Latherin is an intrinsically surfactant protein of ~23 kDa found in the sweat and saliva of horses. Its function is probably to enhance the translocation of sweat water from the skin to the surface of the pelt for evaporative cooling. Its role in saliva may be to enhance the wetting, softening and maceration of the dry, fibrous food for which equines are adapted. Latherin is unusual in its relatively high content of aliphatic amino acids (~25 % leucines) that might contribute to its surfactant properties. Latherin is related to the palate, lung, and nasal epithelium carcinoma-associated proteins (PLUNCs) of mammals, at least one of which is now known to exhibit similar surfactant activity to latherin. No structures of any PLUNC protein are currently available. 15N,13C-labelled recombinant latherin was produced in Escherichia coli, and essentially all of the resonances were assigned despite the signal overlap due to the preponderance of leucines. The most notable exceptions include a number of residues located in an apparently dynamic loop region between residues 145 and 154. The assignments have been deposited with BMRB accession number 19067.
Latherin; Surfactant protein; Horse; Sweat; Saliva; Allergen; NMR
As-p18 is produced and secreted by larvae of the parasitic nematode Ascaris suum as they develop within their eggs. The protein is a member of the fatty acid binding protein (FABP) family found in a wide range of eukaryotes, but is distinctive in that it is secreted from the synthesizing cell and has predicted additional structural features not previously seen in other FABPs. As-p18 and similar proteins found only in nematodes have therefore been designated ‘nemFABPs’. Sequence-specific 1H, 13C and 15N resonance assignments were established for the 155 amino acid recombinant protein (18.3 kDa) in complex with oleic acid, using a series of three-dimensional triple-resonance heteronuclear NMR experiments. The secondary structure of As-p18 is predicted to be very similar to other FABPs, but the protein has extended loops that have not been observed in other FABPs whose structures have so far been solved.
As-p18; Fatty acid binding protein; nemFABP; Nematode; Parasite; Ascaris suum
The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.
Parasitic nematode; Necator americanus; Fatty-acid and retinol-binding protein; Na-FAR-1; NMR
As-p18, an unusual fatty-acid-binding protein from a parasitic nematode, was expressed in bacteria, purified and crystallized. The use of a microfocus beamline was essential for data collection.
As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of ‘microdomains’. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.
fatty-acid-binding proteins; parasitic nematodes; Ascaris suum; microfocus beamlines
Na-FAR-1, a fatty acid- and retinol-binding protein, was expressed in bacteria, purified and crystallized. Crystals grew in two different morphologies under the same conditions.
Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.
fatty acid- and retinol-binding proteins; parasitic nematodes; Necator americanus; Na-FAR-1
Ranasmurfin is an unusual blue protein isolated from the nests of a Malaysian tree frog, Polypedates leucomystax, showing the rich chemical diversity displayed by biomolecular foams. Many species of tropical frogs use foams to protect delicate eggs and developing embryos against environmental challenges. These nests act as miniature ecosystems containing a spectrum of novel proteins and other macromolecules with functions related to foam stabilization and adhesion, resistance to microbial degradation, predation, or dehydration, providing a biocompatible environment for embryonic development.Thisworkformspartofourwiderstudyofthe intriguing physical and chemical properties of biofoams as unusual examples of biological soft matter.
Frogs that build foam nests floating on water face the problems of over-dispersion of the secretions used and eggs being dangerously exposed at the foam : air interface. Nest construction behaviour of túngara frogs, Engystomops pustulosus, has features that may circumvent these problems. Pairs build nests in periodic bursts of foam production and egg deposition, three discrete phases being discernible. The first is characterized by a bubble raft without egg deposition and an approximately linear increase in duration of mixing events with time. This phase may reduce initial over-dispersion of foam precursor materials until a critical concentration is achieved. The main building phase is marked by mixing events and start-to-start intervals being nearly constant in duration. During the final phase, mixing events do not change in duration but intervals between them increase in an exponential-like fashion. Pairs joining a colonial nesting abbreviate their initial phase, presumably by exploiting a pioneer pair's bubble raft, thereby reducing energy and material expenditure, and time exposed to predators. Finally, eggs are deposited only in the centre of nests with a continuously produced, approximately 1 cm deep egg-free cortex that protectively encloses hatched larvae in stranded nests.
Amphibia; túngara frog; Engystomops pustulosus; foam nest; nest construction behaviour
Naturally occurring foam constituent and surfactant proteins with intriguing structures and functions are now being identified from a variety of biological sources. The ranaspumins from tropical frog foam nests comprise a range of proteins with a mixture of surfactant, carbohydrate binding and antimicrobial activities that together provide a stable, biocompatible, protective foam environment for developing eggs and embryos. Ranasmurfin, a blue protein from a different species of frog, displays a novel structure with a unique chromophoric crosslink. Latherin, primarily from horse sweat, but with similarities to salivary, oral and upper respiratory tract proteins, illustrates several potential roles for surfactant proteins in mammalian systems. These proteins, together with the previously discovered hydrophobins of fungi, throw new light on biomolecular processes at air–water and other interfaces. This review provides a perspective on these recent findings, focussing on structure and biophysical properties.
Foam; Frog; Nest; Latherin; Ranaspumin; Ranasmurfin
Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L4) H. polygyrus cDNA expression library using antibodies raised against L4 stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.
Heligmosomoides polygyrus; host-parasitic relationship; Hp-FAR-1; hp-far-1; lifecycle; molecular biology; nematode; retinol binding
Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg ml−1), and therefore probably acts as a wetting agent to facilitate evaporative cooling through a waterproofed pelt. Neutron reflection experiments indicate that this detergent-like activity is associated with the formation of a dense protein layer, about 10 Å thick, at the air-water interface. However, biophysical characterization (circular dichroism, differential scanning calorimetry) in solution shows that latherin behaves like a typical globular protein, although with unusual intrinsic fluorescence characteristics, suggesting that significant conformational change or unfolding of the protein is required for assembly of the air-water interfacial layer. RT-PCR screening revealed latherin transcripts in horse skin and salivary gland but in no other tissues. Recombinant latherin produced in bacteria was also found to be the target of IgE antibody from horse-allergic subjects. Equids therefore may have adapted an oral/salivary mucosal protein for two purposes peculiar to their lifestyle, namely their need for rapid and efficient heat dissipation and their specialisation for masticating and processing large quantities of dry food material.
The foam nests of the túngara frog (Engystomops pustulosus) form a biocompatible incubation medium for eggs and sperm while resisting considerable environmental and microbiological assault. We have shown that much of this behaviour can be attributed to a cocktail of six proteins, designated ranaspumins (Rsn-1 to Rsn-6), which predominate in the foam. These fall into two discernable classes based on sequence analysis and biophysical properties. Rsn-2, with an amphiphilic amino acid sequence unlike any hitherto reported, exhibits substantial detergent-like surfactant activity necessary for production of foam, yet is harmless to the membranes of eggs and spermatozoa. A further four (Rsn-3 to Rsn-6) are lectins, three of which are similar to fucolectins found in teleosts but not previously identified in a land vertebrate, though with a carbohydrate binding specificity different from previously described fucolectins. The sixth, Rsn-1, is structurally similar to proteinase inhibitors of the cystatin class, but does not itself appear to exhibit any such activity. The nest foam itself, however, does exhibit potent cystatin activity. Rsn-encoding genes are transcribed in many tissues of the adult frogs, but the full cocktail is present only in oviduct glands. Combinations of lectins and cystatins have known roles in plants and animals for defence against microbial colonization and insect attack. Túngara nest foam displays a novel synergy of selected elements of innate defence plus a specialized surfactant protein, comprising a previously unreported strategy for protection of unattended reproductive stages of animals.
túngara frog; Engystomops pustulosus; foam nests; lectins; surfactant protein; cystatin activity
Summary: Infection of humans with the nematode worm parasite Anisakis simplex was first described in the 1960s in association with the consumption of raw or undercooked fish. During the 1990s it was realized that even the ingestion of dead worms in food fish can cause severe hypersensitivity reactions, that these may be more prevalent than infection itself, and that this outcome could be associated with food preparations previously considered safe. Not only may allergic symptoms arise from infection by the parasites (“gastroallergic anisakiasis”), but true anaphylactic reactions can also occur following exposure to allergens from dead worms by food-borne, airborne, or skin contact routes. This review discusses A. simplex pathogenesis in humans, covering immune hypersensitivity reactions both in the context of a living infection and in terms of exposure to its allergens by other routes. Over the last 20 years, several studies have concentrated on A. simplex antigen characterization and innate as well as adaptive immune response to this parasite. Molecular characterization of Anisakis allergens and isolation of their encoding cDNAs is now an active field of research that should provide improved diagnostic tools in addition to tools with which to enhance our understanding of pathogenesis and controversial aspects of A. simplex allergy. We also discuss the potential relevance of parasite products such as allergens, proteinases, and proteinase inhibitors and the activation of basophils, eosinophils, and mast cells in the induction of A. simplex-related immune hypersensitivity states induced by exposure to the parasite, dead or alive.
A novel blue protein from frog nests has been crystallized.
Ranasmurfin, a previously uncharacterized ∼13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 Å with P21 symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 Å, β = 93.3°. Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.
Egg quality is a phenotype of, and can profoundly influence fitness in, both mother and offspring. However, the physiological mechanisms that underlie this maternal effect are poorly understood. Carotenoids are hypothesized to enhance antioxidant activity and immune function, and are responsible for the pigmentation of egg yolk. The proximate basis and consequences of this maternal investment, however, have not previously been studied in wild birds. In this supplemental feeding study of lesser black-backed gulls, Larus fuscus, carotenoid-fed females are shown to have increased integument pigmentation, higher plasma concentrations of carotenoids and antioxidant activity, and lower plasma concentrations of immunoglobulins (Igs) in comparison with controls. In turn, carotenoid-fed females produced eggs containing high carotenoid but low Ig concentrations (i.e. passive immunity), whereas control females produced eggs containing low carotenoid but high Ig concentrations. Within-clutch patterns of these resources varied over the laying sequence in a similar manner in both carotenoid-fed and control nests. Our results suggest that carotenoids could be one resource responsible for egg quality maternal effects in birds. We discuss the possible implications of carotenoid-mediated effects on phenotype for fitness in mothers and their offspring.
Helminth infections are among the most potent stimulators of Th2-type immune responses and have been widely demonstrated to modify responsiveness to both nonparasite antigens and other infectious agents in a nonspecific manner in infected animals. We investigated the immunomodulatory properties of pseudocoelomic body fluid from adult Ascaris suum gastrointestinal helminths (ABF) and its defined allergen (ABA-1) by examining their effects on the immune response to a heterologous antigen, ovalbumin. Our results indicate that ABF has potent immunomodulatory activity and that the effects observed are consistent with skewing towards a Th2-type response rather than induction of anergy. Our findings show that the immunomodulatory activities of ABF are associated with components other than the major constituent and putative allergen, ABA-1. Furthermore, the allergic responses to ABA-1 are not a result of an intrinsic allergenicity of the protein but are more a reflection of the wider induction of a Th2 response by the infection. Importantly, the induction of interleukin-10 by ABF also suggests that T regulatory cells may play a role in immunomodulation of immune responses by parasitic helminths.
Children putatively immune to the large roundworm Ascaris lumbricoides were identified in an area of Nigeria where infection is hyperendemic. Immunity was associated with higher levels of serum ferritin, C-reactive protein, and eosinophil cationic protein, indicating ongoing acute phase or inflammatory processes. In contrast, children who were susceptible to the infection had little serological evidence of inflammation despite their high parasite burdens. Immunoglobulin G (IgG) antibody activity in all subclasses was present in high titer in most children but appeared to have no protective function. Despite exceptionally high total IgE levels, there was no evidence that atopic responses to local common allergens was associated with natural immunity to Ascaris. Among those individuals who produced IgG antibody to recombinant ABA-1 allergen of Ascaris, the naturally immune group had significantly more IgE antibody to the allergen than did those susceptible to the infection. IgE antibody responses in conjunction with innate inflammatory processes therefore appear to associate with natural immunity to ascariasis.
Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP) family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious.
We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs).
This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.
Echinococcus granulosus is the causative agent of hydatidosis, a zoonotic infection that affects humans and livestock, representing a public health and economic burden in many countries. Since the parasites are unable to synthesise most of their lipids de novo, they must acquire them from the host and then deliver them by carrier proteins to specific destinations. E. granulosus produces in abundance proteins of the fatty acid binding protein (FABP) family, one of which, EgFABP1 has been characterised at the structural and ligand binding levels, but it has not been studied in terms of the mechanism of its interaction with membranes. We have investigated the lipid transport properties and protein-membrane interaction characteristics of EgFABP1 by applying biophysical techniques. We found that EgFABP1 interacts with membranes by a mechanism which involves direct contact with them to exchange their cargo. Given that the protein has been found in the secretions of the parasite, the implications of its direct interactions with host membranes should be considered.
Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.
The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.
This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.
Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris, and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structure.