PE/PIP2 vesicles. Incubation of lysate or cytosol with YopT caused inhibition of the RhoA-rhotekin interaction but led to increased RhoA-RhoGDI interaction. It is suggested that inhibition of the interaction between RhoA and effectors is the underlying mechanism of the YopT action on the cytoskeleton.     

Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes

The ezrin/radixin/moesin (ERM) family of actin-binding proteins act both as linkers between the actin cytoskeleton and plasma membrane proteins and as signal transducers in responses involving cytoskeletal remodelling. The Rho family of GTPases also regulate cytoskeletal organisation, and several molecular pathways linking ERM proteins and Rho GTPases have been described. This review discusses recent findings on ERM protein function in leucocytes and how these may be integrated with Rho GTPase signalling.     

Protection of Aotus monkeys from malaria infection by immunization with recombinant hybrid proteins.

On the basis of investigations of the malarial blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum, we chose two Escherichia coli-expressed hybrid proteins containing selected partial sequences of these antigens. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding P. falciparum polypeptides. In two independent trials with 13 animals, immunization of Aotus monkeys with either of the two hybrid proteins administered in a well-tolerated oil-based formulation protected the animals from an experimental P. falciparum infection.     

The EDTA-treated expanded satellite tobacco necrosis virus: biochemical properties and crystallization

The calcium ions normally present in the protein shell of the satellite tobacco necrosis virus (STNV) have been removed by EDTA. The radius of the EDTA-treated virion was found to be dependent upon pH. The radial increase was 2% at pH 5.0,4.5% at pH 6.5, and 7% at pH 7.0 as determined by analytical ultracentrifugation and X-ray crystallography. The virion could be recontracted by lowering the pH or by the addition of divalent cations, calcium ions being the most efficient. At pH 7.0 and above, the EDTA-treated virion was sensitive to ribonucleases and to trypsin. The cleavage site for trypsin is at Arg 28, which is near the N terminus of the shell domain of the subunit, but at a point which is well buried in the native structure. Crystals of the expanded STNV particles at pH 5.0 and 6.5 have been produced.     

Intracellular pH regulation in U937 human monocytes: roles of V-ATPase and Na+/H+ exchange

The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal V-ATPase and Na+/H+ exchanger (NHE) in U937 human monocytes. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H+-pump and NHE, respectively. Baseline pHi was approximately 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0-7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5-7.4; pHi declined approximately 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5-7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na+/H+ exchange (EIPA-sensitive component of apparent H+ efflux) was the predominant mechanism for H+ extrusion at acid-loaded pHi values < 7.0. At acid-loaded pHi values > or = 7.0, the V-ATPase (bafilomycin-sensitive component) and NHE contributed almost equally to H+ extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H+-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes.