Conformational changes of an exchangeable apolipoprotein, apolipophorin III from Locusta migratoria, at low pH: correlation with lipid binding.

Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into smaller discoidal complexes is enhanced as a function of decreasing pH, with maximal transformation occurring at pH 3.5. Over the entire pH range studied, apoLp-III retains nearly all of its secondary structure content. Whereas no changes in fluorescence emission maximum of the two Trp residues in apoLp-III were observed in the pH range from 7.0 to 4.0, a further decrease in pH resulted in a strong red shift. Near-UV circular dichroism spectra of apoLp-III showed well-defined extrema (at 286 and 292 nm) between pH 7.0 and pH 4.0, which were attributed to Trp115. Below pH 4.0, these extrema collapsed, indicating a less rigid environment for Trp115. Similarly, the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate in the presence of apoLp-III increased 4-fold below pH 4.0, indicating exposure of hydrophobic sites in the protein in this pH range. Taken together, the data suggest two conformational states of the protein. In the first state between pH 7.0 and pH 4.0, apoLp-III retains a nativelike helix bundle structure. The second state, found between pH 3.0 and pH 4.0, is reminiscent of a molten globule, wherein tertiary structure contacts are disrupted without a significant loss of secondary structure content. In both states DMPC vesicle transformation is enhanced by lowering the solution pH, reaching an optimum in the second state. The correlation between tertiary structure and lipid binding activity suggests that helix bundle organization is a determinant of apoLp-III lipid binding activity.     

Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen-related enzyme norcoclaurine synthase

The enzyme NCS [(S)-norcoclaurine synthase; EC 4.2.1.78] found in the common meadow rue, Thalictrum flavum, and other plant species, is involved in the biosynthesis of BIAs (benzylisoquinoline alkaloids). This group of plant secondary metabolites comprises pharmacologically-active compounds such as morphine and codeine. NCS catalyses the condensation of 4-HPAA (4-hydroxyphenylacetaldehyde) and dopamine to (S)-norcoclaurine, the common precursor of all plant BIAs. Although enzymatic properties of NCS and mechanistic aspects of the reaction have been studied in detail, no structural information on NCS was available so far. The enzyme shows significant sequence homology to members of the PR10 proteins (class 10 of pathogenesis-related proteins) such as the major birch pollen allergen Bet v 1. Our CD and NMR spectroscopic data indicated high similarity of the NCS and the Bet v 1 fold and allowed us to model NCS using Bet v 1 as a template. Virtually complete backbone assignment of the NCS sequence was used to study substrate binding by NMR titration experiments. Although binding of 4-HPAA seems to induce side-chain rearrangements in an extensive part of the protein, the putative distinct interaction site for dopamine could be clearly identified. The oligomerization state of NCS that reportedly plays an important role in enzyme functionality was determined to be concentration-dependent by SEC (size-exclusion chromatography) as well as NMR relaxation measurements, and the enzyme was found to be predominantly a monomer at the low micromolar concentrations used for activity assays.     

Elimination of a misfolded folding intermediate by a single point mutation

We present an analysis of the folding behavior of the 159-residue major birch pollen allergen Bet v 1. The protein contains a water-filled channel running through it. Consequently, the protein has a hydrophobic shell, rather than a hydrophobic core. During the folding of the protein from either the urea-, pH-, or SDS-denatured state, Bet v 1 transiently populates a partially folded intermediate state. This state appears to be misfolded, since it has to unfold at least partially to fold to the native state. The misfolded intermediate is not, however, a result of the water-filled channel in Bet v 1. The intermediate completely disappears in the mutant Tyr --> Trp120, in which the channel is still present. Tyr120 appears to behave as a "negative gatekeeper" which attenuates efficient folding. The close structural homologue, the apple allergen Mal d 1, also folds without any detectable folding intermediates. However, the position of the transition state on the reaction coordinate, which is a measure of its overall compactness relative to the denatured and native states, is reduced dramatically from ca. 0.9 in Bet v 1 to around 0.5 in Mal d 1. We suggest that this large shift in the transition state structure is partly due to different local helix propensities. Given that individual mutations can have such large effects on folding, one should not a priori expect structurally homologous proteins to fold by the same mechanism.     

Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen,characterized by X-ray crystallography and site-directed mutagenesis

Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab' fragment. The epitope occupies approximately 10% of the molecular surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid substitution, Glu(45)-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.     

Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes.

A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.     

The Influence of Recombinant Production on the Immunologic Behavior of Birch Pollen Isoallergens

Background

Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.

Methodology

Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.

Conclusion

The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.     

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS.Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.     

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Binding of Synapsin I to Synaptic Vesicles: Clues from the Study of its Interactions with Liposomes

Abstract

Synapsin I is a major brain phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation-dependent fashion. The binding of synapsin I to synaptic vesicles involves interactions with the phospholipid and protein components of the vesicle membrane. The highly hydrophobic NH₂-terminal head region of the protein binds with high-affinity to acidic phospholipids and penetrates the hydrophobic core of the membrane, whereas the basic COOH-terminal tail region does not significantly contribute to this binding. The interaction with phospholipids increases the amount of α-helix in the secondary structure of synapsin I, but does not markedly affect the microenvironment of tryptophan and cysteine residues present in the head region. The results suggest that synapsin I binds to synaptic vesicle phospholipids through amphiphilic and positively charged domains present in its NH2-terminal region and that such an interaction contributes to the high-affinity binding of synapsin I to synaptic vesicles.     

NMR resonance assignments of the major apple allergen Mal d 1

The major apple allergen Mal d 1 is the predominant cause of apple (Malus domestica) allergies in large parts of Europe and Northern America. Allergic reactions against this 17.5 kDa protein are the consequence of initial sensitization to the structurally homologous major allergen from birch pollen, Bet v 1. Consumption of apples can subsequently provoke immunologic cross-reactivity of Bet v 1-specific antibodies with Mal d 1 and trigger severe oral allergic syndroms, affecting more than 70 % of all individuals that are sensitized to birch pollen. While the accumulated immunological data suggest that Mal d 1 has a three-dimensional fold that is similar to Bet v 1, experimental structural data for this protein are not available to date. In a first step towards structural characterization of Mal d 1, backbone and side chain ¹H, ¹³C and ¹⁵N chemical shifts of the isoform Mal d 1.0101 were assigned. The NMR-chemical shift data show that this protein is composed of seven β-strands and three α-helices, which is in accordance with the reported secondary structure of the major birch pollen allergen, indicating that Mal d 1 and Bet v 1 indeed have similar three-dimensional folds. The next stage in the characterization of Mal d 1 will be to utilize these resonance assignments in solving the solution structure of this protein.     

Effect of pH on the pore forming activity and conformational stability of ostreolysin, a lipid raft-binding protein from the edible mushroom Pleurotus ostreatus

Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all-beta-structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 degrees C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predominantly beta-sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native alpha-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8.     

TRAPP stably associates with the Golgi and is required for vesicle docking

Bet3p, a component of a large novel complex called TRAPP, acts upstream of endoplasmic reticulum (ER)-Golgi SNAREs. Unlike the SNAREs, which reside on multiple compartments, Bet3p is localized exclusively to Golgi membranes. While other proteins recycle from the Golgi to the ER, Bet3p and other TRAPP subunits remain associated with this membrane under conditions that block anterograde traffic. We propose that the persistent localization of TRAPP to the Golgi may be important for its role in docking vesicles to this membrane. Consistent with this proposal, we find that transport vesicles fail to bind to Golgi membranes in vitro in the absence of Bet3p. Binding is restored by the addition of cytosol containing Bet3p. These findings indicate that TRAPP stably associates with the Golgi and is required for vesicle docking.     

Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.     

Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein

We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.     

Post-Messinian evolutionary relationships across the Sicilian channel: Mitochondrial and nuclear markers link a new green toad from Sicily to African relatives

Background

The major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database.

Results

Structural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.

Conclusion

The ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.     

Annexin A4 binding to anionic phospholipid vesicles modulated by pH and calcium

Annexin A4 belongs to a class of Ca(2+)-binding proteins for which different functions in the cell have proposed, e.g. involvement in exocytosis and in the coagulation process. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study the interaction of annexin A4 with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) or from phosphatidic acid (PA) is investigated at neutral and acidic pH. Annexin A4 strongly binds to either lipid at acidic pH, whereas at neutral pH only weak binding to PA and no binding to PS occurs. Addition of 40 microM Ca(2+) leads to a strong binding to the lipids also at neutral pH. This is caused by the different electric charge of the protein below and above its isoelectric point. Binding of annexin A4 induces dehydration of the vesicle surface. The strength of the effects is much greater at pH 4 than at pH 7.4. At pH 7.4 annexin A4 reduces the Ca(2+)-threshold concentration necessary to induce fusion of PA LUV. The Ca(2+) induced fusion of PS LUV is not affected by annexin A4 at pH 7.4. At pH 4 annexin A4 induces fusion of either vesicles without Ca(2+). Despite the low binding extents at neutral pH annexin A4 induces a Ca(2+) independent leakage of PS- or PA-LUV. The leakage extent is increased at acidic pH. From the data two suggestions are made: (1) At pH 4 annexin A4 (at least partially) penetrates into the bilayer in contrast to the preferred location at the vesicle surface at neutral pH. The conformation of annexin A4 seems to be different at the two conditions. (2) At neutral pH, Annexin A4 seems to be able to bind two PA vesicles simultaneously; however, only one PS vesicle at the same time. This behavior might be related to a recently described double Ca(2+) binding site, which appears to be uniquely suited for PS.     

The major birch pollen allergen, Bet v 1, shows ribonuclease activity

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units · mg^?1.