A hydrophobic ligand-binding protein of the Taenia solium metacestode mediates uptake of the host lipid: implication for the maintenance of parasitic cellular homeostasis

Parasitic organisms are incapable of de novo fatty acid synthesis due to a down-regulated expression of enzymes involved in the oxygen-dependent pathway. We investigated the uptake of host lipids by a 150-kDa hydrophobic ligand-binding protein (HLBP) of Taenia solium metacestode, an agent causative of neurocysticercosis. The protein was found to be a hetero-oligomeric complex consisting of multiple subunits (M(r) 7, 10, and 15 kDa within pH 8.0-9.7), which may originate from four unique genes of 7- and 10-kDa gene families with 2-3 polymorphic alleles/paralogs. The 15-kDa protein represented glycosylated forms of the 10-kDa. With high binding affinity to lipid analogs, these subunits evidenced high-level sequence identity with other cestode HLBPs and form a novel clade associated with excretory-secretory type HLBP. In vitro experiments with viable worms suggested that the excreted 150-kDa protein might bind to lipids, and participate in the translocation of host lipids across the syncytial membrane. This process was substantially inhibited by the specific anti-150 kDa antibodies. The protein was localized in the parasite syncytium and in the lipid droplets within host granuloma wall, where significant lipase activity was expressed. HLBP-mediated uptake of the host lipid may be critical for the parasite survival and thus could be targeted by chemotherapeutics and/or vaccine.     

Echinococcus granulosus antigen B hydrophobic ligand binding properties

Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.     

Conformational choreography of a molecular switch region in myelin basic protein--molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5 kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5 kDa myelin basic protein (residues Glu80-Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80-Val91 and extended poly-proline type II from Thr92-Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5 kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.     

Subunit Composition of the Zinc Proteins α- and β-Lipovitellin from Chicken

Chicken α- and β-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per α-lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per β-lipovitellin, respectively. The subunits of β-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of α- and β-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from α- and β-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of α- and β-Lv, respectively, and two peptides of the 30-kDa subunit of α- and β-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of α- and β-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of α-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Fatty acid compositions of Taenia solium metacestode and its surrounding tissues

Fatty acids (FAs) are the main energy sources of living organisms and are the major components of cellular and organelle membranes. Their compositions also affect the flexibility/rigidity of cells and cell vitality. The Taenia solium metacestode (TsM) causes neurocysticercosis (NC), which is one of the most common helminthic infections of the central nerve system. We investigated the FA composition of the cyst fluid (CF) and parenchyma of the TsM, together with those of the granuloma and swine tissue surrounding the granuloma. The FA fractions of the TsM CF and swine tissue showed a composition and proportional contents comparable to each other, in which C18:0 (stearic acid), C18:1n9c (oleic acid), C20:4 (arachidonic acid) and C16:0 (palmitic acid) constituted the major fractions. However, the relative amount of individual FAs of the TsM parenchyma and granuloma differed from those of TsM CF and swine tissue, which contained enriched C16:0 and a lower amount of C20:4. Saturated FAs were the major constituents in parenchyma and granuloma, 50.4% and 46.1%, respectively. Conversely, monounsaturated FAs were the major constituents of CF and swine tissue, 38.7% and 40.3%, respectively. Our results strongly suggest that host-derived FAs might translocate across the parasite syncytial membrane and be stored in the CF.     

Import of the barley PSI-F subunit into the thylakoid lumen of isolated chloroplasts

A full-length cDNA clone encoding the PSI-F subunit of barley photosystem I has been isolated and sequenced. The open reading frame encodes a precursor polypeptide with a deduced molecular mass of 24837 Da. The barley PSI-F precursor contains a bipartite presequence with characteristics similar to the presequences of proteins destined to the thylakoid lumen. In vitro import studies demonstrate that an in vitro synthesized precursor is transported across the chloroplast envelope and directed to the thylakoid membrane, where it accumulates in a protease-resistant form. Incubation of the precursor with a chloroplast stromal extract results in processing to a form intermediate in size between the precursor and mature forms. Hydrophobicity analysis of the barley PSI-F protein reveals a hydrophobic region predicted to be a membrane spanning -helix. The hydrophobic nature of PSI-F combined with a bipartite presequence is unusual. We postulate that the second domain in the bipartite presequence of the PSI-F precursor proteins is required to ensure the proper orientation of PSI-F in the thylakoid membrane. The expression of the PsaF gene is light-induced similar to other barley photosystem I genes.Abbreviations 16K 23K and 33K proteins, the 16 kDa, 23 kDa and 33 kDa subunits of the photosystem II oxygen-evolving complex - PSI-N and PSI-F photosystem I subunit N and F - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Interaction of differently oriented lipids in monolayer: mixed monolayers of 16-(9-anthroyloxy)palmitic acid with phosphatidylcholine and cholesterol

16-(9-Anthroyloxy)palmitic acid (16-AP) is a bifunctional molecule with carboxyl and 9-anthroyloxy groups attached at both ends of the hydrocarbon chain. At the air-water interface, in a monolayer, the 16-AP molecule has horizontal and vertical orientations, depending on the surface pressure of the monolayer. The miscibilities of 16-AP with dimyristoylphosphatidylcholine (DMPC), cholesterol (CH), and fatty acids in mixed monolayers were evaluated in investigations of monolayer phase transitions. Lipid molecules with flexible hydrocarbon chains, i.e., DMPC and fatty acids, formed homogeneous mixed monolayers with horizontally oriented 16-AP. On the other hand, the rigid molecule, CH, could not accommodate the horizontally oriented 16-AP in a monolayer, and there was a phase separation from 16-AP. In biological and reconstituted membranes, preferential binding of phospholipid to the integral protein and exclusion of cholesterol in close vicinity of the membrane protein have been recognized. On the basis of this work, it can be expected that flexible lipids readily accommodate the rough hydrophobic surface of integral proteins and stabilize the structure of the protein, while rigid lipids such as cholesterol are removed from the immediate environment of the membrane protein, if the protein does not interact specifically with the rigid lipids.     

Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode

Background

Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive.

Methodology/Principal Findings

We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol.

Conclusions/Significance

The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

Polypeptide composition of the Photosystem I complex and the Photosystem I core protein from Synechococcus sp. PCC 6301.

The polypeptide composition of the Photosystem I complex from Synechococcus sp. PCC 6301 was determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. The PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaK and PsaL proteins, as well as three polypeptides with apparent masses less than 8 kDa and small amounts of the 12.6 kDa GlnB (PII) protein, wee present in the Photosystem I complex. No proteins homologous to the PsaG and PsaH subunits of eukaryotic Photosystem I complexes were detected. When the Photosystem I complex was treated with 6.8 M urea and ultrafiltered using a 100 kDa cutoff membrane, the resulting Photosystem I core protein was found to be depleted of the PsaC, PsaD and PsaE proteins. The filtrate contained the missing proteins, along with five proteolytically-cleaved polypeptides with apparent masses of less than 16 kDa and with N-termini identical to that of the PsaD protein. The PsaF and PsaL proteins, along with the three less than 8 kDa polypeptides, were not released from the Photosystem I complex to any significant extent, but low-abundance polypeptides with N-termini identical to those of PsaF and PsaL were found in the filtrate with apparent masses slightly smaller than those found in the native Photosystem I complex. When the filtrate was incubated with FeCl3, Na2S and beta-mercaptoethanol in the presence of the isolated Photosystem I core protein, the PsaC, PsaD and PsaE proteins were rebound to reconstitute a Photosystem I complex functional in light-induced electron flow from P700 to FA/FB. In the absence of the iron-sulfur reconstitution agents, there was little rebinding of the PsaC, psaD or PsaE proteins to the Photosystem I core protein. No binding of the truncated PsaD polypeptides occurred, either in the presence or absence of the iron-sulfur reagents. The reconstitution of the FA/FB iron-sulfur clusters thus appears to be a necessary precondition for rebinding of the PsaC, psaD and psaE proteins to the Photosystem I core protein.     

Identification of a 14-kDa laminin binding protein (HLBP14) in human melanoma cells that is identical to the 14-kDa galactoside binding lectin.

The carbohydrate moieties present on laminin play a crucial role in the multiple biological activities of this basement membrane glycoprotein. We report the identification of a human laminin binding protein with an apparent molecular mass of 14 kDa on sodium dodecyl sulfate-polyacrylamide gels that was found, after purification and amino acid microsequencing, to be identical to the previously described 14-kDa galactoside binding soluble L-14 lectin. We have designated this human laminin binding protein as HLBP14. HLBP14 was purified from human melanoma cells in culture by laminin affinity chromatography and gel electroelution. We demonstrate that HLBP14 binds specifically to the poly-N-acetyllactosamine residues of murine laminin and does not bind to other glycoproteins that do not contain such structures, such as fibronectin. HLBP14 was eluted from a murine laminin column by lactose, N-acetyllactosamine, and galactose but not by other control saccharides, including glucose, fucose, mannose, and melibiose. It did not bind to laminin treated with endo-beta-galactosidase. Lactose also eluted HLBP14 off a human laminin affinity column, implying that human laminin also contains poly-N-acetyllactosamine residues. On immunoblots, polyclonal antibodies raised against HLBP14 recognized HLBP14 as well as 31- and 67-kDa molecules that are also laminin binding proteins, indicating that these proteins share common epitopes. L-14, a dimeric lactose binding lectin, is expressed in a wide variety of tissues. Although the expression of this molecule has been linked to a variety of biological events, the elucidation of its specific functions has been elusive. The observation that HLBP14, a human cancer cell laminin binding protein, is identical to L-14 strongly suggests that the functions attributed to this lectin could be mediated, at least in part, through its ability to interact with the poly-N-acetyllactosamine residues of laminin. HLBP14 could potentially play a role during tumor invasion and metastasis by modulating the interactions between cancer cells and laminin.     

The Analysis of Participation of Individual Proteins in the Protein Interactome Formation

It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.     

Structure of the three-chain unit of the bovine epidermal keratin filament

The characteristic α-type X-ray diffraction pattern displayed by bovine epidermal keratin filaments can be ascribed to the presence of segments of triple-chain coiled coil α-helix in the repeating three-chain unit of the filaments.Limited proteolysis of filaments polymerized in vitro or a citrate-soluble protein derived from them with crystalline trypsin releases two types of α-helix-enriched particles which provide information on the structure of the three-chain unit. The smaller, particle 2, of molecular weight 42,500, α-helix content of 92% and dimensions of 180 Å × 20 Å, consists of three chains aligned side-by-side that presumably form a coiled coil. The high yields of particle 2 allow the conclusion that all of the α-helix of the epidermal keratin filament is present in the form of these discrete three-chain α-helical segments. The larger, particle 1, recovered during the earlier stages of digestion has a molecular weight of 100,000 to 110,000, α-helix content of 75%, average dimensions of 400 Å × 20 Å and also consists of three chains aligned side-by-side. It contains two α-helical segments corresponding to particle 2 which are located at the amino -terminal and carboxyl-terminal ends and are separated by a region of non-helix. Particle 1 contains all of the α-helix and therefore is the major portion of the three-chain unit of the keratin filament. The products resulting from reaction of intact filament subunits with N-bromosuccinimide suggest that particle 1 is formed during digestion by removal of regions of non-helix from each end of this unit.The structure of the three-chain unit of the bovine epidermal keratin filament may thus be viewed as three polypeptide subunits aligned side-by-side with two discrete coiled coil α-helical segments interspersed with regions of non-helix.     

Folding simulations of three proteins having all α-helix,all β-strand and α/β-structures

We performed folding simulations of three proteins using four force fields, AMBER parm96, AMBER parm99, CHARMM 27 and OPLS-AA/L, in order to examine the features of these force fields. We studied three proteins, protein A (all α-helix), cold-shock protein (all β-strand) and protein G (α/β-structures), for the folding simulations. For the simulation, we used the simulated annealing molecular dynamics method, which was performed 50 times for each protein using the four force fields. The results showed that the secondary-structure-forming tendencies are largely different among the four force fields. AMBER parm96 favours β-bridge structures and extended β-strand structures, and AMBER parm99 favours α-helix structures and 3₁₀-helix structures. CHARMM 27 slightly favours α-helix structures, and there are also π-helix and β-bridge structures. OPLS-AA/L favours α-helix structures and 3₁₀-helix structures.     

Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: Sequence analysis and over-expression in Escherichia coli

Abstract The antigenic properties of the surface layer (S-layer) proteins of various Campylobacter rectus strains including 24 clinical isolates and the type strain ATCC 33238 were examined. S-layer proteins were extracted from whole cells by acid treatment according to the method of McCoy et al. (Infect. Immun. 11, 517–525, 1975). The acid extracts from 23 of the isolates and ATCC 33238 contained two major proteins with molecular masses of 130 kDa and 150 kDa, both of which were identified as subunits of the S-layer after comparison with the protein profiles of acid-treated (S-layer-deficient) cells. An S-layer protein from one isolate (CI-808) demonstrated a different molecular mass (160 kDa). Both the 150-kDa proteins of ATCC 33238 and isolate CI-306 and the 160-kDa protein of CI-808 were purified by ion-exchange chromatography in the presence of urea. In Ouchterlony immunodiffusion experiments with these purified proteins and rabbit antiserum raised to each purified protein, both common and strain-specific antigenic determinants were identified in the C. rectus S-layer proteins.     

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.     

Characterization of hydrophobic-ligand-binding proteins of Taenia solium that are expressed specifically in the adult stage

SUMMARY Taenia solium, a causative agent of taeniasis and cysticercosis, has evolved a repertoire of lipid uptake mechanisms. Proteome analysis of T. solium excretory-secretory products (TsESP) identified 10 kDa proteins displaying significant sequence identity with cestode hydrophobic-ligand-binding-proteins (HLBPs). Two distinct 362- and 352-bp-long cDNAs encoding 264- and 258-bp-long open reading frames (87 and 85 amino acid polypeptides) were isolated by mining the T. solium expressed sequence tags and a cDNA library screening (TsHLBP1 and TsHLBP2; 94% sequence identity). They clustered into the same clade with those found in Moniezia expansa and Hymenolepis diminuta. Genomic structure analysis revealed that these genes might have originated from a common ancestor. Both the crude TsESP and bacterially expressed recombinant proteins exhibited binding activity toward 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), which was competitively inhibited by oleic acid. The proteins also bound to cis-parinaric acid (cPnA) and 16-(9-anthroyloxy) palmitic acid (16-AP), but showed no binding activity against 11-[(5-dimethylaminonaphthalene-1-sulfonyl) amino] undecanoic acid (DAUDA) and dansyl-DL-α-aminocaprylic acid (DACA). Unsaturated fatty acids (FAs) showed greater affinity than saturated FAs. The proteins were specifically expressed in adult worms throughout the strobila. The TsHLBPs might be involved in uptake and/or sequestration of hydrophobic molecules provided by their hosts, thus contributing to host-parasite interface interrelationships.     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin